Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The monoclonal antibody MPM-2 recognizes a subset of M phase phosphoproteins in a phosphorylation-dependent manner. It is believed that phosphorylation at MPM-2 antigenic sites could regulate mitotic events since most of the MPM-2 antigens identified to date have M phase functions. In addition, many of these proteins are substrates of the mitotic regulator Pin1, a peptidyl-prolyl isomerase which is present throughout the cell cycle and which is thought to alter its mitotic targets by changing their conformation. In interphase cells, most MPM-2 reactivity is confined to nuclear speckles. We report here that a hyperphosphorylated form of the RNA polymerase II largest subunit is the major MPM-2 interphase antigen. These findings were made possible by the availability of another monoclonal antibody, CC-3, that was previously used to identify a 255 kDa nuclear matrix protein associated with spliceosomal components as a hyperphosphorylated form of the RNA polymerase II largest subunit. MPM-2 recognizes a phosphoepitope of the large subunit that becomes hyperphosphorylated upon heat shock in contrast to the phosphoepitope defined by CC-3, whose reactivity is diminished by the heat treatment. Therefore, these two antibodies may discriminate between distinct functional forms of RNA polymerase II. We also show that RNA polymerase II large subunit interacts with Pin1 in HeLa cells. Pin1 may thus regulate transcriptional and post-transcriptional events by catalyzing phosphorylation-dependent conformational changes of the large RNA polymerase II subunit.
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PMID:A hyperphosphorylated form of RNA polymerase II is the major interphase antigen of the phosphoprotein antibody MPM-2 and interacts with the peptidyl-prolyl isomerase Pin1. 1039 5

A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein. Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3'-end formation. Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD. Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts. We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain. These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism. This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3'-end formation and transcription termination.
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PMID:Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation. 1053 63

The Ess1/Pin1 peptidyl-prolyl isomerase (PPIase) is thought to control mitosis by binding to cell cycle regulatory proteins and altering their activity. Here we isolate temperature-sensitive ess1 mutants and identify six multicopy suppressors that rescue their mitotic-lethal phenotype. None are cell cycle regulators. Instead, five encode proteins involved in transcription that bind DNA, modify chromatin structure or are regulatory subunits of RNA polymerase II. A sixth suppressor, cyclophilin A, is a member of a distinct family of PPIases that are targets of immuno suppressive drugs. We show that the expression of some but not all genes is decreased in ess1 mutants, and that Ess1 interacts with the C-terminal domain (CTD) of RNA polymerase II in vitro and in vivo. The results forge a strong link between PPIases and the transcription machinery and suggest a new model for how Ess1/Pin1 controls mitosis. In this model, Ess1 binds and isomerizes the CTD of RNA polymerase II, thus altering its interaction with proteins required for transcription of essential cell cycle genes.
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PMID:The Ess1 prolyl isomerase is linked to chromatin remodeling complexes and the general transcription machinery. 1089 26

We showed previously that the WW domain of the prolyl isomerase, Ess1, can bind the phosphorylated carboxyl-terminal domain (phospho-CTD) of the largest subunit of RNA Polymerase II. Analysis of phospho-CTD binding by four other WW domain-containing Saccharomyces cerevisiae proteins indicates the splicing factor, Prp40, and the RNA polymerase II ubiquitin ligase, Rsp5, can also bind the phospho-CTD. The identification of Prp40 as a phospho-CTD binding protein represents the first demonstration of direct interaction between a documented splicing factor and the phospho-CTD. Domain dissection studies reveal that phospho-CTD binding occurs at multiple locations in Prp40, including sites in both the WW and FF domain regions. Because the conserved repeats of the CTD make it an ideal ligand for multi-site binding events, the implications of multi-site binding are discussed. Our data suggest a mechanism by which the phospho-CTD of elongating RNA polymerase II facilitates commitment complex formation by juxtaposing the 5' and 3' splice sites.
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PMID:The splicing factor, Prp40, binds the phosphorylated carboxyl-terminal domain of RNA polymerase II. 1097 20

