Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Ag-NOR proteins are defined as markers of "active" ribosomal genes. They correspond to a set of proteins specifically located in the nucleolar organizer regions (NORs), but have not yet been clearly identified. We adapted the specific detection method of the Ag-NOR proteins to Western blots in order to identify these proteins. Using a purified protein, Western blots, and immunological characterization, the present study brings the first direct evidence leading to the identity of one Ag-NOR protein. We found that nucleolin is specifically revealed by Ag-NOR staining. Using different nucleolin fragments generated by CNBr cleavage and by overexpression in Escherichia coli, we demonstrate that the amino-terminal domain of nucleolin and not the carboxy-part of the protein is involved in silver staining. Moreover, as the pattern of staining does not vary using casein kinase II- and cdc2-phosphorylated nucleolin or dephosphorylated nucleolin, we conclude that the reduction of the silver ions is not linked to the phosphorylation state of the molecule. We propose that the concentration of acidic amino acids in the amino-terminal domain of nucleolin is responsible for Ag-NOR staining. This hypothesis is also supported by the finding that poly L-glutamic acid peptides are silver stained. These results provide data that can be used to explain the specificity of Ag-NOR staining. Furthermore, we clearly establish that proteolysis of the amino-terminal Ag-NOR-sensitive part of nucleolin occurs in vitro, leading to the accumulation of the carboxy-terminal Ag-NOR-negative part of the protein. We argue that this cleavage occurs in vivo as already proposed, bearing in mind that nucleolin is present in the fibrillar and in the granular component of the nucleolus, whereas no Ag-NOR staining is observed in the latter nucleolar component.
 ...
PMID:Nucleolin is an Ag-NOR protein; this property is determined by its amino-terminal domain independently of its phosphorylation state. 138 90

In a screen of mouse erythroleukemia cDNA expression libraries with anti-phosphotyrosine antibodies, designed to isolate tyrosine kinase coding sequences, we identified several cDNAs encoding proteins identical or very similar to known protein-tyrosine kinases. However, two frequently isolated cDNAs, clk and nek, encode proteins which are most closely related to protein kinases involved in regulating progression through the cell cycle, and contain motifs generally considered diagnostic of protein-serine/threonine kinases. The clk gene product contains a C-terminal cdc2-like kinase domain, most similar to the FUS3 catalytic domain. The Clk protein, expressed in bacteria, becomes efficiently phosphorylated in vitro on tyrosine as well as serine/threonine, and phosphorylates the exogenous substrate poly(glu, tyr) on tyrosine. Direct biochemical evidence indicates that both protein-tyrosine and protein-serine/threonine kinase activities are intrinsic to the Clk catalytic domain. These results suggest the existence of a novel class of protein kinases, with an unusual substrate specificity, which may be involved in cell cycle control.
 ...
PMID:A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators. 182 55

Activation of the cdc2 protein kinase at different stages of the cell cycle is regulated by post-translational modifications and interactions with cyclins. We show that in vitro translated human cdc2 binds very poorly to A and B cyclins, unless it has been preincubated with a Xenopus egg extract. This results in the phosphorylation of cdc2 which allows binding to cyclins. The replacement of Thr161, a residue conserved and phosphorylated in other protein kinases, with valine inhibits cdc2 association with A and B cyclins. In addition, mutations in the amino-terminus of cdc2 and within the conserved 'PSTAIR' region strongly inhibit binding. The Thr161Val mutation causes a lethal phenotype in the fission yeast Schizosaccharomyces pombe, while replacement of Thr161 with glutamic acid, potentially mimicking phosphorylation, causes uncoordination of mitosis and multiple cytokinesis. These results suggest that a threonine phosphorylation/dephosphorylation cycle is involved in regulating cdc2 function.
 ...
PMID:cdc2 phosphorylation is required for its interaction with cyclin. 183 85

