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 product (pRb) of the retinoblastoma gene (RB-1) prevents S-phase entry during the cell cycle, and inactivation of this growth-suppressive function is presumed to result from pRb hyperphosphorylation during late G1 phase. Complexes of the cyclin-dependent kinase, cdk4, and each of three different D-type cyclins, assembled in insect Sf9 cells, phosphorylated a pRb fusion protein in vitro at sites identical to those phosphorylated in human T cells. Only D-type cyclins activated cdk4 enzyme activity, whereas cyclins A, B1, and E did not. When Sf9 cells were coinfected with baculovirus vectors encoding human pRb and murine D-type cyclins, cyclins D2 and D3, but not D1, bound pRb with high stoichiometry in intact cells. Introduction of a vector encoding cdk4, together with those expressing pRb and D-type cyclins, induced pRb hyperphosphorylation and dissociation of cyclins D2 and D3, whereas expression of a kinase-defective cdk4 mutant in lieu of the wild-type catalytic subunit yielded ternary complexes. The transcription factor E2F-1 also bound to pRb in insect cells, and coexpression of cyclin D-cdk4 complexes, but neither subunit alone, triggered pRb phosphorylation and prevented its interaction with E2F-1. The D-type cyclins may play dual roles as cdk4 regulatory subunits and as adaptor proteins that physically target active enzyme complexes to particular substrates.
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PMID:Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. 844 99

We report the distribution of phosphorylation sites in murine lamins A and C (A-type lamins) in vitro and in vivo followed by reverse-phase high-performance liquid chromatography and microsequencing of peptides spanning the almost complete lamin sequence. We show that two distinct protein kinases, cell-division-cycle-2 kinase (cdc2 kinase) and protein kinase C (PKC), phosphorylate murine A-type lamins at the non-alpha-helical amino- and carboxy-terminal domains in vitro and in vivo. Cdc2 kinase, but not PKC, is capable of inducing depolymerization of the nuclear lamina in permeabilized cells. Accordingly, lamins were proposed to be direct in vivo substrates of cdc2 kinase and PKC with different effects on the lamina dynamics. Analysis of the original A-type lamins revealed phosphorylation of residues Ser5 and Ser392. Residue Ser392 was substoichiometrically phosphorylated in the substrate and by cdc2 kinase in vitro. PKC phosphorylated peptides with its kinase-specific motifs surrounding Ser5, Thr199, Thr416, Thr480 and Ser625. In vivo, a mitosis-specific phosphorylation at the cdc2-kinase-specific phosphoacceptor site Ser392 and of the N-terminal peptide was identified. An interphase-specific phosphorylation at Ser525 matching the PKC consensus sequence and of peptides phosphorylated by unknown kinases was determined. The results lead us to propose that different cyclin-dependent kinase activities act as lamin kinases in mitosis and in interphase. Other kinases may cooperate with cdc2 kinase during reversible disassembly in mitosis and may modulate the supramolecular assembly of lamin filaments.
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PMID:Identification of novel phosphorylation sites in murine A-type lamins. 847 40