The WW domain is a protein module found in a wide range of signaling proteins. It is one of the smallest protein modules that folds as a monomer without disulfide bridges or cofactors. WW domains bind proteins containing short linear peptide motifs that are proline-rich or contain at least one proline. Although the WW domain was initially considered a 'cytoplasmic module', the proteins containing WW domains have also been localized in the cell nucleus. Moreover, these proteins have been documented to participate in co-activation of transcription and modulation of RNA polymerase II activity. The carboxy-terminal domain (CTD) of RNA polymerase II acts as an assembly platform for distinct WW domain-containing proteins that affect the function of the RNA polymerase II. The formation of complexes between CTD and WW domain-containing proteins is regulated by phosphorylation of the CTD. Since the CTD sequence is highly repetitive and a target of several post-translational modifications and conformational changes, it presents a unique structure capable of enormous molecular diversity. The WW domain has been implicated in several human diseases including Alzheimer's disease. The WW domain-containing iso-prolyl isomerase named Pin1, a protein known to be essential for cell cycle progression, was shown to be active in restoration of the microtubule-binding activity of Tau, a protein of neurofibrillar tangles found in the brains of Alzheimer's patients. It is the WW domain of Pin1 that interacts directly with Tau protein. In addition, the WW domain-containing adapter protein FE65 was shown to regulate processing of Alzheimer's amyloid precursor protein. It is expected that by understanding the details of the WW domain-mediated protein-protein interactions, we will be able to illuminate numerous signaling pathways which control certain aspects of transcription and cell cycle.
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PMID:Functions of WW domains in the nucleus. 1122 34

The yeast prolyl isomerase, Ess1, has recently been shown to interact via its WW domain with the hyperphosphorylated form of the RNA polymerase II C-terminal domain (CTD). We have investigated folding of the Ess1 WW domain and its binding to peptides representing the CTD by circular dichroism and fluorescence. Ess1 WW folds and unfolds reversibly, but in the absence of ligand is only marginally stable with a melting temperature of 19 degrees C. The WW domain is stabilized by the addition of anionic ligands, namely, chloride, inorganic phosphate, phosphoserine, and phosphorylated CTD peptides. Dissociation constants were measured to be 70--100 microM for CTD peptides phosphorylated at one serine, and 16--21 microM for peptides with two or more phosphorylated serines. Weaker or no affinity was observed for nonphosphorylated CTD peptides. There is surprisingly little difference in the affinity for peptides phosphorylated at Ser 2 or Ser 5 of the consensus repeat, or for peptides with different patterns of multiple phosphorylation. The binding of Ess1 to phosphorylated CTD peptides is consistent with a model wherein the WW domain positions Ess1 to catalyze isomerization of the many pSer--Pro peptide bonds in the phosphorylated CTD. We suggest that cis/trans isomerization of prolyl peptide bonds plays a crucial role in CTD function during eukaryotic transcription.
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PMID:Phosphorylation of RNA polymerase II CTD fragments results in tight binding to the WW domain from the yeast prolyl isomerase Ess1. 1145 85

We identify and characterize several phosphorylated forms of the hSpt5 subunit of the DRB sensitivity-inducing factor (DSIF). A 175-kDa phosphorylated form of hSpt5 is bound to nuclei of interphase HeLa cells. This form is rapidly dephosphorylated when cultured cells are exposed to various drugs belonging to distinct chemical families. All these compounds are known to inhibit the protein kinase Cdk9, which phosphorylates in vitro hSpt5 and Rpb1, the largest subunit of RNA polymerase II. The efficiency to promote the dephosphorylation of both proteins matches their capacity to inhibit purified Cdk9 kinase, suggesting that Cdk9 is the major kinase phosphorylating hSpt5 and Rpb1 in vivo. We show that Cdk9 phosphorylates both the CTR1 and the CTR2 domains of recombinant hSpt5. These domains contain numerous serine-proline and threonine-proline residues similar to those found in the carboxyl-terminal domain (CTD) of Rpb1. The structural homology between hSpt5 CTRs and the Rpb1 CTD is further highlighted by the presence on both proteins of a phosphoepitope recognized by the monoclonal antibody CC-3. Of particular interest, the peptidyl-prolyl isomerase Pin1 interacts with Cdk9-phosphorylated hSpt5. Cdk9 dependent phosphorylation of Rpb1 and hSpt5 followed by Pin1 interaction might thus contribute to the regulation of transcription, pre-mRNA maturation, and the dynamics of these proteins in interphase and mitosis.
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PMID:The peptidyl-prolyl isomerase Pin1 interacts with hSpt5 phosphorylated by Cdk9. 1157 23

