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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Despite the growing body of evidence supporting prolactin (PRL) actions in human breast cancer, little is known regarding PRL regulation of its own receptor in these cells. Ligand-initiated endocytosis is a key process in the regulation of receptor availability and signaling cascades that may lead to oncogenic actions. Although exposure to exogenous PRL accelerates degradation of the long isoform of the PRL receptor (lPRLR), neither the signals initiated by PRL that lead to lPRLR internalization and subsequent down-regulation, nor the relationship to downstream pathways are understood in breast cancer cells. In this study, we showed that PRL-induced down-regulation of the lPRLR was reduced by inhibition of src family kinases (SFKs), but not Janus kinase 2, in MCF-7 cells. Inhibition of SFKs also resulted in accumulation of a PRL-induced PRLR fragment containing the extracellular domain, which appeared to be generated from newly synthesized PRLR. lPRLR was constitutively associated with SFKs in lipid rafts. PRL-induced SFK activation led to recruitment of the guanosine triphosphatase, dynamin-2, to an internalization complex, resulting in endocytosis. Inhibition of endocytosis by small interfering RNA-mediated knockdown of dynamin-2 blocked PRL-induced down-regulation of lPRLR, confirming that internalization is essential for this process. Endocytosis also was required for optimal phosphorylation of ERK1/2 and Akt, but not for Janus kinase 2 or signal transducer and activator of transcription 5, indicating that internalization selectively modulates signaling cascades. Together, these data indicate that SFKs are key mediators of ligand-initiated lPRLR internalization, down-regulation, and signal transduction in breast cancer cells, and underscore the importance of target cell context in receptor trafficking and signal transduction.
Mol Endocrinol 2009 Feb
PMID:SRC family kinases accelerate prolactin receptor internalization, modulating trafficking and signaling in breast cancer cells. 1905 63

The association between novel Src homology 2-containing protein (NSP) and Crk-associated substrate (Cas) family members contributes to integrin and receptor tyrosine kinase signalling and is involved in conferring anti-oestrogen resistance to human breast carcinomas. The precise role of this association in tumorigenesis remains controversial, and the molecular basis for the complex NSP and Cas protein form is unknown. Here we present a pluridisciplinary approach, including small-angle X-ray scattering, that provides first insights into the structure of the complex formed between breast cancer anti-oestrogen resistance 3 (BCAR3, an NSP family member) and human enhancer of filamentation 1 (HEF1, also named NEDD9 or Cas-L, a Cas family protein). Our analysis corroborates a four-helix bundle structure for the NSP-binding domain of HEF1 and a Cdc25-like guanine nucleotide exchange factor (GEF) fold for the Cas-binding domain of BCAR3. Using residues located on helix 2 of the four-helix bundle, HEF1 binds very tightly to a site on BCAR3 that is remote from the putative guanosine triphosphatase binding site of the GEF domain, but similar to a site implicated in allosteric regulation of the homologous SOS (Son of Sevenless) GEF domain. Thus, the association between NSP and Cas proteins might not only create a very stable link between these molecules, co-localising their cellular functions, but also modulate the function of the NSP GEF domains. Such modulation may explain, at least in part, the controversial results published for NSP GEF function.
J Mol Biol 2009 Feb 13
PMID:Structural insights into the association between BCAR3 and Cas family members, an atypical complex implicated in anti-oestrogen resistance. 1910 5