p16INK4A, a specific inhibitor of cyclin-dependent kinase (cdk)4 and cdk6, is a candidate tumor suppressor in malignancies with wild-type retinoblastoma (Rb). Loss of p16INK4A frees these cdks from inhibition, permitting constitutive phosphorylation of Rb and inactivation of its growth suppressive properties. Consistent with this model, Rb-positive non-small cell lung cancers (NSCLCs) have little or no detectable p16INK4A protein, whereas Rb-negative lung cancers have abundant p16INK4A. However, only some NSCLCs have homozygous deletions or nonsense mutations in a remaining p16INK4A allele, suggesting that other mechanisms must account for absent or low levels of p16INK4A protein. Here, we analyzed 9 Rb-positive NSCLC cell lines for the controls governing p16INK4A activity. Four lines had homozygous deletions of p16INK4A (SK-LU-1, SK-MES-1, A-427, and SW900), and three had a point mutation in a single allele. First, in H520 cells, the previously reported deletion at codon 45 results in a frameshift that produces no detectable protein. Second, in Calu-3 cells, a His to Tyr substitution at codon 83 produced a variant with a shortened half-life that was unable to form complexes with cdk4 or cdk6. Third, in H661 cells, the previously reported point mutation in the second intron splice donor site resulted in a smaller p16INK4A protein. Although this variant formed complexes with cdk4 and cdk6, it had a profoundly reduced half-life, producing low steady-state levels of p16INK4A and abundant levels of free cdks. Finally, Calu-1 and Calu-6 cells transcribed no detectable mRNA encoding authentic p16INK4A. These cell lines displayed methylation of the CpG island surrounding the first exon of p16INK4A and expressed abundant levels of a nontranslated mRNA containing an alternative first exon (E1 beta), as did all other cell lines in which the p16INK4A locus was not deleted. These data indicate that Rb-positive NSCLC cells have evolved a variety of pathways to suppress p16INK4A expression. Reintroduction of p16INK4A into these cell lines by retroviral transfer resulted in a reduced growth rate, increased abundance of hypophosphorylated Rb, accumulation of cells in G1, and a less transformed morphology in Rb-positive, but not Rb-negative cells, suggesting that loss of p16INK4A is essential for maintenance of the transformed phenotype.
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PMID:Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines. 852 14

The cyclin-dependent kinase (CDK) inhibitor p27 binds and inhibits the kinase activity of several CDKs. Here we report an analysis of the behavior and partners of p27 in Swiss 3T3 mouse fibroblasts during normal mitotic cell cycle progression, as well as in cells arrested at different stages in the cycle by growth factor deprivation, lovastatin treatment, or ultraviolet (UV) irradiation. We found that the level of p27 is elevated in cells arrested in G0 by growth factor deprivation or contact inhibition. In G0, p27 was predominantly monomeric, although some portion was associated with residual cyclin A.Cdk2. During G1, all of p27 was associated with cyclin D1.Cdk4 and was then redistributed to cyclin A.Cdk2 as cells entered S phase. The loss of the monomeric p27 pool as cyclins accumulate in G1 is consistent with the in vivo and in vitro data showing that p27 binds better to cyclin.CDK complexes than to monomeric CDKs. In growing cells, the majority of p27 was associated with cyclin D1 and the level of p27 was significantly lower than the level of cyclin D1. In cells arrested in G1 with lovastatin, cyclin D1 was degraded and p27 was redistributed to cyclin A.Cdk2. In contrast to p21 (which is a p27-related CDK inhibitor and is induced by UV irradiation), the level of p27 was reduced after UV irradiation, but because cyclin D1 was degraded more rapidly than p27, there was a transient increase in binding of p27 to cyclin A.Cdk2. These data suggest that cyclin D1.Cdk4 acts as a reservoir for p27, and p27 is redistributed from cyclin D1.Cdk4 to cyclin A.Cdk2 complexes during S phase, or when cells are arrested by growth factor deprivation, lovastatin treatment, or UV irradiation. It is likely that a similar principle of redistribution of p27 is used by the cell in other instances of cell cycle arrest.
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PMID:Redistribution of the CDK inhibitor p27 between different cyclin.CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation. 853 16

