MCB

Polyamines regulate the stability of JunD mRNA by modulating the competitive binding of its 3'-untranslated region to HuR and AUF1.

Publication Date: 2010 Aug 30 PMID: 20805360
Authors: Zou, T. - Rao, J. N. - Liu, L. - Xiao, L. - Yu, T. X. - Jiang, P. - Gorospe, M. - Wang, J. Y.
Journal: Mol Cell Biol

Polyamines critically regulate all mammalian cell growth and proliferation by mechanisms including the repression of growth-inhibitory proteins such as JunD. Decreasing the levels of cellular polyamines stabilizes JunD mRNA without affecting its transcription, but the exact mechanism whereby polyamines regulate JunD mRNA degradation has not been elucidated. RNA-binding proteins HuR and AUF1 associate with labile mRNAs bearing AU-rich elements located in the 3'-untranslated regions (3'-UTRs) and modulate their stability. Here, we show that JunD mRNA is a target of HuR and AUF1 and that polyamines modulate JunD mRNA degradation by altering the competitive binding of HuR and AUF1 to the JunD 3'-UTR. Depletion of cellular polyamines enhanced HuR binding to JunD mRNA and decreased the levels of JunD transcript associated with AUF1, thus stabilizing JunD mRNA. The silencing of HuR increased AUF1 binding to the JunD mRNA, decreased the abundance of [HuR/JunD mRNA] complexes, rendered the JunD mRNA unstable, and prevented increases in JunD mRNA and protein in polyamine-deficient cells. Conversely, increasing the cellular polyamines repressed JunD mRNA interaction with HuR and enhanced its association with AUF1, resulting in an inhibition of JunD expression. These results indicate that polyamines modulate the stability of JunD mRNA in IECs through HuR and AUF1 and provide new insight into the molecular functions of cellular polyamines.

post to: CiteULike

Cortactin modulates RhoA activation and expression of Cip/Kip cyclin dependent kinase inhibitors to promote cell cycle progression in 11q13-amplified head and neck squamous cell carcinoma cells.

Publication Date: 2010 Aug 30 PMID: 20805359
Authors: Croucher, D. R. - Rickwood, D. - Tactacan, C. M. - Musgrove, E. A. - Daly, R. J.
Journal: Mol Cell Biol

The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this manuscript we report that stable shRNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21(WAF1/Cip1), p27(Kip1) and p57(Kip2) and inhibition of S-phase entry. These effects were associated with increased binding of p21(WAF1/Cip1) and p27(Kip1) to cyclin D1- and E1-containing complexes and decreased Rb phosphorylation. Cortactin regulated expression of p21(WAF1/Cip1) and p27(Kip1) at the transcriptional and post-transcriptional level, respectively. The direct roles of p21(WAF1/Cip1), p27(Kip1) and p57(Kip2) downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific siRNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27(Kip1) and p57(Kip2) for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.

post to: CiteULike

REGULATION OF MEMORY CD8 T CELL DIFFERENTIATION BY CDK INHIBITOR p27Kip1.

Publication Date: 2010 Aug 30 PMID: 20805358
Authors: Singh, A. - Jatjek, A. - Plisch, E. H. - Srinivasan, R. - Svaren, J. - Suresh, M.
Journal: Mol Cell Biol

Induction of potent T cell memory is the goal of vaccinations, but the molecular mechanisms that regulate the formation of memory CD8 T cells are not well understood. Despite the recognition that controls of cellular proliferation and apoptosis govern the number of memory T cells, the cell cycle regulatory mechanisms that control these key cellular processes in CD8 T cells during an immune response are poorly defined. Here, we have identified the cyclin-dependent kinase inhibitor p27(Kip1) as a critical regulator of the CD8 T cell homeostasis at all phases of the T cell response to an acute viral infection in mice. By acting as a timer for cell cycle exit, p27(Kip1) curtailed the programmed expansion of interleukin-2-producing memory precursors and markedly limited the magnitude and quality of CD8 T cell memory. In the absence of p27(Kip1), CD8 T cells showed superior recall responses shortly after vaccination with recombinant Listeria monocytogenes. Additionally, we show that p27(Kip1) constrains proliferative renewal of memory CD8 T cells, especially of the effector memory subset. These findings provide critical insights into the cell cycle regulation of CD8 T-cell homeostasis and suggest that modulation of p27(Kip1) could bolster vaccine-induced T cell memory and protective immunity.

post to: CiteULike

The Ubiquitin Carboxyl Hydrolase BAP1 Forms a Ternary Complex with YY1 and HCF-1 and is a Critical Regulator of Gene Expression.

Publication Date: 2010 Aug 30 PMID: 20805357
Authors: Yu, H. - Mashtalir, N. - Daou, S. - Hammond-Martel, I. - Ross, J. - Sui, G. - Hart, G. W. - Rauscher, F. J. 3rd - Drobetsky, E. - Milot, E. - Shi, Y. - Affar, E. B.
Journal: Mol Cell Biol

The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with, and deubiquitinate the transcriptional regulator Host Cell Factor-1 (HCF-1). Here, we show that BAP1 assembles multi-protein complexes containing numerous transcription factors and cofactors including HCF-1 and the transcription factor Yin Yang 1 (YY1). Through its coiled coil motif, BAP1 directly interacts with the zinc fingers of YY1. Moreover, HCF-1 interacts with the middle region of YY1 encompassing the glycine-lysine-rich domain and is essential for the formation of a ternary complex with YY1 and BAP1 in vivo. BAP1 activates transcription in an enzymatic activity-dependent manner and regulates the expression of a variety of genes involved in numerous cellular processes. We further show that BAP1 and HCF-1 are recruited by YY1 to the promoter of cox7c gene, which encodes a mitochondrial protein used here as a model of BAP1-activated gene expression. Our findings (i) establish a direct link between BAP1 and transcriptional control of genes regulating cell growth and proliferation and (ii) shed light on a novel mechanism of transcription regulation involving ubiquitin signaling.

post to: CiteULike

Chromatin Remodeling Around NFRs Leads to Repression of Non-Coding RNA Transcription.

Publication Date: 2010 Aug 30 PMID: 20805356
Authors: Yadon, A. N. - Van De Mark, D. - Basom, R. - Delrow, J. - Whitehouse, I. - Tsukiyama, T.
Journal: Mol Cell Biol

Nucleosome free regions (NFRs) at the 5'- and 3'-end of genes are general sites of transcription initiation for mRNA and non-coding RNA (ncRNA). The presence of NFRs within transcriptional regulatory regions and the conserved location of transcription start sites at NFRs strongly suggest the regulation of NFRs profoundly affects transcription initiation. To date, multiple factors are known to facilitate transcription initiation by positively regulating the formation and/or size of NFRs in vivo. However, mechanisms to repress transcription by negatively regulating the size of NFRs have not been identified. We identified four distinct classes of NFRs located at the 5'-and 3'-end of genes, within ORFs, and far from ORFs. The ATP-dependent chromatin remodeling enzyme Isw2 was enriched at all classes of NFRs. Analysis of RNA levels also demonstrated Isw2 is required to repress ncRNA transcription from many of these NFRs. Thus by the systematic annotation of NFRs across the yeast genome and analysis of ncRNA transcription, we establish, for the first time, a mechanism by which NFR size is negatively regulated to repress ncRNA transcription from NFRs. Finally, we provide evidence suggesting one biological consequence for repression of ncRNA, by Isw2 or the exosome, prevents transcriptional interference of mRNA.

post to: CiteULike

Inhibition of Human Peptide Deformylase Disrupts Mitochondrial Function.

Publication Date: 2010 Aug 30 PMID: 20805355
Authors: Escobar-Alvarez, S. - Gardner, J. - Sheth, A. - Manfredi, G. - Yang, G. - Ouerfelli, O. - Heaney, M. L. - Scheinberg, D. A.
Journal: Mol Cell Biol

Deformylases are metalloproteases in bacteria, plants, and humans that remove the N-formyl-methionine off peptides in vitro. The human homolog, HsPDF, resides in the mitochondria, along with its putative formylated substrates; however the cellular function of HsPDF remains elusive. Here we report on the function of HsPDF in mitochondrial translation and OXPHOS complex biogenesis. Functional HsPDF appears to be necessary for the accumulation of mitochondrial DNA-encoded proteins and assembly of new respiratory complexes containing these proteins. Consequently, inhibition of HsPDF reduces respiratory function and cellular ATP levels, causing dependence on aerobic glycolysis for cell survival. A series of structurally different HsPDF inhibitors and control peptidase inhibitors confirmed that inhibition of HsPDF decreases mtDNA-encoded protein accumulation. Therefore, HsPDF appears to have a role in maintenance of mitochondrial respiratory function, and this function is analogous to that of chloroplast PDF.

post to: CiteULike

The {beta}/Gcd7 subunit of guanine nucleotide exchange factor eIF2B is crucial for binding eIF2 in vivo.

Publication Date: 2010 Aug 30 PMID: 20805354
Authors: Dev, K. - Qiu, H. - Dong, J. - Zhang, F. - Barthlme, D. - Hinnebusch, A. G.
Journal: Mol Cell Biol

eIF2B is the guanine nucleotide exchange factor (GEF) for eukaryotic translation initiation factor 2 (eIF2), which stimulates formation of the eIF2.GTP.Met-tRNAi(Met) ternary complex (TC) in a manner inhibited by phosphorylated eIF2 (eIF2(alphaP)). While eIF2B contains five subunits, the epsilon/Gcd6 subunit is sufficient for GEF activity in vitro. The delta/Gcd2 and beta/Gcd7 subunits function with alpha/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(alphaP)-GDP, but the essential functions of delta/Gcd2 and beta/Gcd7 are not well understood. We show that the depletion of wild-type beta/Gcd7, three lethal beta/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in beta/Gcd7 and eIF2alpha all impair eIF2 binding to eIF2B without reducing epsilon/Gcd6 abundance in the native eIF2B.eIF2 holocomplex. Additionally, beta/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2alpha (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, beta/Gcd7 can overcome the toxicity of eIF2(alphaP) and rescue native eIF2B function when overexpressed with delta/Gcd2 or gamma/Gcd1. In aggregate, these findings provide compelling evidence that beta/Gcd7 is crucial for substrate binding by eIF2B in vivo.

post to: CiteULike

Doubles Game: Src-Stat3 versus p53-PTEN in Cellular Migration and Invasion.

Publication Date: 2010 Aug 23 PMID: 20733006
Authors: Mukhopadhyay, U. K. - Mooney, P. - Jia, L. - Eves, R. - Raptis, L. - Mak, A. S.
Journal: Mol Cell Biol

We have recently shown that Src induces the formation of podosomes and cell invasion by suppressing endogenous p53, while enhanced p53 strongly represses the Src-induced invasive phenotype. However, the mechanism by which Src and p53 play antagonistic roles in cell invasion is unknown. Here, we show that Stat3 oncogene is a required downstream effector of Src in inducing podosome structures and related invasive phenotypes. Stat3 promotes Src-phenotypes through the suppression of p53, and the p53-inducible caldesmon, a known podosome antagonist. In contrast, enhanced p53 attenuates Stat3 function and Src-induced podosome formation by up-regulating PTEN, the tumor suppressor. PTEN, through the inactivation of Src/Stat3 function also stabilizes podosome-antagonizing p53/caldesmon axis and thereby further enhancing the anti-invasive potential of the cell. Furthermore, the protein phosphatase activity of PTEN plays a major role in the negative regulation of the Src/Stat3 pathway and represses podosome formation. Our data suggests that cellular invasiveness is dependent upon the balance between two opposing forces, pro-invasive oncogenes Src-Stat3 and anti-invasive tumor suppressors p53-PTEN.

post to: CiteULike

Stimulation of Sirt1-regulated FoxO protein function by the ligand-bound vitamin D receptor.

