A newly isolated gene, ESS1, was shown to encode a protein required for vegetative growth in Saccharomyces cerevisiae. The nucleotide sequence of ESS1 revealed a 172 amino acid open reading frame predicting a highly basic, 19.5 kilodalton product. Although the gene was isolated by cross-hybridization with the vertebrate v-sis oncogene, the primary amino acid sequence bears only a slight resemblance to the p28sis protein. ESS1 is single copy in the yeast genome and transcriptionally active during logarithmic growth. It is located on the right arm of chromosome X, 6 centimorgans distal to ilv3. The genetic map location indicates it is not allelic to any previously characterized mutation in this organism. Both inactivation of ESS1 by gene disruption and overexpression by fusion to a heterologous promoter are detrimental to growth in both haploid and diploid cell types. Under non-permissive conditions, the terminal phenotype of strains containing a suppressible amber mutation within ESS1 is one of aberrant multibudded structures. Examination of this morphology indicates that loss of ESS1 function may lead to a defect in cytokinesis or cell separation (Hanes, 1989).
In a genetic screen aimed at the identification of trans-acting factors involved in mRNA 3'-end processing of budding yeast, two temperature-sensitive mutants were isolated with an apparent defect in the 3'-end formation of a plasmid-derived pre-mRNA. Surprisingly, both mutants were rescued by the essential gene ESS1/PTF1 that encodes a putative peptidylprolyl-cis/trans-isomerase (PPIase). Such enzymes, which catalyze the cis/trans-interconversion of peptide bonds N-terminal of prolines, have been suggested to play a role in protein folding or trafficking. Ptf1p shows PPIase activity in vitro, displaying an unusual substrate specificity for peptides with phosphorylated serine and threonine residues preceding proline. Both mutations were found to result in amino acid substitutions of highly conserved residues within the PPIase domain, causing a marked decrease in PPIase activity of the mutant enzymes. These results are suggestive of a thus far unknown involvement of a PPIase in mRNA 3'-end formation in Saccharomyces cerevisiae (Hani, 1999).
Complementation of a temperature sensitive mutant of the yeast Saccharomyces cerevisiae resulted in the isolation of PTF1 (processing/termination factor 1), an essential gene encoding a putative 3'-end processing or transcription termination factor of pre-mRNAs. Ptf1p shows significant homology to a newly discovered family of PPIases. This family is characterized by its insensitivity to immunosuppressive drugs and the lack of homology with cyclophilins and FK-506 binding proteins. Should Ptf1p display PPIase activity, it would be the first characterized, eukaryotic member of this putative family, which is essential for growth (Hani, 1995).
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. 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 (Morris, 1999).
Three families of prolyl isomerases have been identified: cyclophilins, FK506-binding proteins (FKBPs) and parvulins. All 12 cyclophilins and FKBPs are dispensable for growth in yeast, whereas the one parvulin homolog, Ess1, is essential. Cyclophilin A becomes essential when Ess1 function is compromised. Overexpression of cyclophilin A suppresses ess1 conditional and null mutations, and cyclophilin A enzymatic activity is required for suppression. These results indicate that cyclophilin A and Ess1 function in parallel pathways and act on common targets by a mechanism that requires prolyl isomerization. Using genetic and biochemical approaches, it has been found that one of these targets is the Sin3-Rpd3 histone deacetylase complex. Cyclophilin A increases and Ess1 decreases disruption of gene silencing by this complex. Conditions that favor acetylation over deacetylation suppress ess1 mutations. These findings support a model in which Ess1 and cyclophilin A modulate the activity of the Sin3-Rpd3 complex, and excess histone deacetylation causes mitotic arrest in ess1 mutants (Arevalo-Rodriguez, 2000).
The Ess1/Pin1 peptidyl-prolyl isomerase (PPIase) is thought to control mitosis by binding to cell cycle regulatory proteins and altering their activity. Temperature-sensitive ess1 mutants have been isolated and six multicopy suppressors have been identified that rescue their mitotic-lethal phenotype. None are cell cycle regulators. Instead, five of these 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. Expression of some but not all genes is decreased in ess1 mutants, and 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 (Wu, 2000).
The NIMA kinase is essential for progression through mitosis in Aspergillus nidulans, and there is evidence for a similar pathway in other eukaryotic cells. The human protein Pin1, a peptidyl-prolyl cis/trans isomerase (PPIase) that interacts with NIMA, is described. PPIases are important in protein folding, assembly and/or transport, but none has so far been shown to be required for cell viability. Pin1 is nuclear PPIase containing a WW protein interaction domain, and is structurally and functionally related to Ess1/Ptf1, an essential protein in budding yeast. PPIase activity is necessary for Ess1/Pin1 function in yeast. Depletion of Pin1/Ess1 from yeast or HeLa cells induces mitotic arrest, whereas HeLa cells overexpressing Pin1 arrest in the G2 phase of the cell cycle. Pin1 is thus an essential PPIase that regulates mitosis, presumably by interacting with NIMA and attenuating its mitosis-promoting activity (Lu, 1996).