The cellular transcription factor DRTF1/E2F is implicated in the control of early cell cycle progression due to its interaction with important regulators of cellular proliferation, such as pocket proteins (for example, the retinoblastoma tumour suppressor gene product), cyclins and cyclin-dependent kinase subunits. In mammalian cells DRTF1/E2F is a heterodimeric DNA binding activity which arises when a DP protein interacts with an E2F protein. Here, we report an analysis of DRTF1/E2F in Drosophila cells, and show that many features of the pathway which regulate its transcriptional activity are conserved in mammalian cells, such as the interaction with pocket proteins, binding to cyclin A and cdk2, and its modulation by viral oncoproteins. We show that a Drosophila DP protein which can interact co-operatively with E2F proteins is a physiological DNA binding component of Drosophila DRTF1/E2F. An analysis of the expression patterns of a Drosophila DP and E2F protein indicated that DmDP is developmentally regulated and in later embryonic stages preferentially expressed in proliferating cells. In contrast, the expression of DmE2F-1 in late stage embryos occurs in a restricted group of neural cells, whereas in early embryos it is widely expressed, but in a segmentally restricted fashion. Some aspects of the mechanisms which integrate early cell cycle progression with the transcription apparatus are thus conserved between Drosophila and mammalian cells. The distinct expression patterns of DmDP and DmE2F-1 suggest that the formation of DP/E2F heterodimers, and hence DRTF1/E2F, is subject to complex regulatory cues.
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PMID:Functional conservation of the cell cycle-regulating transcription factor DRTF1/E2F and its pathway of control in Drosophila melanogaster. 853 34

PHO4, a transcription factor required for induction of the PHO5 gene in response to phosphate starvation, is phosphorylated by the PHO80-PHO85 cyclin-CDK (cyclin-dependent kinase) complex when yeast are grown in phosphate-rich medium. PHO4 was shown to be concentrated in the nucleus when yeast were starved for phosphate and was predominantly cytoplasmic when yeast were grown in phosphate-rich medium. The sites of phosphorylation on PHO4 were identified, and phosphorylation was shown to be required for full repression of PHO5 transcription when yeast were grown in high phosphate. Thus, phosphorylation of PHO4 by PHO80-PHO85 turns off PHO5 transcription by regulating the nuclear localization of PHO4.
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PMID:Regulation of PHO4 nuclear localization by the PHO80-PHO85 cyclin-CDK complex. 853 22

The retinoblastoma protein (pRb) functions as a negative regulator of the cell cycle and is essential to maintain certain cell types in a post-mitotic state during terminal differentiation. In the ocular lens, inactivation of this protein is sufficient to cause lens fiber cells, which are normally post-mitotic, to enter the cell cycle. The current studies address whether regulation of the cell cycle during lens fiber differentiation in normal lenses or in lenses in which pRB has been inactivated is accompanied by changes in expression of cyclin and cyclin-dependent kinase genes. In the normal lens, our experiments using in-situ hybridization reveal that the expression of cyclin A, cyclin B1, cdc2 and cdk2 is restricted to the proliferative epithelial cells, with no expression in the differentiating fiber cells. Cyclins D1 and D2 and cdk4 show a less restrictive pattern and are expressed in some of the post-mitotic cells. Lenses from RB-deficient embryos, in contrast, show inappropriate expression in the fiber cells of cyclins A, B1 and E, as well as cdc2 and cdk2. The lens fiber cells in these embryos express protein markers for differentiation, such as beta- and gamma-crystallins, even though the cells do not withdraw from the cell cycle. These results indicate that the regulated expression of multiple cell cycle regulatory genes during lens fiber cell differentiation requires the presence of pRb.
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PMID:Regulation of cyclin and cyclin-dependent kinase gene expression during lens differentiation requires the retinoblastoma protein. 855 1

The cyclin-dependent kinase (CDK)-activating kinase, CAK, from mammals and amphibians consists of MO15/CDK7 and cyclin H, a complex which has been identified also as a RNA polymerase II C-terminal domain (CTD) kinase. While the Schizosaccharomyces pombe cdc2 gene product also requires an activating phosphorylation, the enzyme responsible has not been identified. We have isolated an essential S.pombe gene, mop1, whose product is closely related to MO15 and to Saccharomyces cerevisiae Kin28. The functional similarity of Mop1 and MO15 is reflected in the ability of MO15 to rescue a mop1 null allele. This suggests that Mop1 would be a CDK, and indeed Mop1 associates with a previously characterized cyclin H-related cyclin Mcs2 of S.pombe. Also, Mop1 and Mcs2 can associate with the heterologous partners human cyclin H and MO15, respectively. Moreover, the rescue of a temperature-sensitive mcs2 strain by expression of mop1+ demonstrates a genetic interaction between mop1 and mcs2. In a functional assay, immunoprecipitated Mop1-Mcs2 acts both as an RNA polymerase II CTD kinase and as a CAK. The CAK activity of Mop1-Mcs2 distinguishes it from the related CDK-cyclin pair Kin28-Ccl1 from S.cerevisiae, and supports the notion that Mop1-Mcs2 may represent a homolog of MO15-cyclin H in S.pombe with apparent dual roles as a RNA polymerase CTD kinase and as a CAK.
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PMID:Schizosaccharomyces pombe Mop1-Mcs2 is related to mammalian CAK. 855 36