Publication Date: 2010 Aug 23 PMID: 20733005
Authors: An, B. S. - Tavera-Mendoza, L. E. - Dimitrov, V. - Wang, X. - Calderon, M. R. - Wang, H. J. - White, J. H.
Journal: Mol Cell Biol

Hormonal vitamin D, 1,25-dihydroxyvitamin D (1,25D), signals through the nuclear vitamin D receptor (VDR). 1,25D regulates cell proliferation and differentiation, and has been identified as a cancer chemopreventive agent. FoxO proteins are transcription factors that control cell proliferation and survival. They function as tumor suppressors and are associated with longevity in several organisms. Accumulating data has revealed that 1,25D and FoxO proteins regulate similarly common target genes. We show here that the ligand-bound VDR regulates post-translational modification and function of FoxO proteins. 1,25D treatment enhances binding of FoxO3a and FoxO4 within 4h to promoters of FoxO target genes, and blocks mitogen-induced FoxO protein nuclear export. The VDR associates directly with FoxO proteins and regulators, the Sirtuin 1 (Sirt1) class III histone deacetylase (HDAC) and protein phosphatase 1. In addition, phosphatase activity and trichostatin A-resistant HDAC activity coimmunoprecipitate with the VDR. 1,25D treatment rapidly (<4h) induces FoxO deacetylation and dephosphorylation, consistent with activation. In contrast, ablation of VDR expression enhances FoxO3a phosphorylation, as does knockdown of Sirt1, consistent with the coupling of FoxO acetylation and phosphorylation. 1,25D regulation of common VDR/FoxO target genes is attenuated by blockade of phosphatase activity, or by siRNA-mediated knockdown of Sirt 1 or FoxO protein expression. Finally, 1,25D-dependent cell cycle arrest is blocked in FoxO3a-deficient cells, indicating that FoxO proteins are key downstream mediators of the antiproliferative actions of 1,25D. These studies link 1,25D signaling through the VDR directly to Sirt1 and FoxO function and provide a molecular basis for its cancer chemopreventive actions.

post to: CiteULike

Drosophila Transcription Factor Tramtrack69 Binds MEP1 to Recruit the Chromatin Remodeler NuRD.

Publication Date: 2010 Aug 23 PMID: 20733004
Authors: Reddy, B. A. - Bajpe, P. K. - Bassett, A. - Moshkin, Y. M. - Kozhevnikova, E. - Bezstarosti, K. - Demmers, J. A. - Travers, A. A. - Verrijzer, C. P.
Journal: Mol Cell Biol

ATP-dependent chromatin remodeling complexes (remodelers) are essential regulators of chromatin structure and gene transcription. How remodelers can act in a gene-selective manner has remained enigmatic. A yeast two-hybrid screen for proteins binding the Drosophila transcription factor Tramtrack69 (TTK69) identified MEP1. Proteomic characterization revealed that MEP1 is a tightly associated subunit of the NuRD remodeler, harboring the Mi2 enzymatic core ATPase. In addition, we identified the fly homolog of human Deleted in Oral Cancer 1 (DOC1), also known as CDK2 Associated Protein 1 (CDK2AP1), as a bona fide NuRD subunit. Biochemical and genetic assays supported the functional association between MEP1, Mi2 and TTK69. Genome-wide expression analysis established that TTK69, MEP1 and Mi2 cooperate closely to control transcription. The TTK69 transcriptome profile correlates poorly with remodelers other than NuRD, emphasizing the selectivity of remodeler action. On the genes examined, TTK69 is able to bind chromatin in the absence of NuRD, but targeting of NuRD is dependent on TTK69. Thus, there appears to be a hierarchical relationship in which transcription factor binding precedes remodeler recruitment.

post to: CiteULike

Loss of Par-1a/MARK3/C-TAK1 kinase leads to reduced adiposity, resistance to hepatic steatosis and defective gluconeogenesis.

Publication Date: 2010 Aug 23 PMID: 20733003
Authors: Lennerz, J. K. - Hurov, J. B. - White, L. S. - Lewandowski, K. T. - Prior, J. L. - Planer, G. J. - Gereau, R. W. 4th - Piwnica-Worms, D. - Schmidt, R. E. - Piwnica-Worms, H.
Journal: Mol Cell Biol

Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs and mammals. The mammalian Par-1 family consists of four members. Knockout studies in mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning and memory as well as adiposity, insulin hypersensitivity and glucose metabolism. Here we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1), which exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity, displayed attenuated weight gain, complete resistance to hepatic steatosis and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy and increased glycogen synthase levels in Par-1a(-/-), but not in control or Par-1b(-/-) mice. Intercrossing of Par-1a(-/-) with Par-1b(-/-) mice revealed that at least one of the four alleles is necessary for embryonic survival. Severity of phenotypes followed a rank-order whereby loss of one Par-1b allele in Par-1a(-/-) mice conveyed milder phenotypes than loss of one Par-1a allele in Par-1b(-/-) mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice.

post to: CiteULike

Protein misfolding induces hypoxic preconditioning via a subset of the unfolded protein response machinery.

Publication Date: 2010 Aug 23 PMID: 20733002
Authors: Mao, X. R. - Crowder, C. M.
Journal: Mol Cell Biol

Prolonged cellular hypoxia results in energy failure and ultimately cell death. However, less severe hypoxia can induce a cytoprotective response termed hypoxic preconditioning (HP). The unfolded protein response pathway (UPR) has been known for some time to respond to hypoxia and regulate hypoxic sensitivity; however, the role of the UPR, if any, in HP has been essentially unexplored. We have previously shown that a sublethal hypoxic exposure of the nematode C. elegans induces a protein chaperone component of the UPR. Here, we show that HP induces the UPR and that pharmacological induction of misfolded proteins is itself sufficient to stimulate a delayed protective response to hypoxic injury that requires the UPR pathway proteins IRE-1, XBP-1, and ATF-6. HP also required IRE-1 but not XBP-1 nor ATF-6; instead GCN-2, which is known to suppress translation and induce an adaptive transcriptional response under conditions of UPR activation or amino acid deprivation, was required for HP. Phosphorylation of the translation factor EIF2alpha, an established mechanism of GCN-2 mediated translational suppression, was not necessary for HP. These data suggest a model where hypoxia-induced misfolded proteins trigger activation of IRE-1, which along with GCN-2 controls an adaptive response essential to HP.

post to: CiteULike

Insulin regulates adipocyte lipolysis via an Akt-independent signaling pathway.

Publication Date: 2010 Aug 23 PMID: 20733001
Authors: Choi, S. M. - Tucker, D. F. - Gross, D. N. - Easton, R. M. - Dipilato, L. M. - Dean, A. S. - Monks, B. R. - Birnbaum, M. J.
Journal: Mol Cell Biol

After a meal, insulin suppresses lipolysis through activation of its downstream kinase, Akt, resulting in inhibition of protein kinase A (PKA), the main positive effector of lipolysis. During insulin resistance, this process is ineffective, leading to a characteristic dyslipidemia and worsening of impaired insulin action and obesity. Here, we describe a non-canonical Akt-independent, phosphoinositide-3 kinase (PI3K)-dependent pathway that regulates adipocyte lipolysis using restricted subcellular signaling. This pathway selectively alters PKA phosphorylation of its major lipid droplet-associated substrate, perilipin. In contrast, phosphorylation of another PKA substrate, hormone sensitive lipase (HSL), remains Akt-dependent. Furthermore, insulin regulates total PKA activity in an Akt-dependent manner. These findings indicate that localized changes in insulin action are responsible for the differential phosphorylation of PKA substrates. Thus, we identify a pathway by which insulin regulates lipolysis through spatially compartmentalized modulation of PKA.

post to: CiteULike

PKC{delta}-mediated phosphorylation of phospholipase D controls integrin-mediated cell spreading.

Publication Date: 2010 Aug 23 PMID: 20733000
Authors: Chae, Y. C. - Kim, K. L. - Ha, S. H. - Kim, J. - Suh, P. G. - Ryu, S. H.
Journal: Mol Cell Biol

Integrin signaling plays critical roles in cell adhesion, spreading, and migration and it is generally accepted that to regulate these integrin functions accurately, localized actin remodeling is required. However, the molecular mechanisms that control the targeting of actin regulation molecules to proper sites are unknown. We previously demonstrated that integrin-mediated cell spreading and migration on fibronectin is dependent on the localized activation of phospholipase D (PLD). However, the mechanism underlying PLD activation by integrin is largely unknown. Here, we demonstrate that protein kinase Cdelta (PKCdelta) is required for integrin-mediated PLD signaling. After integrin stimulation, PKCdelta is activated and translocated to the edge of lamellipodia, where it colocalizes with PLD2. The abrogation of PKCdelta activity inhibited integrin-induced PLD activation and cell spreading. Finally, we show that Thr566 of PLD2 is directly phosphorylated by PKCdelta, and that PLD2 mutation in this region prevents PLD2 activation, PLD2 translocation to the edge of lamellipodia, Rac translocation and cell spreading after integrin activation. Together, these results suggest that PKCdelta is a primary regulator of integrin-mediated PLD activation via the direct phosphorylation of PLD, which is essential for directing integrin-induced cell spreading.

post to: CiteULike

RFC recruits DNA polymerase {delta} to sites of NER but is not required for PCNA recruitment.

Publication Date: 2010 Aug 16 PMID: 20713449
Authors: Overmeer, R. M. - Gourdin, A. M. - Giglia-Mari, A. - Kool, H. - Houtsmuller, A. B. - Siegal, G. - Fousteri, M. I. - Mullenders, L. H. - Vermeulen, W.
Journal: Mol Cell Biol

Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and post-incision complexes. The post-incision step of NER includes gap filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation and live cell protein dynamic studies we show that RFC is implicated in post-incision NER in mammalian cells. siRNA mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment, yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.

post to: CiteULike

Down-regulation of thrombomodulin, a novel target of Snail, induces tumorigenesis through epithelial-mesenchymal transition.

Publication Date: 2010 Aug 16 PMID: 20713448
Authors: Kao, Y. C. - Wu, L. W. - Shi, C. S. - Chu, C. H. - Huang, C. W. - Kuo, C. P. - Sheu, H. M. - Shi, G. Y. - Wu, H. L.
Journal: Mol Cell Biol

The expression of thrombomodulin (TM), a calcium-dependent adhesion molecule, is frequently down-regulated in various cancer types. However, the mechanism responsible for the low expression level of TM in tumorigenesis is unknown. Here, an inverse expression of TM and Snail was detected in different cancer cell lines. We further confirmed this inverse relation using epithelial-mesenchymal transition cell model in HaCaT and A431 cells. We demonstrated that Snail suppressed TM expression by binding to E-box (CACCTG) in TM promoter. Moreover, TM knockdown by short hairpin RNA disrupted E-cadherin-mediated cell junctions and contributed to tumorigenesis. In calcium switch assay, E-cadherin lost the ability to associate with beta-catenin and accumulated in cytoplasm in TM knockdown cells. Meanwhile, wound healing and invasive assays showed that TM knockdown promoted cell motility. A subcutaneous injection of TM knockdown transfectants into immunocompromised mice induced squamous cell carcinoma-like tumors. Besides, forced expression of murine TM in TM knockdown cells made the cells re-assume epithelial-like morphology and increased calcium-dependent association of E-cadherin and beta-catenin. In conclusion, TM, a novel downstream target of Snail in epithelial-mesenchymal transition, is required for maintaining epithelial morphology and functions as a tumor suppressor.

post to: CiteULike

Tetrahymena telomerase protein p65 induces conformational changes throughout TER and rescues TERT and TER assembly mutants.