ERF (ETS2 Repressor Factor) is a novel member of the ets family of genes, which was isolated by virtue of its interaction with the ets binding site (EBS) within the ETS2 promoter. The 2.7 kb ubiquitously expressed ERF mRNA encodes a 548 amino acid phosphoprotein that exhibits strong transcriptional repressor activity on promoters that contain an EBS. The localization of the DNA-binding domain of the protein at the N-terminus and th repression domain at the C-terminus is reminiscent of the organization of ELK1-like members of the ets family; however, there is no significant homology between ERF and ELK1 or any other ets member outside the DNA-binding domain. The repressor activity of ERF can antagonize the activity of other ets genes that are known transcriptional activators. Furthermore, ERF can suppress the ets-dependent transforming activity of the gag-myb-ets fusion oncogene of ME26 virus. Although ERF protein levels remain constant throughout the cell cycle, the phosphorylation level of the protein is altered as a function of the cell cycle and after mitogenic stimulation. The ERF protein is also hyperphosphorylated in cells transformed by the activated Ha-ras and v-src genes and the transcription repressor activity of ERF is decreased after co-transfection with activated Ha-ras or the kinase domain of the c-Raf-1 gene, indicating that ERF activity is probably regulated by the ras/MAPK pathway. Consistent with the in vivo phosphorylation and inactivation by ras, ERF is efficiently phosphorylated in vitro by Erk2 and cdc2/cyclin B kinases, at sites similar to those detected in vivo. Furthermore, a single mutation at position 526 results in the loss of a specific phosphopeptide both in in vivo and in vitro (by Erk2) labeling. Substitution of Thr526 for glutamic acid also decreases the repression ability of ERF. Our data suggest a model in which modulation of ERF activity is involved in the transcriptional regulation of genes activated during entry into G1 phase. Obstruction of the ERF repressor function by the transactivating members of the ets family of genes (i.e.gag-myb-ets) may be essential for the control of genes involved in cell proliferation and may also underlie their tumorigenic effects.
 ...
PMID:ERF: an ETS domain protein with strong transcriptional repressor activity, can suppress ets-associated tumorigenesis and is regulated by phosphorylation during cell cycle and mitogenic stimulation. 758 8

Entry into mitosis requires activation of cdc2 kinase brought on by its association with cyclin B, phosphorylation of the conserved threonine (Thr-167 in Schizosaccharomyces pombe) in the T loop, and dephosphorylation of the tyrosine residue at position 15. Exit from mitosis, on the other hand, is induced by inactivation of cdc2 activity via cyclin destruction. It has been suggested that in addition to cyclin degradation, dephosphorylation of Thr-167 may also be required for exit from the M phase. Here we show that Saccharomyces cerevisiae cells expressing cdc28-E169 (a CDC28 allele in which the equivalent threonine, Thr-169, has been replaced by glutamic acid) are able to degrade mitotic cyclin Clb2, inactivate the Cdc28/Clb2 kinase, and disassemble the anaphase spindles, suggesting that they exit mitosis normally. The cdc28-E169 allele is active with respect to its mitotic functions, since it complements the mitosis-defective cdc28-1N allele. Whereas replacement of Thr-169 with serine affects neither Start nor the mitotic activity of Cdc28, replacement with glutamic acid or alanine renders Cdc28 inactive for Start-related functions. Coimmunoprecipitation experiments show that although Cdc28-E169 associates with mitotic cyclin Clb2, it fails to associate with the G1 cyclin Cln2. Thus, an unmodified threonine at position 169 in Cdc28 is important for interaction with G1 cyclins. We propose that in S. cerevisiae, dephosphorylation of Thr-169 is not required for exit from mitosis but may be necessary for commitment to the subsequent division cycle.
 ...
PMID:Dephosphorylation of threonine 169 of Cdc28 is not required for exit from mitosis but may be necessary for start in Saccharomyces cerevisiae. 875 58

In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues. It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function its activation by the dephosphorylation of an equivalent tyrosine (Tyr-19) serves. We report here that cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles. The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1. We also find that the overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs. These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs. The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct. On the basis of these results, we propose that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation.
 ...
PMID:Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28. 888 67

Many protein kinases are regulated by phosphorylation in the activation loop, which is required for enzymatic activity. Glutamic acid can substitute for phosphothreonine in some proteins activated by phosphorylation, but this substitution (T169E) at the site of activation loop phosphorylation in the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28p blocks biological function and protein kinase activity. Using cycles of error-prone DNA amplification followed by selection for successively higher levels of function, we identified mutant versions of Cdc28p-T169E with high biological activity. The enzymatic and biological activity of the mutant Cdc28p was essentially normally regulated by cyclin, and the mutants supported normal cell cycle progression and regulation. Therefore, it is not a requirement for control of the yeast cell cycle that Cdc28p be cyclically phosphorylated and dephosphorylated. These CDC28 mutants allow viability in the absence of Cak1p, the essential kinase that phosphorylates Cdc28p-T169, demonstrating that T169 phosphorylation is the only essential function of Cak1p. Some growth defects remain in suppressed cak1 cdc28 strains carrying the mutant CDC28 genes, consistent with additional nonessential roles for CAK1.
 ...
PMID:Molecular evolution allows bypass of the requirement for activation loop phosphorylation of the Cdc28 cyclin-dependent kinase. 956 11