The human rotamase or peptidyl-prolyl cis-trans isomerase Pin1 is a conserved mitotic regulator essential for the G2/M transition of the eukaryotic cell cycle. The 1.35 A crystal structure of Pin1 complexed with an AlaPro dipeptide is reported and the initial characterization of Pin1's functional properties. The crystallographic structure as well as pH titration studies and mutagenesis of an active site cysteine suggest a catalytic mechanism that includes general acid-base and covalent catalysis during peptide bond isomerization. Pin1 displays a preference for an acidic residue N-terminal to the isomerized proline bond due to interaction of this acidic side chain with a basic cluster. This raises the possibility of phosphorylation-mediated control of Pin1-substrate interactions in cell cycle regulation (Ranganathan, 1997).
The cis/trans peptidyl-prolyl isomerase, Pin1, is a regulator of mitosis that is well conserved from yeast to man. Depletion of Pin1-binding proteins from Xenopus egg extracts results in hyperphosphorylation and inactivation of the key mitotic regulator, Cdc2/cyclin B. This phenotype is a consequence of Pin1 interaction with critical upstream regulators of Cdc2/cyclin B, including the Cdc2-directed phosphatase, Cdc25, and its known regulator, Plx1. Although Pin1 can interact with Plx1 during interphase and mitosis, only the phosphorylated, mitotically active form of Cdc25 is able to bind Pin1, an event that has been recapitulated using in vitro phosphorylated Cdc25. Taken together, these data suggest that Pin1 may modulate cell cycle control through interaction with Cdc25 and its activator, Plx1 (Crenshaw, 1998).
Phosphorylation of mitotic proteins on the Ser/Thr-Pro motifs has been shown to play an important role in regulating mitotic progression. Pin1 is a novel essential peptidyl-prolyl isomerase (PPIase) that inhibits entry into mitosis and is also required for proper progression through mitosis, but its substrate(s) and function(s) remain to be determined. In both human cells and Xenopus extracts, Pin1 interacts directly with a subset of mitotic phosphoproteins on phosphorylated Ser/Thr-Pro motifs in a phosphorylation-dependent and mitosis-specific manner. Many of these Pin1-binding proteins are also recognized by the monoclonal antibody MPM-2, and they include the important mitotic regulators Cdc25, Myt1, Wee1, Plk1, and Cdc27. The importance of this Pin1 interaction was tested by constructing two Pin1 active site point mutants that fail to bind a phosphorylated Ser/Thr-Pro motif in mitotic phosphoproteins. Wild-type, but not mutant, Pin1 inhibits both mitotic division in Xenopus embryos and entry into mitosis in Xenopus extracts. The interaction between Pin1 and Cdc25 was examined in detail. Pin1 not only binds the mitotic form of Cdc25 on the phosphorylation sites important for its activity in vitro and in vivo, but it also inhibits its activity, offering one explanation for the ability of Pin1 to inhibit mitotic entry. Pin1 is a phosphorylation-dependent PPIase that can recognize specifically the phosphorylated Ser/Thr-Pro bonds present in mitotic phosphoproteins. Thus, Pin1 likely acts as a general regulator of mitotic proteins that have been phosphorylated by Cdc2 and other mitotic kinases (Shen, 1998).
The peptidyl prolil cis/trans isomerase Ess1/Pin1 is essential for mitosis progression in yeast cells and is hypothesized to perform the same role in mammalian cells. To investigate the function of Pin1 in mammalian cells, mice lacking Pin1 were created. These mice undergo normal development. Although the embryonic Pin1-/- fibroblasts grow normally, they prove significantly deficient in their ability to restart proliferation in response to serum stimulation after G(0) arrest. These results suggest that Pin1 is required for cell cycle progression from G(0) arrest as well as mitosis progression in normal mammalian cells (Fujimori, 1999).
The reversible protein phosphorylation on serine or threonine residues that precede proline (pSer/Thr-Pro) is a key signaling mechanism for the control of various cellular processes, including cell division. The pSer/Thr-Pro moiety in peptides exists in the two completely distinct cis and trans conformations whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1. Previous results suggest that Pin1 might regulate the conformation and dephosphorylation of its substrates. However, it is not known whether phosphorylation-dependent prolyl isomerization occurs in a native protein and/or affects dephosphorylation of pSer/Thr-Pro motifs. The major Pro-directed phosphatase PP2A is conformation-specific and effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs both in Cdc25C and tau to facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions, and prolyl isomerase activity of Pin1 is essential for cell division in vivo. Thus, phosphorylation-specific prolyl isomerization catalyzed by Pin1 is a novel mechanism essential for regulating dephosphorylation of certain pSer/Thr-Pro motifs (Zhou, 2000).
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