Publication Date: 2010 Aug 16 PMID: 20713447
Authors: Berman, A. J. - Gooding, A. R. - Cech, T. R.
Journal: Mol Cell Biol

The biogenesis of the Tetrahymena telomerase ribonucleoprotein particle (RNP) is enhanced by p65, a La family protein. Single-molecule and biochemical studies have uncovered a hierarchical assembly of the RNP, wherein the binding of p65 to stems I and IV of telomerase RNA (TER) causes a conformational change that facilitates the subsequent binding of telomerase reverse transcriptase (TERT) to TER. We used purified p65 and variants of TERT and TER to investigate the conformational rearrangements that occur during RNP assembly. Nuclease protection assays and mutational analysis revealed that p65 interacts with and stimulates conformational changes in regions of TER beyond stem IV. Several TER mutants exhibited telomerase activity only in the presence of p65, revealing the importance of p65 in promoting the correct RNP assembly pathway. In addition, p65 rescued TERT assembly mutants but not TERT activity mutants. Together, these results suggest that p65 stimulates telomerase assembly and activity in two ways. First, by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER. Second, p65 acts as a molecular buttress within the assembled RNP, mutually stabilizing TER and TERT in catalytically active conformations.

post to: CiteULike

Regulation of nucleolar chromatin by B23/nucleophosmin jointly depends upon its RNA binding activity and transcription factor UBF.

Publication Date: 2010 Aug 16 PMID: 20713446
Authors: Hisaoka, M. - Ueshima, S. - Murano, K. - Nagata, K. - Okuwaki, M.
Journal: Mol Cell Biol

Histone chaperones regulate the density of incorporated histone proteins around DNA transcription sites and therefore constitute an important site-specific regulatory mechanism for the control of gene expression. At present the targeting mechanism conferring this site specificity is unknown. We previously reported that the histone chaperone B23/nucleophosmin associates with ribosomal RNA chromatin (r-chromatin) to stimulate rRNA transcription. Here, we report on the mechanism for site-specific targeting of B23 to the r-chromatin. We observed that, during mitosis, B23 was released from chromatin upon inactivation of its RNA binding activity by cdc2 kinase-mediated phosphorylation. The phosphorylation status of B23 was also shown to strongly affect its chromatin binding activity. We further found that r-chromatin binding of B23 was a necessary condition for B23 histone chaperone activity in vivo. In addition, we found that depletion of UBF (an rRNA transcription factor) decreased the chromatin binding affinity of B23, which in turn led to an increase in histone density at the r-chromatin. These two major strands of evidence suggest a novel cell cycle dependent mechanism for the site-specific regulation of histone density via joint RNA- and transcription factor-mediated recruitment of histone chaperones to specific chromosome loci.

post to: CiteULike

Transcriptional Mediator Subunit MED1/TRAP220 in Stromal Cells Is Involved in Hematopoietic Stem/Progenitor Cell Support through Osteopontin Expression.

Publication Date: 2010 Aug 16 PMID: 20713445
Authors: Sumitomo, A. - Ishino, R. - Urahama, N. - Inoue, K. - Yonezawa, K. - Hasegawa, N. - Horie, O. - Matsuoka, H. - Kondo, T. - Roeder, R. G. - Ito, M.
Journal: Mol Cell Biol

MED1/TRAP220, a subunit of the transcriptional Mediator/TRAP complex, is crucial for various biological events through its interaction with distinct activators such as nuclear receptors and GATA family activators. In hematopoiesis, MED1 plays a pivotal role in optimal nuclear receptor-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we present evidence that MED1 in stromal cells is involved in supporting hematopoietic stem and/or progenitor cells (HSPCs) through osteopontin (OPN) expression. We found that the proliferation of bone marrow (BM) cells cocultured with MED1 knockout (Med1(-/-)) mouse embryonic fibroblasts (MEFs) was significantly suppressed when compared to the control. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) was attenuated for BM cells cocultured with Med1(-/-) MEFs. The vitamin D receptor (VDR)- and Runx2-mediated expression of OPN, as well as Mediator recruitment to the Opn promoter, was specifically attenuated in the Med1(-/- )MEFs. Addition of OPN to these MEFs restored the growth of cocultured BM cells and the number of LTC-ICs, both of which were attenuated by the addition of the anti-OPN antibody to Med1(+/+) MEFs and to BM stromal cells. Consequently, MED1 in niche appears to play an important role in supporting HSPCs, by upregulating VDR- and Runx2-mediated transcription on the Opn promoter.

post to: CiteULike

Srs2 plays a critical role in reversible G2 arrest upon chronic and low doses of UV irradiation via two distinct homologous recombination-dependent mechanisms in post-replication repair deficient cells.

Publication Date: 2010 Aug 16 PMID: 20713444
Authors: Hishida, T. - Hirade, Y. - Haruta, N. - Kubota, Y. - Iwasaki, H.
Journal: Mol Cell Biol

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously, it was shown that the loss of RAD6-dependent error-free post-replication repair (PRR) results in DNA damage checkpoint-mediated G2 arrest in cells exposed to chronic low-dose ultraviolet radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G2 arrest or maintenance in PRR-deficient cells, but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G2 arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.

post to: CiteULike

Molecular dissection of TERRA biogenesis unveils the presence of distinct and multiple regulatory pathways.

Publication Date: 2010 Aug 16 PMID: 20713443
Authors: Porro, A. - Feuerhahn, S. - Reichenbach, P. - Lingner, J.
Journal: Mol Cell Biol

Telomeres are transcribed into telomeric repeat containing RNA (TERRA), large heterogeneous non-coding (nc) transcripts, which form part of telomeric heterochromatin. Despite a large number of functions that have been ascribed to TERRA, little is known about its biogenesis. Here, we present the first comprehensive analysis of the molecular structure of TERRA. We identify biochemically distinct TERRA complexes and we describe TERRA regulation during the cell cycle. Moreover, we demonstrate that TERRA 5'-ends contain 7-methylguanosine cap structures and that the poly(A) tail, present on a fraction of TERRA transcripts, contributes to their stability. Poly(A)- TERRA, but not poly(A)+ TERRA is associated with chromatin, possibly reflecting distinct biological roles of TERRA ribonucleoprotein complexes. In support of this idea, poly(A)- and poly(A)+ TERRA molecules end with distinct sequence registers. We also determine that the bulk of 3'-terminal UUAGGG-repeats have an average length of 200 bases, indicating that the length heterogeneity of TERRA likely stems from its subtelomeric regions. Finally, we find that TERRA is regulated during the cell cycle, being lowest in late S phase and peaking in early G1. Our analyses offer the basis for investigating multiple regulatory pathways that affect TERRA synthesis, processing, turnover and function.

post to: CiteULike

Lysine Specific Demethylase 1 (LSD1) Regulates the Embryonic Transcriptome and CoREST Stability.

Publication Date: 2010 Aug 16 PMID: 20713442
Authors: Foster, C. T. - Dovey, O. M. - Lezina, L. - Luo, J. L. - Gant, T. W. - Barlev, N. - Bradley, A. - Cowley, S. M.
Journal: Mol Cell Biol

Lysine specific demethylase 1 (LSD1), which demethylates mono- and di-methylated histone H3-Lys4 as part of a complex including CoREST and histone deacetylases (HDAC), is essential for embryonic development in the mouse beyond e6.5 days. To determine the role of LSD1 during this early period of embryogenesis we have generated loss-of-function genetrap mice and conditional knock-out embryonic stem (ES) cells. Analysis of post-implantation genetrap embryos revealed that LSD1 expression, and therefore function, is restricted to the epiblast. Conditional deletion of LSD1 in mouse ES cells, in vitro counterpart of the epiblast, revealed a reduction in CoREST protein and associated HDAC activity, resulting in a global increase in Histone H3-Lys56 acetylation, but not H3-Lys4 methylation. Despite this biochemical perturbation, LSD1 deleted ES cells proliferate normally and retain stem cell characteristics. Loss of LSD1 causes the aberrant expression of 588 genes, including transcription factors with roles in anterior/posterior patterning and limb development such as brachyury, Hoxb7, Hoxd8 and RARgamma. Brachyury, a key-regulator of mesodermal differentiation, is a direct target gene of LSD1 and is over-expressed in e6.5 day Lsd1 genetrap embryos. Thus, LSD1 regulates the expression and appropriate timing of key developmental regulators, as part of the LSD1/CoREST/HDAC complex, during early embryonic development.

post to: CiteULike

The atypical homeodomain transcription factor Mohawk controls tendon morphogenesis.

Publication Date: 2010 Aug 9 PMID: 20696843
Authors: Liu, W. - Watson, S. S. - Lan, Y. - Keene, D. R. - Ovitt, C. E. - Liu, H. - Schweitzer, R. - Jiang, R.
Journal: Mol Cell Biol

The Mohawk homeobox (Mkx) gene encodes a new atypical homeodomain-containing protein with transcriptional repressor activity. Mkx mRNA exhibited dynamic expression patterns during development of the palate, somite, kidney, and testis, suggesting that it may be an important regulator of multiple developmental processes. To investigate the roles of Mkx in organogenesis, we generated mice carrying a null mutation in this gene. Mkx(-/-) mice survive postnatally and exhibit a unique wavy tail phenotype. Close examination revealed that the mutant mice had smaller tendons than in wildtype littermates and that the rapid postnatal growth of collagen fibrils in tendons was disrupted in Mkx(-/-) mice. Defects in tendon development were detected in the mutant mouse embryos as early as E16.5. Although collagen fibril assembly initially appeared normal, the tendons of Mkx(-/-) embryos expressed significantly reduced amounts of Collagen I, fibromodulin, and tenomodulin, in comparison with control littermates. We found that Mkx mRNA is strongly expressed in differentiating tendon cells during embryogenesis and in the tendon sheath cells in postnatal stages. In addition to defects in tendon collagen fibrillogenesis, Mkx(-/-) mutant mice exhibited abnormal tendon sheaths. These results identify Mkx as an important regulator of tendon development.

post to: CiteULike

CD69 association with Jak3/Stat5 proteins regulates Th17 cell differentiation.

Publication Date: 2010 Aug 9 PMID: 20696842
Authors: Martin, P. - Gomez, M. - Lamana, A. - Cruz-Adalia, A. - Ramirez-Huesca, M. - Ursa, M. A. - Yanez-Mo, M. - Sanchez-Madrid, F.
Journal: Mol Cell Biol

T cell differentiation involves the early decision to commit to a particular pattern of response to an antigen. Herein, we show that the leukocyte activation antigen CD69 limits differentiation to pro-inflammatory helper T cells (Th17). Upon antigen stimulation in vitro, CD4(+) T cells from CD69 deficient mice generate an expansion of Th17 cells and the induction of greater mRNA expression of IL-17, IL-23R and the orphan nuclear receptor RORgammat. In vivo studies with CD69 deficient mice bearing OTII TCRs specific for OVA peptide showed a high proportion of antigen specific Th17 subpopulation in the draining lymph nodes, as well as in CD69-deficient mice immunized with type II collagen. Biochemical analysis demonstrates that CD69 cytoplasmic tail associates with Jak3/Stat5 signalling pathway, which regulates the transcription of RORgammat and consequently, the differentiation towards the Th17 lineage. Functional experiments in Th17 cultures demonstrate that the selective inhibition of Jak3 activation enhanced the transcription of RORgammat. Moreover, the addition of exogenous IL-2 restores Stat5 phosphorylation and inhibits the enhanced Th17 differentiation in CD69-deficient cells. These results support the early activation receptor CD69 as an intrinsic modulator of the T cell differentiation program that conditions immune inflammatory processes.

post to: CiteULike

RhoA Phosphorylation Induces Rac1 Release from GDI{alpha} and Stimulation of Vascular Smooth Muscle Cell Migration.

