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

Viral oncoproteins that inactivate the retinoblastoma tumor suppressor protein (pRb) family both block skeletal muscle differentiation and promote cell cycle progression. To clarify the dependence of terminal differentiation on the presence of the different pRb-related proteins, we have studied myogenesis using isogenic primary fibroblasts derived from mouse embryos individually deficient for pRb, p107, or p130. When ectopically expressed in fibroblasts lacking pRb, MyoD induces an aberrant skeletal muscle differentiation program characterized by normal expression of early differentiation markers such as myogenin and p21, but attenuated expression of late differentiation markers such as myosin heavy chain (MHC). Similar defects in MHC expression were not observed in cells lacking either p107 or p130, indicating that the defect is specific to the loss of pRb. In contrast to wild-type, p107-deficient, or p130-deficient differentiated myocytes that are permanently withdrawn from the cell cycle, differentiated myocytes lacking pRb accumulate in S and G2 phases and express extremely high levels of cyclins A and B, cyclin-dependent kinase (Cdk2), and Cdc2, but fail to readily proceed to mitosis. Administration of caffeine, an agent that removes inhibitory phosphorylations on inactive Cdc2/cyclin B complexes, specifically induced mitotic catastrophe in pRb-deficient myocytes, consistent with the observation that the majority of pRb-deficient myocytes arrest in S and G2. Together, these findings indicate that pRb is required for the expression of late skeletal muscle differentiation markers and for the inhibition of DNA synthesis, but that a pRb-independent mechanism restricts entry of differentiated myocytes into mitosis.
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PMID:Skeletal muscle cells lacking the retinoblastoma protein display defects in muscle gene expression and accumulate in S and G2 phases of the cell cycle. 889

Normal somatic cells of higher organisms do not divide indefinitely. After a finite number of divisions, normal cells irreversibly cease proliferation by a process termed replicative or cellular senescence. Replicative senescence is controlled by multiple, dominant-acting genes about which very little is known. The only genes known to reactivate DNA synthesis in senescent cells are viral oncogenes encoding proteins that bind and inactivate the p53 and retinoblastoma (pRb) tumor suppressor proteins. SV40 T antigen is the best studied of these viral oncoproteins. T[K1] is a T antigen point mutant that selectively is defective in binding pRb and the pRb-related proteins p107 and p130. We show that T[K1] stimulated quiescent human fibroblasts to synthesize DNA nearly as well as wild-type T but was incapable of stimulating senescent cells. We tested several growth regulatory genes that are repressed in senescent cells for ability to restore activity to T[K1]. These included c-fos, c-jun, Id-1, Id-2, E2F-1, and cdc2. Only the helix-loop-helix (HLH) protein, Id-1, restored the ability of T[K1] to reactivate DNA synthesis in senescent cells. This activity of Id-1 was not shared by Id-2, a related protein, and depended on an intact HLH domain. It did not appear that Id-1 interacted directly with pRb or p107. Constitutive Id-1 expression failed to rescue proliferating cells from growth inhibition by pRb, p107, or p130, and failed to interact with pRb in the yeast two hybrid system. Because Id proteins negatively regulate basic-HLH (bHLH) transcription factors, we suggest that senescent cells express one or more bHLH factor that cooperates with pRb, or pRb-related proteins, to suppress proliferation.
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PMID:The helix-loop-helix protein Id-1 and a retinoblastoma protein binding mutant of SV40 T antigen synergize to reactivate DNA synthesis in senescent human fibroblasts. 893 78

Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A-cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphorylation of the retinoblastoma protein by cyclin A- but not cyclin B-associated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 and E2F1 likewise prevented their phosphorylation by cyclin A-associated kinases in vitro. The cdk inhibitor p21 was found to contain two functional copies of this recognition motif, as determined by in vitro kinase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate recognition motif. Furthermore, these data suggest that p21-like cdk inhibitors function, at least in part, by blocking the interaction of substrates with cyclin-cdk2 complexes.
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PMID:Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors. 894 16

Progression through the cell cycle is dependent on the sequential expression of cyclins, which combine with cyclin-dependent kinases (cdks) to form active kinases. The transition from G1 to S phase is dependent on D cyclins in complex with cdk4 or cdk6 and cyclin E complexed with cdk2. One target of G1 cyclins is the retinoblastoma susceptibility protein (Rb). Rb is a transcriptional repressor that is selectively targeted to genes through interaction with the E2F family of cell cycle transcription factors. Rb is a member of a family of proteins that include p107 and p130. The three proteins share a region known as the pocket that is important for binding E2F and is also the binding site for oncoproteins from DNA tumor viruses that inactivate Rb. We have found that two conserved domains within the Rb pocket (A and B) interact to form a transcriptional repressor motif (K. N. B. Chow and D. C. Dean, Mol. Cell. Biol. 16:4862-4868, 1996). Here we demonstrate that p107 also has an A-B repressor motif, and using domain swapping and coimmunoprecipitation assays, we compare A and B from Rb and p107. Finally and most importantly, we demonstrate that the A-B interaction which forms the repressor motif is blocked by G1 cdk phosphorylation, thereby blocking repressor activity. This A-B repressor motif is then the first example of a cdk-regulated transcriptional repressor.
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PMID:The Rb family contains a conserved cyclin-dependent-kinase-regulated transcriptional repressor motif. 894 73