In chondrocytes, PTHrP maintains them in a proliferative state and prevents premature hypertrophy. The mechanism by which PTHrP does this is not fully understood. Both Runx2 and Runx3 are required for chondrocyte maturation. We recently demonstrated that cyclin D1 induces Runx2 protein phosphorylation and degradation. In the present studies, we tested the hypothesis that PTHrP regulates both Runx2 and Runx3 protein stability through cyclin D1. We analyzed the effects of cyclin D1 on Runx3 protein stability and function using COS cells, osteoprogenitor C3H10T1/2 cells and chondrogenic RCJ3.1C5.18 cells. We found that cyclin D1 induced Runx3 degradation in a dose-dependent manner and that both Myc-tagged Runx3 and endogenous Runx3 interact directly with CDK4 in COS and RCJ3.1C5.18 cells. A conserved CDK recognition site was identified in the C-terminal region of Runx3 by sequence analysis (residues 356-359). Pulse-chase experiments showed that the mutation of Runx3 at Ser356 to alanine (SA-Runx3) increased the half-life of Runx3. By contrast, the mutation at the same serine residue to glutamic acid (SE-Runx3) accelerated Runx3 degradation. In addition, SA-Runx3 was resistant to cyclin D1-induced degradation. GST-Runx3 was strongly phosphorylated by CDK4 in vitro. By contrast, CDK4 had no effect on the phosphorylation of SA-Runx3. Although both wild-type and SE-Runx3 were ubiquitylated, this was not the case for SA-Runx3. Runx3 degradation by cyclin D1 was completely blocked by the proteasome inhibitor PS1. In C3H10T1/2 cells, SA-Runx3 had a greater effect on reporter activity than SE-Runx3. The same was true for ALP activity in these cells. To investigate the role of cyclin D1 in chondrocyte proliferation and hypertrophy, we analyzed the growth plate morphology and expression of chondrocyte differentiation marker genes in Ccnd1-knockout mice. The proliferating and hypertrophic zones were significantly reduced and expression of chondrocyte differentiation marker genes and ALP activity were enhanced in 2-week-old Ccnd1-knockout mice. PTHrP significantly suppressed protein levels of both Runx2 and Runx3 in primary chondrocytes derived from wild-type mice. By contrast, the suppressive effect of PTHrP on Runx2 and Runx3 protein levels was completely abolished in primary chondrocytes derived from Ccnd1-knockout mice. Our findings demonstrate that the cell cycle proteins cyclin D1 and CDK4 induce Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradation. PTHrP suppresses Runx2 and Runx3 protein levels in chondrocytes through cyclin D1. These results suggest that PTHrP might prevent premature hypertrophy in chondrocytes, at least in part by inducing degradation of Runx2 and Runx3 in a cyclin-D1-dependent manner.
 ...
PMID:PTHrP prevents chondrocyte premature hypertrophy by inducing cyclin-D1-dependent Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradation. 1935 20

Saccharomyces cerevisiae Actin-Binding Protein 1 (Abp1p) is a member of the Abp1 family of proteins, which are in diverse organisms including fungi, nematodes, flies, and mammals. All proteins in this family possess an N-terminal Actin Depolymerizing Factor Homology (ADF-H) domain, a central Proline-Rich Region (PRR), and a C-terminal SH3 domain. In this study, we employed sequence analysis to identify additional conserved features of the family, including sequences rich in proline, glutamic acid, serine, and threonine amino acids (PEST), which are found in all family members examined, and two motifs, Conserved Fungal Motifs 1 and 2 (CFM1 and CFM2), that are conserved in fungi. We also discovered that, similar to its mammalian homologs, Abp1p is phosphorylated in its PRR. This phosphorylation is mediated by the Cdc28p and Pho85p kinases, and it protects Abp1p from proteolysis mediated by the conserved PEST sequences. We provide evidence for an intramolecular interaction between the PRR region and SH3 domain that may be affected by phosphorylation. Although deletion of CFM1 alone caused no detectable phenotype in any genetic backgrounds or conditions tested, deletion of this motif resulted in a significant reduction of growth when it was combined with a deletion of the ADF-H domain. Importantly, this result demonstrates that deletion of highly conserved domains on its own may produce no phenotype unless the domains are assayed in conjunction with deletions of other functionally important elements within the same protein. Detection of this type of intragenic synthetic lethality provides an important approach for understanding the function of individual protein domains or motifs.
 ...
PMID:The importance of conserved features of yeast actin-binding protein 1 (Abp1p): the conditional nature of essentiality. 2266 26