Publication Date: 2010 Aug 9 PMID: 20696841
Authors: Rolli-Derkinderen, M. - Toumaniantz, G. - Pacaud, P. - Loirand, G.
Journal: Mol Cell Biol

Although over-activation of RhoA is recognized as a common component of vascular disorders, the molecular mechanisms regulating RhoA activity in vascular smooth muscle cells (VSMC) are still unclear. We have previously shown that in VSMC, RhoA was phosphorylated on Ser188 by nitric oxide (NO)-stimulated cGMP-dependent kinase (PKG), which leads to RhoA-Rho kinase pathway inhibition. Here we showed that expression of phosphoresistant RhoA mutants prevented the stimulation of VSMC migration and adhesion induced by NO-PKG-pathway activation. In contrast, under basal conditions, phosphomimetic RhoA mutants stimulated VSMC adhesion and migration through a signaling pathway requiring Rac1 and the Rho exchange factor Vav3. RhoA phosphorylation or phosphomimetic RhoA mutants induced Rac1 activation but did not activate Vav3. Indeed, phosphorylated RhoA or phosphomimetic mutants trapped GDIalpha, leading to the release of Rac1 and its translocation to the membrane where it was then activated by the basal Vav3 nucleotide exchange activity. In vivo, RhoA phosphorylation induced by PKG activation in the aorta of rat treated by sildenafil induced dissociation of Rac1 from GDIalpha and activation of Rac1 signaling pathway. These results suggest that the phosphorylation of RhoA represents a novel potent and physiological GDIalpha-displacement-factor that leads to Rac1 activation and regulation of Rac1-dependent VSMC functions.

post to: CiteULike

Mechanism of Hypoxia Induced NF-kappaB.

Publication Date: 2010 Aug 9 PMID: 20696840
Authors: Culver, C. - Sundqvist, A. - Mudie, S. - Melvin, A. - Xirodimas, D. - Rocha, S.
Journal: Mol Cell Biol

NF-kappaB activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here, we report that induction of IKK activity under hypoxic conditions occurs through a distinct pathway to other common inducers of NF-kappaB, being dependent upon the calcium calmodulin kinase, CaMK2. Similar to inflammatory inducers of NF-kappaB, this process still requires IKK and the IKK kinase TAK1, but the TAK1 associated proteins TAB1 or TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC ubiquitin conjugation system. Unlike other inducers of NF-kappaB, IKK mediated phosphorylation of IkappaBalpha, does not result in its degradation. We show that this results from IkappaBalpha sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48 linked polyubiquitination. Furthermore, inhibition of specific sumo proteases is sufficient to release RelA from IkappaBalpha and activate NF-kappaB target genes. These results define a novel pathway regulating NF-kappaB activation, important to its physiological role in human health and disease.

post to: CiteULike

Cell type-specific activation and repression of PU.1 by a complex of discrete, functionally specialized cis-regulatory elements.

Publication Date: 2010 Aug 9 PMID: 20696839
Authors: Zarnegar, M. A. - Chen, J. - Rothenberg, E. V.
Journal: Mol Cell Biol

The transcription factor PU.1 is critical for multiple hematopoietic lineages, but different leukocyte types require strictly distinct patterns of PU.1 regulation. PU.1 is required early for T-lineage development, but then must be repressed by a stage-specific mechanism correlated with commitment. Other lineages require steady, low expression or upregulation. Until now, only the promoter plus a distal upstream regulatory element (URE) could be invoked to explain nearly all Sfpi1 (PU.1) activation and repression, including bifunctional effects of Runx1. However, the URE is dispensable for most Sfpi1 downregulation in early T-cells, and we show that it retains enhancer activity in immature T-lineage cells even where endogenous Sfpi1 is repressed. We now present evidence for another complex of conserved noncoding elements that mediate discrete, cell type-specific regulatory features of Sfpi1, including a myeloid-specific activating element and a separate, pro-T cell specific silencer element. These elements yield opposite, cell-type specific responses to Runx1. T-cell specific repression requires Runx1 acting through multiple nonconsensus sites in the silencer core. These newly characterized sites recruit Runx1 binding in early T-cells in vivo and define a functionally specific scaffold for dose-dependent, Runx-mediated repression.

post to: CiteULike

Dosage-sensitive regulation of cohesin chromosome binding and dynamics by Nipped-B, Pds5 and Wapl.

Publication Date: 2010 Aug 9 PMID: 20696838
Authors: Gause, M. - Misulovin, Z. - Bilyeu, A. - Dorsett, D.
Journal: Mol Cell Biol

The cohesin protein complex holds sister chromatids together to ensure proper chromosome segregation upon cell division, and also regulates gene transcription. Partial loss of the Nipped-B protein that loads cohesin onto chromosomes, or the Pds5 protein required for sister chromatid cohesion, alters gene expression and organism development, without affecting chromosome segregation. Knowing if reduced Nipped-B or Pds5 dosage changes how much cohesin binds chromosomes, or the stability with which it binds, is critical information for understanding how cohesin regulates transcription. We addressed this question by in vivo fluorescence recovery after photobleaching (FRAP) in Drosophila salivary glands. Cohesin, Nipped-B, and Pds5 all bind chromosomes in both weak and stable modes with residence half-lives of some twenty seconds and six minutes, respectively. Reducing Nipped-B dosage decreases the amount of stable cohesin without affecting its chromosomal residence time, and reducing Pds5 dosage increases the amount of stable cohesin. This argues that Nipped-B and Pds5 regulate transcription by controlling how much cohesin binds DNA in the stable mode, and not binding affinity. We also find that Nipped-B, Pds5, and the Wapl protein that interacts with Pds5 all play unique roles in cohesin chromosome binding.

post to: CiteULike

Architectural epigenetics: Mitotic retention of mammalian transcriptional regulatory information.

Publication Date: 2010 Aug 9 PMID: 20696837
Authors: Zaidi, S. K. - Young, D. W. - Montecino, M. - Lian, J. B. - Stein, J. L. - van Wijnen, A. J. - Stein, G. S.
Journal: Mol Cell Biol

Epigenetic regulatory information must be retained during mammalian cell division to sustain phenotype-specific and physiologically-responsive gene expression in the progeny cells. Histone modifications, DNA methylation, and RNA-mediated silencing are well-defined epigenetic mechanisms that control cellular phenotype by regulating gene expression. Recent results suggest that mitotic retention of nuclease hypersensitivity, selective histone marks, as well as lineage-specific transcription factor occupancy of promoter elements, contribute to epigenetic control of sustained cellular identity in progeny cells. We propose that these mitotic epigenetic signatures collectively constitute architectural epigenetics, a novel and essential mechanism that conveys regulatory information to sustain control of phenotype and proliferation in progeny cells by 'bookmarking' genes for activation or suppression.

post to: CiteULike

Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

Publication Date: 2010 Sep PMID: 20647544
Authors: Zhu, H. - Suk, H. Y. - Yu, R. Y. - Brancho, D. - Olabisi, O. - Yang, T. T. - Yang, X. - Zhang, J. - Moussaif, M. - Durand, J. L. - Jelicks, L. A. - Kim, J. Y. - Scherer, P. E. - Frank, P. G. - Lisanti, M. P. - Calvert, J. W. - Duranski, M. R. - Lefer, D. J. - Huston, E. - Baillie, G. S. - Houslay, M. D. - Molkentin, J. D. - Jin, J. - Chow, C. W.
Journal: Mol Cell Biol

Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

post to: CiteULike

The molecular basis of phospholipase D2-induced chemotaxis: elucidation of differential pathways in macrophages and fibroblasts.

Publication Date: 2010 Sep PMID: 20647543
Authors: Knapek, K. - Frondorf, K. - Post, J. - Short, S. - Cox, D. - Gomez-Cambronero, J.
Journal: Mol Cell Biol

We report the molecular mechanisms that underlie chemotaxis of macrophages and cell migration of fibroblasts, cells that are essential during the body's innate immune response and during wound repair, respectively. Silencing of phospholipase D1 (PLD1) and PLD2 reduced cell migration (both chemokinesis and chemotaxis) by approximately 60% and >80%, respectively; this migration was restored by cell transfection with PLD2 constructs refractory to small interfering RNA (siRNA). Cells overexpressing active phospholipase D1 (PLD1) but, mostly, active PLD2 exhibited cell migration capabilities that were elevated over those elicited by chemoattractants alone. The mechanism for this enhancement is complex. It involves two pathways: one that is dependent on the activity of the lipase (and signals through its product, phosphatidic acid [PA]) and another that involves protein-protein interactions. The first is evidenced by partial abrogation of chemotaxis with lipase activity-defective constructs (PLD2-K758R) and by n-butanol treatment of cells. The second is evidenced by PLD association with the growth factor receptor-bound protein 2 (Grb2) through residue Y(169), located within a Src homology 2 (SH2) consensus site. The association Grb2-PLD2 could be visualized by fluorescence microscopy in RAW/LR5 macrophages concentrated in actin-rich membrane ruffles, making possible that Grb2 serves as a docking or intermediary protein. The Grb2/PLD2-mediated chemotaxis process also depends on Grb2's ability to recognize other motility proteins, like the Wiskott-Aldrich syndrome protein (WASP). Cell transfection with WASP, PLD2, and Grb2 constructs yields the highest levels of cell migration response, particularly in a macrophage cell line (RAW/LR5) and only modestly in the fibroblast cell line COS-7. Further, RAW/LR5 macrophages utilize for cell migration an additional pathway that involves S6 kinase (S6K) through PLD2-Y(296), known to be phosphorylated by epidermal growth factor receptor (EGFR) kinase. Thus, both fibroblasts and macrophages use activity-dependent and activity-independent signaling mechanisms. However, highly mobile cells like macrophages use all signaling machinery available to them to accomplish their required function in rapid immune response, which sets them apart from fibroblasts, cells normally nonmobile that are only briefly involved in wound healing.

post to: CiteULike

The expression profile of the major mouse SPO11 isoforms indicates that SPO11beta introduces double strand breaks and suggests that SPO11alpha has an additional role in prophase in both spermatocytes and oocytes.

Publication Date: 2010 Sep PMID: 20647542
Authors: Bellani, M. A. - Boateng, K. A. - McLeod, D. - Camerini-Otero, R. D.
Journal: Mol Cell Biol

Both in mice and humans, two major SPO11 isoforms are generated by alternative splicing: SPO11alpha (exon 2 skipped) and SPO11beta. Thus, the alternative splicing event must have emerged before the mouse and human lineages diverged and was maintained during 90 million years of evolution, arguing for an essential role for both isoforms. Here we demonstrate that developmental regulation of alternative splicing at the Spo11 locus governs the sequential expression of SPO11 isoforms in male meiotic prophase. Protein quantification in juvenile mice and in prophase mutants indicates that early spermatocytes synthesize primarily SPO11beta. Estimation of the number of SPO11 dimers (betabeta/alphabeta/alphaalpha) in mutants in which spermatocytes undergo a normal number of double strand breaks but arrest in midprophase due to inefficient repair argues for a role for SPO11beta-containing dimers in introducing the breaks in leptonema. Expression kinetics in males suggested a role for SPO11alpha in pachytene/diplotene spermatocytes. Nevertheless, we found that both alternative transcripts can be detected in oocytes throughout prophase I, arguing against a male-specific function for this isoform. Altogether, our data support a role for SPO11alpha in mid- to late prophase, presumably acting as a topoisomerase, that would be conserved in male and female meiocytes.

post to: CiteULike

Targeted 2'-O methylation at a nucleotide within the pseudoknot of telomerase RNA reduces telomerase activity in vivo.