p190-A and -B Rho GAPs (guanosine triphosphatase activating proteins) are the only cytoplasmatic proteins containing FF domains. In p190-A Rho GAP, the region containing the FF domains has been implicated in binding to the transcription factor TFII-I. Moreover, phosphorylation of Tyr308 within the first FF domain inhibits this interaction. Because the structural determinants governing this mechanism remain unknown, we sought to solve the structure of the first FF domain of p190-A Rho GAP (RhoGAPFF1) and to study the potential impact of phosphorylation on the structure. We found that RhoGAPFF1 does not fold with the typical (alpha1-alpha2-3(10)-alpha 3) arrangement of other FF domains. Instead, the NMR data obtained at 285 K show an alpha1-alpha2-alpha 3-alpha 4 topology. In addition, we observed that specific contacts between residues in the first loop and the fourth helix are indispensable for the correct folding and stability of this domain. The structure also revealed that Tyr308 contributes to the domain hydrophobic core. Furthermore, the residues that compose the target motif of the platelet-derived growth factor receptor alpha kinase form part of the alpha 3 helix. We observed that the phosphorylation reaction requires a previous step including domain unfolding, a process that occurs at 310 K. In the absence of phosphorylation, the temperature-dependent RhoGAPFF1 folding/unfolding process is reversible. However, phosphorylation causes an irreversible destabilization of the RhoGAPFF1 structure, which probably accounts for the inhibitory effect that it exerts on the TFII-I interaction. Our results link the ability of a protein domain to be phosphorylated with conformational changes in its three-dimensional structure.
J Mol Biol 2009 Jun 05
PMID:NMR structural studies on human p190-A RhoGAPFF1 revealed that domain phosphorylation by the PDGF-receptor alpha requires its previous unfolding. 1939 45

Late expression factor 4 (LEF4) is one of the four identified subunits of Autographa californica nucleopolyhedrosis virus (AcNPV) encoded RNA polymerase that carries out transcription from viral late and very late promoters. This 464-amino acid baculovirus-encoded protein also harbors 5' mRNA capping activity that includes RNA 5' triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities. Hydrolysis of 5' triphosphate RNA and free NTPs is metal ion dependent property of the protein. In the present communication, we describe the structural changes in the recombinant LEF4 protein following ligand binding. Metal ion binding causes some alteration in the conformation around aromatic amino acids whereas there is no effect on tryptophan fluorescence on GTP binding in absence and presence of metal ion. It is found that GTP and divalent cation cofactor produce some prominent changes in the secondary structure of the protein. Electrophoretic mobility shift assay (EMSA) shows that LEF4 is the probable factor that acts as anchor to dock the viral RNA polymerase on the very late polyhedrin promoter (Ppolh) facilitated by other factors.
Mol Cell Biochem 2010 Jan
PMID:Characterization of LEF4 ligand binding property and its role as part of baculoviral transcription machinery. 1963 19

We have previously observed that a chronic drinking water exposure to monomethylarsonous acid [MMA(III)], a cellular metabolite of inorganic arsenic, increases tumor frequency in the skin of keratin VI/ornithine decarboxylase (K6/ODC) transgenic mice. To characterize gene expression profiles predictive of MMA(III) exposure and mode of action of carcinogenesis, skin and papilloma RNA was isolated from K6/ODC mice administered 0, 10, 50, and 100 ppm MMA(III) in their drinking water for 26 weeks. Following RNA processing, the resulting cRNA samples were hybridized to Affymetrix Mouse Genome 430A 2.0 GeneChips(R). Micoarray data were normalized using MAS 5.0 software, and statistically significant genes were determined using a regularized t-test. Significant changes in bZIP transcription factors, MAP kinase signaling, chromatin remodeling, and lipid metabolism gene transcripts were observed following MMA(III) exposure as determined using the Database for Annotation, Visualization and Integrated Discovery 2.1 (DAVID) (Dennis et al., Genome Biol 2003;4(5):P3). MMA(III) also caused dose-dependent changes in multiple Rho guanine nucleotide triphosphatase (GTPase) and cell cycle related genes as determined by linear regression analyses. Observed increases in transcript abundance of Fosl1, Myc, and Rac1 oncogenes in mouse skin support previous reports on the inducibility of these oncogenes in response to arsenic and support the relevance of these genomic changes in skin tumor induction in the K6/ODC mouse model.
J Biochem Mol Toxicol
PMID:Oncogene expression profiles in K6/ODC mouse skin and papillomas following a chronic exposure to monomethylarsonous acid. 2002 57