Promoter elements that are important for the G1-S induction of the human thymidine kinase (htk) promoter reside within the core of the cell cycle regulatory unit, positioned between -110 and -84 upstream of the TATA element. Within this 27-bp region are three GC-rich motifs, which resemble the E2F binding site. By site-directed mutagenesis, we identified a 14-bp region, between -97 and -84, critical for the htk promoter transcriptional activity. Methylation interference studies indicate that the sequences between -97 and -84 are major protein contact points, correlating with the functional significance of this sequence in vivo. Although the core of the cell cycle regulatory unit contains three E2F-like sites and can form minor S-phase-specific complexes containing p107, cyclin A, and cdk2, the major complex that binds to this region is not competed by E2F binding sites. Through DNA affinity chromatography, we identified a set of protein species of approximately 40 kDa that copurified with the htk DNA binding activity. From gel shift assays and Western blot analysis, this protein species is antigenically distinct from E2F-1, E2F-2, E2F-3, and E2F-4. Our studies raise the possibility that other members of the E2F protein family or a novel protein(s) with preferred binding affinity for the htk promoter exert(s) control on the G1 to S regulation of the htk promoter through their interactions with cyclins and kinases.
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PMID:Identification of a set of protein species approximately 40 kDa as high-affinity DNA binding factor(s) to the cell cycle regulatory region of the human thymidine kinase promoter. 895 43

Cyclin E is necessary and rate limiting for the passage of mammalian cells through the G1 phase of the cell cycle. Control of cell cycle progression by cyclin E involves cdk2 kinase, which requires cyclin E for catalytic activity. Expression of cyclin E/cdk2 leads to an activation of cyclin A gene expression, as monitored by reporter gene constructs derived from the human cyclin A promoter. Promoter activation by cyclin E/cdk2 requires an E2F binding site in the cyclin A promoter. We show here that cyclin E/cdk2 kinase can directly bind to E2F/p107 complexes formed on the cyclin A promoter-derived E2F binding site, and this association is controlled by p27KIP1, most likely through direct protein-protein interaction. These observation suggest that cyclin E/cdk2 associates with E2F/p107 complexes in late G1 phase, once p27KIP1 has decreased below a critical threshold level. Since a kinase-negative mutant of cdk2 prevents promoter activation, it appears that transcriptional activation of the cyclin A gene requires an active cdk2 kinase tethered to its promoter region.
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PMID:p27KIP1 blocks cyclin E-dependent transactivation of cyclin A gene expression. 897 21

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G1/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.
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PMID:Cyclin E, a redundant cyclin in breast cancer. 898 90

Cells expressing human papillomavirus type 16 (HPV-16) E7, similar to those which express HPV-16 E6, are resistant to a p53-mediated G1 growth arrest. We examined the p53-mediated DNA damage response pathway in E7-expressing cells to determine the mechanism by which E7-containing cells continue to cycle. In response to DNA damage, no dramatic difference was detected in G1- or S-phase cyclin or cyclin-dependent kinase (Cdk) levels when E7-expressing cells were compared to the parental cell line, RKO. Furthermore, Cdk2 kinase activity was inhibited in both RKO cells and E7-expressing cells, while Cdk2 remained active in E6-expressing cells. However, the steady-state levels of pRB and p107 protein were substantially lower in E7-expressing cells than in the parental RKO cells or E6-expressing cells. There was no reduction in pRB mRNA levels, but the half-life of pRB in E7-expressing cells was markedly shorter. Infection of primary human foreskin keratinocytes with recombinant retroviruses expressing HPV-16 E7 resulted in a decrease in pRB protein levels, indicating this phenomenon is a consequence of E7 expression, not of immortalization or transformation. These data strongly suggest E7 interferes with the stability of pRB and p107 protein. We propose that the removal of these components of the p53-mediated G1 growth arrest pathway in E7-expressing cells contributes to the ability of E7 to overcome a p53-mediated G1 growth arrest.
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PMID:Analysis of the p53-mediated G1 growth arrest pathway in cells expressing the human papillomavirus type 16 E7 oncoprotein. 906 Jun 48

v-Abl is an oncogenic form of the c-Abl nonreceptor tyrosine kinase. v-Abl induces transcription of c-myc, and c-Myc function is a necessary but not sufficient component of the v-Abl transformation program. Previously we showed that the E2F site in the c-myc promoter is a v-Abl response element and that v-Abl appears to induce c-myc by initiating a phosphorylation cascade that ultimately activates E2F-binding proteins. In this work we have investigated the signaling pathway between the v-Abl tyrosine kinase and activated E2F proteins. We show that the Ras GTPase and Raf1 serine/threonine kinase are required in this pathway. However, in contrast to other aspects of v-Abl signaling, induction of c-myc transcription is independent of the Rac GTPase. Our results also establish a requirement for activated cyclin-dependent kinases (cdks), as v-Abl-dependent induction of c-myc transcription is blocked by cdk inhibitor p21 and induction of c-myc is accompanied by activation of cdk2 and cdk4. Finally, we show that v-Abl-dependent induction of c-myc is accompanied by hyperphosphorylation of pRb, p107, and p130. On the basis of these data, we propose a model for the signaling path from v-Abl to c-myc.
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PMID:Induction of c-myc transcription by the v-Abl tyrosine kinase requires Ras, Raf1, and cyclin-dependent kinases. 911 29

Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream effectors of 'pocket' proteins are the components of the E2F family of transcription factors, which regulate the G1/S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undifferentiated and differentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G0/G1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G1/S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1/S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes. In differentiating C2C12 cells, E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream effector p21 are induced. Thus, during myogenic differentiation, formation of E2F4 and pRb2/p130 containing complexes is an early event, but not enough on its own to prevent the reactivation of DNA synthesis. Using a subclone of C3H10T1/2 mouse fibroblasts stably expressing Estrogen Receptor-MyoD (ER-MyoD) chimerae, we found that estrogen directed MyoD activation prevents the reassociation of cyclinA/cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle. Our data indicate that, in differentiating myocytes, one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation.
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PMID:MyoD prevents cyclinA/cdk2 containing E2F complexes formation in terminally differentiated myocytes. 912 66


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