Publication Date: 2010 Sep PMID: 20647541
Authors: Huang, C. - Yu, Y. T.
Journal: Mol Cell Biol

Telomerase RNA is an essential component of telomerase, a ribonucleoprotein enzyme that maintains chromosome ends in most eukaryotes. Here we employ a novel approach, namely, RNA-guided RNA modification, to assess whether introducing 2'-O methylation into telomerase RNA can influence telomerase activity in vivo. We generate specific 2'-O methylation sites in and adjacent to the triple helix (within the conserved pseudoknot structure) of Saccharomyces cerevisiae telomerase RNA (TLC1). We show that 2'-O methylation at U809 reduces telomerase activity, resulting in telomere shortening, whereas 2'-O methylation at A804 or A805 leads to moderate telomere lengthening. Importantly, we also show that targeted 2'-O methylation does not affect TLC1 levels and that 2'-O-methylated TLC1 appears to be efficiently assembled into telomerase ribonucleoprotein. Our results demonstrate that RNA-guided RNA modification is a highly useful approach for modulating telomerase activity.

post to: CiteULike

Las1L is a nucleolar protein required for cell proliferation and ribosome biogenesis.

Publication Date: 2010 Sep PMID: 20647540
Authors: Castle, C. D. - Cassimere, E. K. - Lee, J. - Denicourt, C.
Journal: Mol Cell Biol

Ribosome biogenesis is a highly regulated process ensuring that cell growth (increase in biomass) is coordinated with cell proliferation. The formation of eukaryotic ribosomes is a multistep process initiated by the transcription and processing of rRNA in the nucleolus. Concomitant with this, several preribosomal particles, which transiently associate with numerous nonribosomal factors before mature 60S and 40S subunits are formed and exported in the cytoplasm, are generated. Here we identify Las1L as a previously uncharacterized nucleolar protein required for ribosome biogenesis. Depletion of Las1L causes inhibition of cell proliferation characterized by a G1 arrest dependent on the tumor suppressor p53. Moreover, we demonstrate that Las1L is crucial for ribosome biogenesis and that depletion of Las1L leads to inhibition of rRNA processing and failure to synthesize the mature 28S rRNA. Taken together, our data demonstrate that Las1L is essential for cell proliferation and biogenesis of the 60S ribosomal subunit.

post to: CiteULike

Convergent transcription through a long CAG tract destabilizes repeats and induces apoptosis.

Publication Date: 2010 Sep PMID: 20647539
Authors: Lin, Y. - Leng, M. - Wan, M. - Wilson, J. H.
Journal: Mol Cell Biol

Short repetitive sequences are common in the human genome, and many fall within transcription units. We have previously shown that transcription through CAG repeat tracts destabilizes them in a way that depends on transcription-coupled nucleotide excision repair and mismatch repair. Recent observations that antisense transcription accompanies sense transcription in many human genes led us to test the effects of antisense transcription on triplet repeat instability in human cells. Here, we report that simultaneous sense and antisense transcription (convergent transcription) initiated from two inducible promoters flanking a CAG95 tract in a nonessential gene enhances repeat instability synergistically, arrests the cell cycle, and causes massive cell death via apoptosis. Using chemical inhibitors and small interfering RNA (siRNA) knockdowns, we identified the ATR (ataxia-telangiectasia mutated [ATM] and Rad3 related) signaling pathway as a key mediator of this cellular response. RNA polymerase II, replication protein A (RPA), and components of the ATR signaling pathway accumulate at convergently transcribed repeat tracts, accompanied by phosphorylation of ATR, CHK1, and p53. Cell death depends on simultaneous sense and antisense transcription and is proportional to their relative levels, it requires the presence of the repeat tract, and it occurs in both proliferating and nonproliferating cells. Convergent transcription through a CAG repeat represents a novel mechanism for triggering a cellular stress response, one that is initiated by events at a single locus in the genome and resembles the response to DNA damage.

post to: CiteULike

Single-molecule analysis reveals changes in the DNA replication program for the POU5F1 locus upon human embryonic stem cell differentiation.

Publication Date: 2010 Sep PMID: 20647538
Authors: Schultz, S. S. - Desbordes, S. C. - Du, Z. - Kosiyatrakul, S. - Lipchina, I. - Studer, L. - Schildkraut, C. L.
Journal: Mol Cell Biol

Human embryonic stem cells (hESCs), due to their pluripotent nature, represent a particularly relevant model system to study the relationship between the replication program and differentiation state. Here, we define the basic properties of the replication program in hESCs and compare them to the programs of hESC-derived multipotent cells (neural rosette cells) and primary differentiated cells (microvascular endothelial cells [MECs]). We characterized three genomic loci: two pluripotency regulatory genes, POU5F1 (OCT4) and NANOG, and the IGH locus, a locus that is transcriptionally active specifically in B-lineage cells. We applied a high-resolution approach to capture images of individual replicated DNA molecules. We demonstrate that for the loci studied, several basic properties of replication, including the average speed of replication forks and the average density of initiation sites, were conserved among the cells analyzed. We also demonstrate, for the first time, the presence of initiation zones in hESCs. However, significant differences were evident in other aspects of replication for the DNA segment containing the POU5F1 gene. Specifically, the locations of centers of initiation zones and the direction of replication fork progression through the POU5F1 gene were conserved in two independent hESC lines but were different in hESC-derived multipotent cells and MECs. Thus, our data identify features of the replication program characteristic of hESCs and define specific changes in replication during hESC differentiation.

post to: CiteULike

A novel mechanism for SUMO system control: regulated Ulp1 nucleolar sequestration.

Publication Date: 2010 Sep PMID: 20647537
Authors: Sydorskyy, Y. - Srikumar, T. - Jeram, S. M. - Wheaton, S. - Vizeacoumar, F. J. - Makhnevych, T. - Chong, Y. T. - Gingras, A. C. - Raught, B.
Journal: Mol Cell Biol

The small ubiquitin-related modifiers (SUMOs) are evolutionarily conserved polypeptides that are covalently conjugated to protein targets to modulate their subcellular localization, half-life, or activity. Steady-state SUMO conjugation levels increase in response to many different types of environmental stresses, but how the SUMO system is regulated in response to these insults is not well understood. Here, we characterize a novel mode of SUMO system control: in response to elevated alcohol levels, the Saccharomyces cerevisiae SUMO protease Ulp1 is disengaged from its usual location at the nuclear pore complex (NPC) and sequestered in the nucleolus. We further show that the Ulp1 region previously demonstrated to interact with the karyopherins Kap95 and Kap60 (amino acids 150 to 340) is necessary and sufficient for nucleolar targeting and that enforced sequestration of Ulp1 in the nucleolus significantly increases steady-state SUMO conjugate levels, even in the absence of alcohol. We have thus characterized a novel mechanism of SUMO system control in which the balance between SUMO-conjugating and -deconjugating activities at the NPC is altered in response to stress via relocalization of a SUMO-deconjugating enzyme.

post to: CiteULike

Analysis of Leigh syndrome mutations in the yeast SURF1 homolog reveals a new member of the cytochrome oxidase assembly factor family.

Publication Date: 2010 Sep PMID: 20624914
Authors: Bestwick, M. - Jeong, M. Y. - Khalimonchuk, O. - Kim, H. - Winge, D. R.
Journal: Mol Cell Biol

Three missense SURF1 mutations identified in patients with Leigh syndrome (LS) were evaluated in the yeast homolog Shy1 protein. Introduction of two of the Leigh mutations, F(249)T and Y(344)D, in Shy1 failed to significantly attenuate the function of Shy1 in cytochrome c oxidase (CcO) biogenesis as seen with the human mutations. In contrast, a G(137)E substitution in Shy1 results in a nonfunctional protein conferring a CcO deficiency. The G(137)E Shy1 mutant phenocopied shy1Delta cells in impaired Cox1 hemylation and low mitochondrial copper. A genetic screen for allele-specific suppressors of the G(137)E Shy1 mutant revealed Coa2, Cox10, and a novel factor designated Coa4. Coa2 and Cox10 are previously characterized CcO assembly factors. Coa4 is a twin CX(9)C motif mitochondrial protein localized in the intermembrane space and associated with the inner membrane. Cells lacking Coa4 are depressed in CcO activity but show no impairment in Cox1 maturation or formation of the Shy1-stabilized Cox1 assembly intermediate. To glean insights into the functional role of Coa4 in CcO biogenesis, an unbiased suppressor screen of coa4Delta cells was conducted. Respiratory function of coa4Delta cells was restored by the overexpression of CYC1 encoding cytochrome c. Cyc1 is known to be important at an ill-defined step in the assembly and/or stability of CcO. This new link to Coa4 may begin to further elucidate the role of Cyc1 in CcO biogenesis.

post to: CiteULike

Differential NDR/LATS interactions with the human MOB family reveal a negative role for human MOB2 in the regulation of human NDR kinases.

Publication Date: 2010 Sep PMID: 20624913
Authors: Kohler, R. S. - Schmitz, D. - Cornils, H. - Hemmings, B. A. - Hergovich, A.
Journal: Mol Cell Biol

MOB proteins are integral components of signaling pathways controlling important cellular processes, such as mitotic exit, centrosome duplication, apoptosis, and cell proliferation in eukaryotes. The human MOB protein family consists of six distinct members (human MOB1A [hMOB1A], -1B, -2, -3A, -3B, and -3C), with hMOB1A/B the best studied due to their putative tumor-suppressive functions through the regulation of NDR/LATS kinases. The roles of the other MOB proteins are less well defined. Accordingly, we characterized all six human MOB proteins in the context of NDR/LATS binding and their abilities to activate NDR/LATS kinases. hMOB3A/B/C proteins neither bind nor activate any of the four human NDR/LATS kinases. We found that both hMOB2 and hMOB1A bound to the N-terminal region of NDR1. However, our data suggest that the binding modes differ significantly. Our work revealed that hMOB2 competes with hMOB1A for NDR binding. hMOB2, in contrast to hMOB1A/B, is bound to unphosphorylated NDR. Moreover, RNA interference (RNAi) depletion of hMOB2 resulted in increased NDR kinase activity. Consistent with these findings, hMOB2 overexpression interfered with the functional roles of NDR in death receptor signaling and centrosome overduplication. In summary, our data indicate that hMOB2 is a negative regulator of human NDR kinases in biochemical and biological settings.

post to: CiteULike

Type I phosphatidylinositol phosphate kinase beta regulates focal adhesion disassembly by promoting beta1 integrin endocytosis.

Publication Date: 2010 Sep PMID: 20624912
Authors: Chao, W. T. - Ashcroft, F. - Daquinag, A. C. - Vadakkan, T. - Wei, Z. - Zhang, P. - Dickinson, M. E. - Kunz, J.
Journal: Mol Cell Biol

Cell migration requires the regulated disassembly of focal adhesions, but the underlying mechanisms remain poorly defined. We have previously shown that focal adhesion disassembly requires the dynamin 2- and clathrin-dependent endocytosis of ligand-activated beta1 integrins. Here, we identify type I phosphatidylinositol phosphate kinase beta (PIPKIbeta), an enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)), as a key regulator of this process. We found that knockdown of PIPKIbeta by RNA interference blocks the internalization of active beta1 integrins and impairs focal adhesion turnover and cell migration. These defects are caused by the failure to target the endocytic machinery, including clathrin adaptors and dynamin 2, to focal adhesion sites. As a consequence, depletion of PIPKIbeta blocks clathrin assembly at adhesion plaques and prevents complex formation between dynamin 2 and focal adhesion kinase (FAK), a critical step in focal adhesion turnover. Together, our findings identify PIPKIbeta as a novel regulator of focal adhesion disassembly and suggest that PIPKIbeta spatially regulates integrin endocytosis at adhesion sites to control cell migration.

post to: CiteULike

Asp1, a conserved 1/3 inositol polyphosphate kinase, regulates the dimorphic switch in Schizosaccharomyces pombe.