The reversible phosphorylation of serine and threonine residues N-terminal to proline (pSer/Thr-Pro) is an important signaling mechanism in the cell. The pSer/Thr-Pro moiety exists in the two distinct cis and trans conformations, whose conversion is catalyzed by the peptidyl-prolyl isomerase (PPIase) Pin1. Among others, Pin1 binds to the phosphorylated C-terminal domain (CTD) of the largest subunit of the RNA polymerase II, but the biochemical and functional relevance of this interaction is unknown. Here we confirm that the CTD phosphatase Fcp1 can suppress a Pin1 mutation in yeast. Furthermore, this genetic interaction requires the phosphatase domain as well as the BRCT domain of Fcp1, suggesting a critical role of the Fcp1 localization. Based on these observations, we developed a new in vitro assay to analyze the CTD dephosphorylation by Fcp1 that uses only recombinant proteins and mimics the in vivo situation. This assay allows us to present strong evidence that Pin1 is able to stimulate CTD dephosphorylation by Fcp1 in vitro, and that this stimulation depends on Pin1's PPIase activity. Finally, Pin1 significantly increased the dephosphorylation of the CTD on the Ser(5)-Pro motif, but not on Ser(2)-Pro in yeast, which can be explained with Pin1's substrate specificity. Together, our results indicate a new role for Pin1 in the regulation of CTD phosphorylation and present a further example for prolyl isomerization-dependent protein dephosphorylation.
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PMID:Pin1 modulates the dephosphorylation of the RNA polymerase II C-terminal domain by yeast Fcp1. 1190 69

The C-terminal domain of the RNA polymerase (RNAP) II largest subunit (CTD) plays critical roles both in transcription of mRNA precursors and in the processing reactions needed to form mature mRNAs. The CTD undergoes dynamic changes in phosphorylation during the transcription cycle, and this plays a significant role in coordinating its multiple activities. But how these changes themselves are regulated is not well understood. Here we show that the peptidyl-prolyl isomerase Pin1 influences the phosphorylation status of the CTD in vitro by inhibiting the CTD phosphatase FCP1 and stimulating CTD phosphorylation by cdc2/cyclin B. This is reflected in vivo by accumulation of hypophosphorylated RNAP II in pin1-/- cells, and of a novel hyper-hyperphosphorylated form in cells induced to overexpress Pin1. This hyper-hyperphosphorylated form of RNAP II also accumulates in M-phase cells, in a Pin1-dependent manner, and associates specifically with Pin1. Functionally, we find that Pin1 overexpression specifically inhibits ongoing transcription of mRNA precursors in vivo and both transcription and RNAP II-stimulated pre-mRNA splicing in cell extracts. Pin1 thus plays a significant role in regulating RNAP II CTD structure and function.
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PMID:Pin1 modulates the structure and function of human RNA polymerase II. 1460 23

Reversible phosphorylation of the repetitive C-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit plays a key role in the progression of RNAP through the transcription cycle. The level of CTD phosphorylation is determined by multiple CTD kinases and a CTD phosphatase, FCP1. The phosphorylated CTD binds to a variety of proteins including the cis/trans peptidyl-prolyl isomerase (PPIase) Pin1 and enzymes involved in processing of the primary transcript such as the capping enzyme Hce1 and CA150, a nuclear factor implicated in transcription elongation. Results presented here establish that the dephosphorylation of hyperphosphorylated RNAP II (RNAP IIO) by FCP1 is impaired in the presence of Pin1 or Hce1, whereas CA150 has no influence on FCP1 activity. The inhibition of dephosphorylation is observed with free RNAP IIO generated by different CTD kinases as well as with RNAP IIO engaged in an elongation complex. These findings support the idea that specific phospho-CTD associating proteins can differentially modulate the dephosphorylation of RNAP IIO by steric hindrance and may play an important role in the regulation of gene expression.
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PMID:Dephosphorylation of RNA polymerase II by CTD-phosphatase FCP1 is inhibited by phospho-CTD associating proteins. 1467 52


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