We have identified a second cyclin-dependent kinase (cdk) in fission yeast, crk1, which encodes a 335 amino acid protein that is most closely related to the KIN28 gene product from Saccharomyces cerevisiae and to a cdk activating kinase (CAK) encoded by the MO15 gene from Xenopus laevis, crk1 is essential for viability and delta crk1 cells arrest with septa and condensed chromatin. We show that Crk1 associates with the Mcs2 mitotic catastrophe suppressor, a cyclin H-like molecule, and overexpression of crk1 rescues the cell-cycle arrest defect of a mcs2-75 cdc2-3w cdc25-22 triple mutant at high temperature. The Crk1-Mcs2 complex possesses CAK activity in vitro in that it phosphorylates human Cdk2 on Thr160 which results in its activation in the presence of cyclin A. In addition Crk1-Mcs2 effectively phosphorylates a peptide corresponding to the C-terminal repeat domain (CTD) of RNA polymerase II. We demonstrate that crk1 is allelic to the mcs6 mitotic catastrophe suppressor and that the X.laevis MO15 gene rescues the cell-cycle arrest of an mcs6-13 cdc2-3w cdc25-22 at high temperature. Together these data suggest that the Crk1-Mcs2 complex is a CAK that interacts genetically with Cdc2 in fission yeast.
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PMID:Identification of a cdk-activating kinase in fission yeast. 855 37

Detailed knowledge is available about the molecular makeup of the cell cycle clock in dividing cells. However, comparatively little is known about cell cycle regulation during terminal differentiation. Here we describe a primary cell system in which this question can be addressed. Normal avian erythroid progenitors undergo continuous self-renewal in suspension culture in the presence of growth factors and hormones, allowing us to obtain large cell numbers (10(10)-10(11)). By replacing these "self-renewal factors" with erythropoietin and insulin, the cells can be induced to synchronous, terminal differentiation. During the first 72 h, the cells undergo five cell divisions. Thereafter, they arrest in G1 and complete their maturation into RBC without further divisions. Sixteen to 24 h after induction of differentiation, the cell cycle length decreased from about 20 to 12 h. This shortened doubling time was due to a drastic reduction of G1 (from 12 to 5 h), while S- and G2-phase lengths were not affected. At the same time, the differentiating cells underwent an extensive and concerted switch in their gene expression pattern. During the subsequent four cell divisions, the cell volume decreased from about 300 to less than 70 femtoliters, but the rate of protein synthesis normalized to cell volume remained constant. Interestingly, the shortening of G1 was accompanied by a rapid down-regulation of D-type cyclins and their partner, cyclin-dependent kinase type 4 (cdk4), while expression of S- and G2-M-associated cell cycle regulators (cyclin A and cdk1/cdc2) remained high until the cells arrested in G1 72-96 h after differentiation induction. We conclude that concerted reprogramming of progenitor gene expression during erythroid differentiation is accompanied by profoundly altered cell cycle progression involving the loss or alteration of cell size control at the restriction point.
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PMID:Terminal differentiation of normal chicken erythroid progenitors: shortening of G1 correlates with loss of D-cyclin/cdk4 expression and altered cell size control. 856 72


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