Publication Date: 2010 Sep PMID: 20624911
Authors: Pohlmann, J. - Fleig, U.
Journal: Mol Cell Biol

The ability to undergo dramatic morphological changes in response to extrinsic cues is conserved in fungi. We have used the model yeast Schizosaccharomyces pombe to determine which intracellular signal regulates the dimorphic switch from the single-cell yeast form to the filamentous invasive growth form. The S. pombe Asp1 protein, a member of the conserved Vip1 1/3 inositol polyphosphate kinase family, is a key regulator of the morphological switch via the cAMP protein kinase A (PKA) pathway. Lack of a functional Asp1 kinase domain abolishes invasive growth which is monopolar, while an increase in Asp1-generated inositol pyrophosphates (PP) increases the cellular response. Remarkably, the Asp1 kinase activity encoded by the N-terminal part of the protein is regulated negatively by the C-terminal domain of Asp1, which has homology to acid histidine phosphatases. Thus, the fine tuning of the cellular response to environmental cues is modulated by the same protein. As the Saccharomyces cerevisiae Asp1 ortholog is also required for the dimorphic switch in this yeast, we propose that Vip1 family members have a general role in regulating fungal dimorphism.

post to: CiteULike

Protein tyrosine kinase 6 directly phosphorylates AKT and promotes AKT activation in response to epidermal growth factor.

Publication Date: 2010 Sep PMID: 20606012
Authors: Zheng, Y. - Peng, M. - Wang, Z. - Asara, J. M. - Tyner, A. L.
Journal: Mol Cell Biol

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.

post to: CiteULike

Independent and cooperative roles of adaptor molecules in proximal signaling during FcepsilonRI-mediated mast cell activation.

Publication Date: 2010 Sep PMID: 20606011
Authors: Kambayashi, T. - Okumura, M. - Baker, R. G. - Hsu, C. J. - Baumgart, T. - Zhang, W. - Koretzky, G. A.
Journal: Mol Cell Biol

Activation through FcepsilonRI, a high-affinity IgE-binding receptor, is critical for mast cell function during allergy. The formation of a multimolecular proximal signaling complex nucleated by the adaptor molecules SLP-76 and LAT1 is required for activation through this receptor. Based on previous T-cell studies, current dogma dictates that LAT1 is required for plasma membrane recruitment and function of SLP-76. Unexpectedly, we found that the recruitment and phosphorylation of SLP-76 were preserved in LAT1(-/-) mast cells and that SLP-76(-/-) and LAT1(-/-) mast cells harbored distinct functional and biochemical defects. The LAT1-like molecule LAT2 was responsible for the preserved membrane localization and phosphorylation of SLP-76 in LAT1(-/-) mast cells. Although LAT2 supported SLP-76 phosphorylation and recruitment to the plasma membrane, LAT2 only partially compensated for LAT1-mediated cell signaling due to its decreased ability to stabilize interactions with phospholipase Cgamma (PLCgamma). Comparison of SLP-76(-/-) LAT1(-/-) and SLP-76(-/-) mast cells revealed that some functions of LAT1 could occur independently of SLP-76. We propose that while SLP-76 and LAT1 depend on each other for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions also mediate a positive signaling pathway downstream of FcepsilonRI in mast cells.

post to: CiteULike

A flexible RNA backbone within the polypyrimidine tract is required for U2AF65 binding and pre-mRNA splicing in vivo.

Publication Date: 2010 Sep PMID: 20606010
Authors: Chen, C. - Zhao, X. - Kierzek, R. - Yu, Y. T.
Journal: Mol Cell Biol

The polypyrimidine tract near the 3' splice site is important for pre-mRNA splicing. Using pseudouridine incorporation and in vivo RNA-guided RNA pseudouridylation, we have identified two important uridines in the polypyrimidine tract of adenovirus pre-mRNA. Conversion of either uridine into pseudouridine leads to a splicing defect in Xenopus oocytes. Using a variety of molecular biology methodologies, we show that the splicing defect is due to the failure of U2AF(65) to recognize the pseudouridylated polypyrimidine tract. This negative impact on splicing is pseudouridine specific, as no effect is observed when the uridine is changed to other naturally occurring nucleotides. Given that pseudouridine favors a C-3'-endo structure, our results suggest that it is backbone flexibility that is key to U2AF binding. Indeed, locking the key uridine in the C-3'-endo configuration while maintaining its uridine identity blocks U2AF(65) binding and splicing. This pseudouridine effect can also be applied to other pre-mRNA polypyrimidine tracts. Thus, our work demonstrates that in vivo binding of U2AF(65) to a polypyrimidine tract requires a flexible RNA backbone.

post to: CiteULike

The early isoform of disabled-1 functions independently of Reelin-mediated tyrosine phosphorylation in chick retina.

Publication Date: 2010 Sep PMID: 20606009
Authors: Gao, Z. - Monckton, E. A. - Glubrecht, D. D. - Logan, C. - Godbout, R.
Journal: Mol Cell Biol

The Reelin-Disabled-1 (Dab1) signaling pathway plays a key role in the positioning of neurons during brain development. Two alternatively spliced Dab1 isoforms have been identified in chick retina and brain: Dab1-E, expressed at early stages of development, and Dab1-L (commonly referred to as Dab1), expressed at later developmental stages. The well-studied Dab1-L serves as an adaptor protein linking Reelin signal to its downstream effectors; however, nothing is known regarding the role of Dab1-E. Here we show that Dab1-E is primarily expressed in proliferating retinal progenitor cells whereas Dab1-L is found exclusively in differentiated neuronal cells. In contrast to Dab1-L, which is tyrosine phosphorylated upon Reelin stimulation, Dab1-E is not tyrosine phosphorylated and may function independently of Reelin. Knockdown of Dab1-E in chick retina results in a significant reduction in the number of proliferating cells and promotes ganglion cell differentiation. Our results demonstrate a role for Dab1-E in the maintenance of the retinal progenitor pool and determination of cell fate.

post to: CiteULike

Deficiency in a glutamine-specific methyltransferase for release factor causes mouse embryonic lethality.

Publication Date: 2010 Sep PMID: 20606008
Authors: Liu, P. - Nie, S. - Li, B. - Yang, Z. Q. - Xu, Z. M. - Fei, J. - Lin, C. - Zeng, R. - Xu, G. L.
Journal: Mol Cell Biol

Biological methylation is a fundamental enzymatic reaction for a variety of substrates in multiple cellular processes. Mammalian N6amt1 was thought to be a homologue of bacterial N(6)-adenine DNA methyltransferases, but its substrate specificity and physiological importance remain elusive. Here, we demonstrate that N6amt1 functions as a protein methyltransferase for the translation termination factor eRF1 in mammalian cells both in vitro and in vivo. Mass spectrometry analysis indicated that about 70% of the endogenous eRF1 is methylated at the glutamine residue of the conserved GGQ motif. To address the physiological significance of eRF1 methylation, we disrupted the N6amt1 gene in the mouse. Loss of N6amt1 led to early embryonic lethality. The postimplantation development of mutant embryos was impaired, resulting in degeneration around embryonic day 6.5. This is in contrast to what occurs in Escherichia coli and Saccharomyces cerevisiae, which can survive without the N6amt1 homologues. Thus, N6amt1 is the first glutamine-specific protein methyltransferase characterized in vivo in mammals and methylation of eRF1 by N6amt1 might be essential for the viability of early embryos.

post to: CiteULike

The embryonic transcription cofactor LBH is a direct target of the Wnt signaling pathway in epithelial development and in aggressive basal subtype breast cancers.

Publication Date: 2010 Sep PMID: 20606007
Authors: Rieger, M. E. - Sims, A. H. - Coats, E. R. - Clarke, R. B. - Briegel, K. J.
Journal: Mol Cell Biol

Limb-bud and heart (LBH) is a novel key transcriptional regulator of vertebrate development. However, the molecular mechanisms upstream of LBH and its role in adult development are unknown. Here we show that in epithelial development, LBH expression is tightly controlled by Wnt signaling. LBH is transcriptionally induced by the canonical Wnt pathway, as evident by the presence of conserved functional T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) binding sites in the LBH locus and rapid beta-catenin-dependent upregulation of endogenous LBH by Wnt3a. In contrast, LBH induction by Wnt/beta-catenin signaling is inhibited by Wnt7a, which in limb development signals through a noncanonical pathway involving Lmx1b. Furthermore, we show that LBH is aberrantly overexpressed in mammary tumors of mouse mammary tumor virus (MMTV)-Wnt1-transgenic mice and in aggressive basal subtype human breast cancers that display Wnt/beta-catenin hyperactivation. Deregulation of LBH in human basal breast cancer appears to be Wnt/beta-catenin dependent, as DKK1 and Wnt7a inhibit LBH expression in breast tumor cells. Overexpression studies indicate that LBH suppresses mammary epithelial cell differentiation, an effect that could contribute to Wnt-induced tumorigenesis. Taken together, our findings link LBH for the first time to the Wnt signaling pathway in both development and cancer and highlight LBH as a potential new marker for therapeutically challenging basal-like breast cancers.

post to: CiteULike

The CRL4Cdt2 ubiquitin ligase mediates the proteolysis of cyclin-dependent kinase inhibitor Xic1 through a direct association with PCNA.

Publication Date: 2010 Sep PMID: 20606006
Authors: Kim, D. H. - Budhavarapu, V. N. - Herrera, C. R. - Nam, H. W. - Kim, Y. S. - Yew, P. R.
Journal: Mol Cell Biol

During DNA polymerase switching, the Xenopus laevis Cip/Kip-type cyclin-dependent kinase inhibitor Xic1 associates with trimeric proliferating cell nuclear antigen (PCNA) and is recruited to chromatin, where it is ubiquitinated and degraded. In this study, we show that the predominant E3 for Xic1 in the egg is the Cul4-DDB1-XCdt2 (Xenopus Cdt2) (CRL4(Cdt2)) ubiquitin ligase. The addition of full-length XCdt2 to the Xenopus extract promotes Xic1 turnover, while the N-terminal domain of XCdt2 (residues 1 to 400) cannot promote Xic1 turnover, despite its ability to bind both Xic1 and DDB1. Further analysis demonstrated that XCdt2 binds directly to PCNA through its C-terminal domain (residues 401 to 710), indicating that this interaction is important for promoting Xic1 turnover. We also identify the cis-acting sequences required for Xic1 binding to Cdt2. Xic1 binds to Cdt2 through two domains (residues 161 to 170 and 179 to 190) directly flanking the Xic1 PCNA binding domain (PIP box) but does not require PIP box sequences (residues 171 to 178). Similarly, human p21 binds to human Cdt2 through residues 156 to 161, adjacent to the p21 PIP box. In addition, we identify five lysine residues (K180, K182, K183, K188, and K193) immediately downstream of the Xic1 PIP box and within the second Cdt2 binding domain as critical sites for Xic1 ubiquitination. Our studies suggest a model in which both the CRL4(Cdt2) E3- and PIP box-containing substrates, like Xic1, are recruited to chromatin through independent direct associations with PCNA.

post to: CiteULike

Myocardin-related transcription factor A is a common mediator of mechanical stress- and neurohumoral stimulation-induced cardiac hypertrophic signaling leading to activation of brain natriuretic peptide gene expression.