The small guanosine triphosphatase RhoA has recently been implicated in the pathogenesis of epilepsy in animals. In this study, we investigated the expression of RhoA in human epilepsy. Brain tissue samples from 40 patients with intractable epilepsy and 14 histologically normal temporal lobes from control patients were used to compare RhoA immunoreactivity using immunohistochemistry, immunofluorescent staining, and western blotting. Immunohistochemical staining and western blot analysis showed that RhoA immunoreactivity was increased in the patient group (p < 0.05). Immunofluorescent staining showed that RhoA was mainly expressed on cell membranes and in the cytoplasm. Our findings demonstrate that upregulation of RhoA immunoreactivity occurs in the brains of patients with intractable epilepsy.
J Mol Neurosci 2010 Sep
PMID:Altered expression of the small guanosine triphosphatase RhoA in human temporal lobe epilepsy. 2014 May 37

An interaction network connecting mRNA capping enzymes, the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD), elongation factor Spt5, and the Cdk7 and Cdk9 protein kinases is thought to comprise a transcription elongation checkpoint. A crux of this network is Spt5, which regulates early transcription elongation and has an imputed role in pre-mRNA processing via its physical association with capping enzymes. Schizosaccharomyces pombe Spt5 has a distinctive CTD composed of tandem nonapeptide repeats of the consensus sequence (1)TPAWNSGSK(9). The Spt5 CTD binds the capping enzymes and is a substrate for threonine phosphorylation by the Cdk9 kinase. Here we report that deletion of the S. pombe Spt5 CTD results in slow growth and aberrant cell morphology. The severity of the spt5-DeltaCTD phenotype is exacerbated by truncation of the Pol II CTD and ameliorated by overexpression of the capping enzymes RNA triphosphatase and RNA guanylyltransferase. These results suggest that the Spt5 and Pol II CTDs play functionally overlapping roles in capping enzyme recruitment. We probed structure-activity relations of the Spt5 CTD by alanine scanning of the consensus nonapeptide. The T1A change abolished CTD phosphorylation by Cdk9 but did not affect CTD binding to the capping enzymes. The T1A and P2A mutations elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects and conferred sensitivity to growth inhibition by 6-azauracil that was exacerbated by partial truncations of the Pol II CTD. The T1A phenotypes were rescued by a phosphomimetic T1E change but not by capping enzyme overexpression. These results imply a positive role for Spt5 CTD phosphorylation in Pol Il transcription elongation in fission yeast, distinct from its capping enzyme interactions. Viability of yeast cells bearing both Spt5 CTD T1A and Pol II CTD S2A mutations heralds that the Cdk9 kinase has an essential target other than Spt5 and Pol II CTD-Ser2.
Mol Cell Biol 2010 May
PMID:Separable functions of the fission yeast Spt5 carboxyl-terminal domain (CTD) in capping enzyme binding and transcription elongation overlap with those of the RNA polymerase II CTD. 2023 61

Shigella flexneri Spa15 is a chaperone of the type 3 secretion system, which binds a number of effectors to ensure their stabilization prior to secretion. One of these effectors is IpgB1, a mimic of the human Ras-like Rho guanosine triphosphatase RhoG. In this study, Spa15 alone and in complex with IpgB1 has been studied by double electron electron resonance, an experiment that gives distance information showing the spacial separation of attached spin labels. This distance is explained by determining the crystal structure of the spin-labeled Spa15 where labels are seen to be buried in hydrophobic pockets. The double electron electron resonance experiment on the Spa15 complex with IpgB1 shows that IpgB1 does not bind Spa15 in the same way as is seen in the homologous Salmonella sp. chaperone:effector complex InvB:SipA.
J Mol Biol 2011 Jan 14
PMID:Shigella flexneri Spa15 crystal structure verified in solution by double electron electron resonance. 2107 16

Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2-dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly-disassembly and function during endocytosis.
J Cell Mol Med 2011 Nov
PMID:Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s. 2112 55

RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.
Mol Biol Cell 2011 Mar 01
PMID:RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes. 2120 20


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