Publication Date: 2010 Sep PMID: 20606005
Authors: Kuwahara, K. - Kinoshita, H. - Kuwabara, Y. - Nakagawa, Y. - Usami, S. - Minami, T. - Yamada, Y. - Fujiwara, M. - Nakao, K.
Journal: Mol Cell Biol

Subjecting cardiomyocytes to mechanical stress or neurohumoral stimulation causes cardiac hypertrophy characterized in part by reactivation of the fetal cardiac gene program. Here we demonstrate a new common mechanism by which these stimuli are transduced to a signal activating the hypertrophic gene program. Mechanically stretching cardiomyocytes induced nuclear accumulation of myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), in a Rho- and actin dynamics-dependent manner. Expression of brain natriuretic peptide (BNP) and other SRF-dependent fetal cardiac genes in response to acute mechanical stress was blunted in mice lacking MRTF-A. Hypertrophic responses to chronic pressure overload were also significantly attenuated in mice lacking MRTF-A. Mutation of a newly identified, conserved and functional SRF-binding site within the BNP promoter, or knockdown of MRTF-A, reduced the responsiveness of the BNP promoter to mechanical stretch. Nuclear translocation of MRTF-A was also involved in endothelin-1- and angiotensin-II-induced activation of the BNP promoter. Moreover, mice lacking MRTF-A showed significantly weaker hypertrophic responses to chronic angiotensin II infusion than wild-type mice. Collectively, these findings point to nuclear translocation of MRTF-A as a novel signaling mechanism mediating both mechanical stretch- and neurohumoral stimulation-induced BNP gene expression and hypertrophic responses in cardiac myocytes.

post to: CiteULike

Sortilin facilitates signaling of ciliary neurotrophic factor and related helical type 1 cytokines targeting the gp130/leukemia inhibitory factor receptor beta heterodimer.

Publication Date: 2010 Sep PMID: 20584990
Authors: Larsen, J. V. - Hansen, M. - Moller, B. - Madsen, P. - Scheller, J. - Nielsen, M. - Petersen, C. M.
Journal: Mol Cell Biol

Sortilin is a member of the Vps10p domain family of neuropeptide and neurotrophin binding neuronal receptors. The family members interact with and partly share a variety of ligands and partake in intracellular sorting and protein transport as well as in transmembrane signal transduction. Thus, sortilin mediates the transport of both neurotensin and nerve growth factor and interacts with their respective receptors to facilitate ligand-induced signaling. Here we report that ciliary neurotrophic factor (CNTF), and related ligands targeting the established CNTF receptor alpha, binds to sortilin with high affinity. We find that sortilin may have at least two functions: one is to provide rapid endocytosis and the removal of CNTF, something which is not provided by CNTF receptor alpha, and the other is to facilitate CNTF signaling through the gp130/leukemia inhibitory factor (LIF) receptor beta heterodimeric complex. Interestingly, the latter function is independent of both the CNTF receptor alpha and ligand binding to sortilin but appears to implicate a direct interaction with LIF receptor beta. Thus, sortilin facilitates the signaling of all helical type 1 cytokines, which engage the gp130/LIF receptor beta complex.

post to: CiteULike

Dynamic localization of Fus3 mitogen-activated protein kinase is necessary to evoke appropriate responses and avoid cytotoxic effects.

Publication Date: 2010 Sep PMID: 20584989
Authors: Chen, R. E. - Patterson, J. C. - Goupil, L. S. - Thorner, J.
Journal: Mol Cell Biol

Cellular responses to many external stimuli are mediated by mitogen-activated protein kinases (MAPKs). We investigated whether dynamic intracellular movement contributes to the spatial and temporal characteristics of the responses elicited by a prototypic MAPK, Fus3, in the mating pheromone response pathway in budding yeast (Saccharomyces cerevisiae). Confining Fus3 in the nucleus, via fusion to a histone H2B, reduced MAPK activation and diminished all responses (pheromone-induced gene expression, cell cycle arrest, projection formation, and mating). Elimination of MAPK phosphatases restored more robust outputs for all responses, indicating that nuclear sequestration impedes full MAPK activation but does not abrogate its functional competence. Restricting Fus3 to the plasma membrane, via fusion to a lipid-modified CCaaX motif, led to MAPK hyperactivation yet severely impaired all response outputs. Fus3-CCaaX also caused aberrant cell morphology and a proliferation defect. Unlike similar phenotypes induced by pathway hyperactivation via upstream components, these deleterious effects were independent of the downstream transcription factor Ste12. Thus, appropriate cellular responses require free subcellular MAPK transit to disseminate MAPK activity optimally because preventing dynamic MAPK movement either markedly impaired signal-dependent activation and/or resulted in improper biological outputs.

post to: CiteULike

Opposing roles of Dnmt1 in early- and late-stage murine prostate cancer.

Publication Date: 2010 Sep PMID: 20584988
Authors: Kinney, S. R. - Moser, M. T. - Pascual, M. - Greally, J. M. - Foster, B. A. - Karpf, A. R.
Journal: Mol Cell Biol

Previous studies have shown that tumor progression in the transgenic adenocarcinoma of mouse prostate (TRAMP) model is characterized by global DNA hypomethylation initiated during early-stage disease and locus-specific DNA hypermethylation occurring predominantly in late-stage disease. Here, we utilized Dnmt1 hypomorphic alleles to examine the role of Dnmt1 in normal prostate development and in prostate cancer in TRAMP. Prostate tissue morphology and differentiation status was normal in Dnmt1 hypomorphic mice, despite global DNA hypomethylation. TRAMP; Dnmt1 hypomorphic mice also displayed global DNA hypomethylation, but were characterized by altered tumor phenotype. Specifically, TRAMP; Dnmt1 hypomorphic mice exhibited slightly increased tumor incidence and significantly increased pathological progression at early ages and, conversely, displayed slightly decreased tumor incidence and significantly decreased pathological progression at advanced ages. Remarkably, hypomorphic Dnmt1 expression abrogated local and distant site macrometastases. Thus, Dnmt1 has tumor suppressor activity in early-stage prostate cancer, and oncogenic activity in late stage prostate cancer and metastasis. Consistent with the biological phenotype, epigenomic studies revealed that TRAMP; Dnmt1 hypomorphic mice show dramatically reduced CpG island and promoter DNA hypermethylation in late-stage primary tumors compared to control mice. Taken together, the data reveal a crucial role for Dnmt1 in prostate cancer and suggest that Dnmt1-targeted interventions may have utility specifically for advanced and/or metastatic prostate cancer.

post to: CiteULike

Human Pat1b connects deadenylation with mRNA decapping and controls the assembly of processing bodies.

Publication Date: 2010 Sep PMID: 20584987
Authors: Ozgur, S. - Chekulaeva, M. - Stoecklin, G.
Journal: Mol Cell Biol

In eukaryotic cells, degradation of many mRNAs is initiated by removal of the poly(A) tail followed by decapping and 5'-3' exonucleolytic decay. Although the order of these events is well established, we are still lacking a mechanistic understanding of how deadenylation and decapping are linked. In this report we identify human Pat1b as a protein that is tightly associated with the Ccr4-Caf1-Not deadenylation complex as well as with the Dcp1-Dcp2 decapping complex. In addition, the RNA helicase Rck and Lsm1 proteins interact with human Pat1b. These interactions are mediated via at least three independent domains within Pat1b, suggesting that Pat1b serves as a scaffold protein. By tethering Pat1b to a reporter mRNA, we further provide evidence that Pat1b is also functionally linked to both deadenylation and decapping. Finally, we report that Pat1b strongly induces the formation of processing (P) bodies, cytoplasmic foci that contain most enzymes of the RNA decay machinery. An amino-terminal region within Pat1b serves as an aggregation-prone domain that nucleates P bodies, whereas an acidic domain controls the size of P bodies. Taken together, these findings provide evidence that human Pat1b is a central component of the RNA decay machinery by physically connecting deadenylation with decapping.

post to: CiteULike

miR-375 inhibits differentiation of neurites by lowering HuD levels.

Publication Date: 2010 Sep PMID: 20584986
Authors: Abdelmohsen, K. - Hutchison, E. R. - Lee, E. K. - Kuwano, Y. - Kim, M. M. - Masuda, K. - Srikantan, S. - Subaran, S. S. - Marasa, B. S. - Mattson, M. P. - Gorospe, M.
Journal: Mol Cell Biol

Neuronal development and plasticity are maintained by tightly regulated gene expression programs. Here, we report that the developmentally regulated microRNA miR-375 affects dendrite formation and maintenance. miR-375 overexpression in mouse hippocampus potently reduced dendrite density. We identified the predominantly neuronal RNA-binding protein HuD as a key effector of miR-375 influence on dendrite maintenance. Heterologous reporter analysis verified that miR-375 repressed HuD expression through a specific, evolutionarily conserved site on the HuD 3' untranslated region. miR-375 overexpression lowered both HuD mRNA stability and translation and recapitulated the effects of HuD silencing, which reduced the levels of target proteins with key functions in neuronal signaling and cytoskeleton organization (N-cadherin, PSD-95, RhoA, NCAM1, and integrin alpha1). Moreover, the increase in neurite outgrowth after brain-derived neurotrophic factor (BDNF) treatment was diminished by miR-375 overexpression; this effect was rescued by reexpression of miR-375-refractory HuD. Our findings indicate that miR-375 modulates neuronal HuD expression and function, in turn affecting dendrite abundance.

post to: CiteULike

The C-terminal region of eukaryotic translation initiation factor 3a (eIF3a) promotes mRNA recruitment, scanning, and, together with eIF3j and the eIF3b RNA recognition motif, selection of AUG start codons.

Publication Date: 2010 Sep PMID: 20584985
Authors: Chiu, W. L. - Wagner, S. - Herrmannova, A. - Burela, L. - Zhang, F. - Saini, A. K. - Valasek, L. - Hinnebusch, A. G.
Journal: Mol Cell Biol

The C-terminal domain (CTD) of the a/Tif32 subunit of budding yeast eukaryotic translation initiation factor 3 (eIF3) interacts with eIF3 subunits j/Hcr1 and b/Prt1 and can bind helices 16 to 18 of 18S rRNA, suggesting proximity to the mRNA entry channel of the 40S subunit. We have identified substitutions in the conserved Lys-Glu-Arg-Arg (KERR) motif and in residues of the nearby box6 element of the a/Tif32 CTD that impair mRNA recruitment by 43S preinitiation complexes (PICs) and confer phenotypes indicating defects in scanning and start codon recognition. The normally dispensable CTD of j/Hcr1 is required for its binding to a/Tif32 and to mitigate the growth defects of these a/Tif32 mutants, indicating physical and functional interactions between these two domains. The a/Tif32 CTD and the j/Hcr1 N-terminal domain (NTD) also interact with the RNA recognition motif (RRM) in b/Prt1, and mutations in both subunits that disrupt their interactions with the RRM increase leaky scanning of an AUG codon. These results, and our demonstration that the extreme CTD of a/Tif32 binds to Rps2 and Rps3, lead us to propose that the a/Tif32 CTD directly stabilizes 43S subunit-mRNA interaction and that the b/Prt1-RRM-j/Hcr1-a/Tif32-CTD module binds near the mRNA entry channel and regulates the transition between scanning-conducive and initiation-competent conformations of the PIC.

post to: CiteULike

Islet beta-cell-specific MafA transcription requires the 5'-flanking conserved region 3 control domain.

Publication Date: 2010 Sep PMID: 20584984
Authors: Raum, J. C. - Hunter, C. S. - Artner, I. - Henderson, E. - Guo, M. - Elghazi, L. - Sosa-Pineda, B. - Ogihara, T. - Mirmira, R. G. - Sussel, L. - Stein, R.
Journal: Mol Cell Biol

MafA is a key transcriptional activator of islet beta cells, and its exclusive expression within beta cells of the developing and adult pancreas is distinct among pancreatic regulators. Region 3 (base pairs -8118 to -7750 relative to the transcription start site), one of six conserved 5' cis domains of the MafA promoter, is capable of directing beta-cell-line-selective expression. Transgenic reporters of region 3 alone (R3), sequences spanning regions 1 to 6 (R1-6; base pairs -10428 to +230), and R1-6 lacking R3 (R1-6(DeltaR3)) were generated. Only the R1-6 transgene was active in MafA(+) insulin(+) cells during development and in adult cells. R1-6 also mediated glucose-induced MafA expression. Conversely, pancreatic expression was not observed with the R3 or R1-6(DeltaR3) line, although much of the nonpancreatic expression pattern was shared between the R1-6 and R1-6(DeltaR3) lines. Further support for the importance of R3 was also shown, as the islet regulators Nkx6.1 and Pax6, but not NeuroD1, activated MafA in gel shift, chromatin immunoprecipitation (ChIP), and transfection assays and in vivo mouse knockout models. Lastly, ChIP demonstrated that Pax6 and Pdx-1 also bound to R1 and R6, potentially functioning in pancreatic and nonpancreatic expression. These data highlight the nature of the cis- and trans-acting factors controlling the beta-cell-specific expression of MafA.

post to: CiteULike

Genes of the ecdysone biosynthesis pathway are regulated by the dATAC histone acetyltransferase complex in Drosophila.

Publication Date: 2010 Sep PMID: 20584983
Authors: Pankotai, T. - Popescu, C. - Martin, D. - Grau, B. - Zsindely, N. - Bodai, L. - Tora, L. - Ferrus, A. - Boros, I.
Journal: Mol Cell Biol

Uncovering mechanisms that regulate ecdysone production is an important step toward understanding the regulation of insect metamorphosis and processes in steroid-related pathologies. We report here the transcriptome analysis of Drosophila melanogaster dAda2a and dAda3 mutants, in which subunits of the ATAC acetyltransferase complex are affected. In agreement with the fact that these mutations lead to lethality at the start of metamorphosis, both the ecdysone levels and the ecdysone receptor binding to polytene chromosomes are reduced in these flies. The cytochrome genes (spookier, phantom, disembodied, and shadow) involved in steroid conversion in the ring gland are downregulated, while the gene shade, which is involved in converting ecdysone into its active form in the periphery, is upregulated in these dATAC subunit mutants. Moreover, driven expression of dAda3 at the site of ecdysone synthesis partially rescues dAda3 mutants. Mutants of dAda2b, a subunit of the dSAGA histone acetyltransferase complex, do not share phenotype characteristics and RNA profile alterations with dAda2a mutants, indicating that the ecdysone biosynthesis genes are regulated by dATAC, but not by dSAGA. Thus, we provide one of the first examples of the coordinated regulation of a functionally linked set of genes by the metazoan-specific ATAC complex.

post to: CiteULike

Myristoylation and membrane binding regulate c-Src stability and kinase activity.

Publication Date: 2010 Sep PMID: 20584982
Authors: Patwardhan, P. - Resh, M. D.
Journal: Mol Cell Biol

Myristoylation is critical for membrane association of Src kinases, but a role for myristate in regulating other aspects of Src biology has not been explored. In the c-Abl tyrosine kinase, myristate binds within a hydrophobic pocket at the base of the kinase domain and latches the protein into an autoinhibitory conformation. A similar pocket has been predicted to exist in c-Src, raising the possibility that Src might also be regulated by myristoylation. Here we show that in contrast to the case for c-Abl, myristoylation exerts a positive effect on c-Src kinase activity. We also demonstrate that myristoylation and membrane binding regulate c-Src ubiquitination and degradation. Nonmyristoylated c-Src exhibited reduced kinase activity but had enhanced stability compared to myristoylated c-Src. We then mutated critical residues in the predicted myristate binding pocket of c-Src. Mutation of L360 and/or E486 had no effect on c-Src membrane binding or localization. However, constructs containing a T456A mutation were partially released from the membrane, suggesting that mutagenesis could induce c-Src to undergo an artificial myristoyl switch. All of the pocket mutants exhibited decreased kinase activity. We concluded that myristoylation and the pocket residues regulate c-Src, but in a manner very different from that for c-Abl.

post to: CiteULike

Cross talk between insulin and bone morphogenetic protein signaling systems in brown adipogenesis.

Publication Date: 2010 Sep PMID: 20584981
Authors: Zhang, H. - Schulz, T. J. - Espinoza, D. O. - Huang, T. L. - Emanuelli, B. - Kristiansen, K. - Tseng, Y. H.
Journal: Mol Cell Biol

Both insulin and bone morphogenetic protein (BMP) signaling systems are important for adipocyte differentiation. Analysis of gene expression in BMP7-treated fibroblasts revealed a coordinated change in insulin signaling components by BMP7. To further investigate the cross talk between insulin and BMP signaling systems in brown adipogenesis, we examined the effect of BMP7 in insulin receptor substrate 1 (IRS-1)-deficient brown preadipocytes, which exhibit a severe defect in differentiation. Treatment of these cells with BMP7 for 3 days prior to adipogenic induction restored differentiation and expression of brown adipogenic markers. The high level of adipogenic inhibitor preadipocyte factor 1 (Pref-1) in IRS-1-null cells was markedly reduced by 3 days of BMP7 treatment, and analysis of the 1.3-kb pref-1 promoter revealed 9 putative Smad binding elements (SBEs), suggesting that BMP7 could directly suppress Pref-1 expression, thereby allowing the initiation of the adipogenic program. Using a series of sequential deletion mutants of the pref-1 promoter linked to the luciferase gene and chromatin immunoprecipitation, we demonstrate that the promoter-proximal SBE (-192/-184) was critical in mediating BMP7's suppressive effect on pref-1 transcription. Together, these data suggest cross talk between the insulin and BMP signaling systems by which BMP7 can rescue brown adipogenesis in cells with insulin resistance.

post to: CiteULike

CTCF controls expression and chromatin architecture of the human major histocompatibility complex class II locus.

Publication Date: 2010 Sep PMID: 20584980
Authors: Majumder, P. - Boss, J. M.
Journal: Mol Cell Biol

The major histocompatibility complex class II (MHC-II) locus includes a dense cluster of genes that function to initiate immune responses. Expression of insulator CCCTC binding factor (CTCF) was found to be required for expression of all MHC class II genes associated with antigen presentation. Ten CTCF sites that divide the MHC-II locus into apparent evolutionary domains were identified. To define the role of CTCF in mediating regulation of the MHC II genes, chromatin conformation capture assays, which provide an architectural assessment of a locus, were conducted across the MHC-II region. Depending on whether MHC-II genes and the class II transactivator (CIITA) were being expressed, two CTCF-dependent chromatin architectural states, each with multiple configurations and interactions, were observed. These states included the ability to express MHC-II gene promoter regions to interact with nearby CTCF sites and CTCF sites to interact with each other. Thus, CTCF organizes the MHC-II locus into a novel basal architecture of interacting foci and loop structures that rearranges in the presence of CIITA. Disruption of the rearranged states eradicated expression, suggesting that the formation of these structures is key to coregulation of MHC-II genes and the locus.

post to: CiteULike

Phosphoinositide-dependent kinase 1 provides negative feedback inhibition to Toll-like receptor-mediated NF-kappaB activation in macrophages.

Publication Date: 2010 Sep PMID: 20584979
Authors: Chaurasia, B. - Mauer, J. - Koch, L. - Goldau, J. - Kock, A. S. - Bruning, J. C.
Journal: Mol Cell Biol

Phosphoinositide-dependent kinase 1 (PDK-1) represents an important signaling component in the phosphatidylinositol 3-kinase (PI3K) pathway, which plays an essential role in controlling a coordinated innate immune response. Here, we show that mice with conditional disruption of PDK-1 specifically in myeloid lineage cells (PDK-1(Deltamyel) mice) show enhanced susceptibility to lipopolysaccharide (LPS)-induced septic shock accompanied by exaggerated liver failure. Furthermore, primary macrophages derived from PDK-1(Deltamyel) mice lack LPS- and Pam3CSK4-stimulated AKT activity but exhibit increased mRNA expression and release of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Moreover, LPS- and Pam3CSK4-stimulated primary macrophages exhibit enhanced phosphorylation and degradation of IkappaBalpha. While immediate upstream Toll-like receptor 4 (TLR-4)-induced signaling, including IL-1 receptor (IL-1R)-associated protein kinase (IRAK) phosphorylation, is unaltered in the absence of PDK-1, macrophages from PDK-1(Deltamyel) mice exhibit prolonged ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF-6) in response to LPS stimulation. These experiments reveal a novel PDK-1-dependent negative feedback inhibition of TLR-induced NF-kappaB activation in macrophages in vivo.

post to: CiteULike

Ikaros and Aiolos inhibit pre-B-cell proliferation by directly suppressing c-Myc expression.

Publication Date: 2010 Sep PMID: 20566697
Authors: Ma, S. - Pathak, S. - Mandal, M. - Trinh, L. - Clark, M. R. - Lu, R.
Journal: Mol Cell Biol

Pre-B-cell expansion is driven by signals from the interleukin-7 receptor and the pre-B-cell receptor and is dependent on cyclin D3 and c-Myc. We have shown previously that interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to suppress pre-B-cell proliferation. However, the molecular mechanisms through which Ikaros and Aiolos exert their growth inhibitory effect remain to be determined. Here, we provide evidence that Aiolos and Ikaros bind to the c-Myc promoter in vivo and directly suppress c-Myc expression in pre-B cells. We further show that downregulation of c-Myc is critical for the growth-inhibitory effect of Ikaros and Aiolos. Ikaros and Aiolos also induce expression of p27 and downregulate cyclin D3 in pre-B cells, and the growth-inhibitory effect of Ikaros and Aiolos is compromised in the absence of p27. A time course analysis further reveals that downregulation of c-Myc by Ikaros and Aiolos precedes p27 induction and cyclin D3 downregulation. Moreover, downregulation of c-Myc by Ikaros and Aiolos is necessary for the induction of p27 and downregulation of cyclin D3. Collectively, our studies identify a pre-B-cell receptor signaling induced inhibitory network, orchestrated by Ikaros and Aiolos, which functions to terminate pre-B-cell expansion.

post to: CiteULike

Regulation of the Rho family small GTPase Wrch-1/RhoU by C-terminal tyrosine phosphorylation requires Src.

Publication Date: 2010 Sep PMID: 20547754
Authors: Alan, J. K. - Berzat, A. C. - Dewar, B. J. - Graves, L. M. - Cox, A. D.
Journal: Mol Cell Biol

Wrch-1 is an atypical Rho family small GTPase with roles in migration, epithelial cell morphogenesis, osteoclastogenesis, and oncogenic transformation. Here, we observed rapid relocalization of Wrch-1 from the plasma membrane upon serum stimulation. Studies revealed a requirement for serum-stimulated tyrosine phosphorylation of Wrch-1 at residue Y254 within its C-terminal membrane targeting domain, mediated by the nonreceptor tyrosine kinase Src. Genetic or pharmacological loss of Src kinase activity blocked both phosphorylation and relocalization of Wrch-1. Functionally, Y254 was required for proper Wrch-1 modulation of cystogenesis in three-dimensional culture, and the phospho-deficient mutant, Y254F, was enhanced in Wrch-1-mediated anchorage-independent growth. Mechanistically, C-terminal tyrosine phosphorylation and subsequent relocalization of Wrch-1 downregulated its ability to interact with and activate its effectors by decreasing active Wrch-1-GTP, perhaps by altering proximity to a GEF or GAP. Phospho-deficient Wrch-1(Y254F) remained at the plasma membrane and GTP bound and continued to recruit and activate its effector PAK, even upon serum stimulation. In contrast, a phospho-mimetic mutant, Y254E, was constitutively endosomally localized and GDP bound and failed to recruit PAK unless mutated to be constitutively active/GAP insensitive. C-terminal tyrosine phosphorylation thus represents a new paradigm in posttranslational control of small GTPase localization, activation, and biological function.

post to: CiteULike