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)

We have previously found that loss of C/EBPalpha in hepatocytes of newborn livers leads to increased proliferation, to a reduction in p21 protein levels and to an induction of S phase-specific E2F/p107 complexes. In this paper, we investigated C/EBPalpha-dependent regulation of E2F complexes in a well-characterized cell line, 3T3-L1, and in stable transformants that conditionally express C/EBPalpha. C/EBPalpha and C/EBPbeta proteins are induced in 3T3-L1 preadipocytes during differentiation with different kinetics and potentially may regulate E2F/Rb family complexes. In pre-differentiated cells, three E2F complexes are observed: cdk2/E2F/p107, E2F/p130 and E2F4. cdk2/E2F/p107 complexes are induced in nuclear extracts of 3T3-L1 cells during mitotic expansion, but are not detectable in nuclear extracts at later stages of 3T3-L1 differentiation. The reduction in E2F/p107 complexes is associated with elevation of C/EBPalpha, but is independent of C/EBPbeta expression. Bacterially expressed, purified His-C/EBPalpha is able to disrupt E2F/p107 complexes that are observed at earlier stages of 3T3-L1 differentiation. C/EBPbeta, however, does not disrupt E2F/p107 complexes. A short C/EBPalpha peptide with homology to E2F is sufficient to bring about the disruption of E2F/p107 complexes from 3T3-L1 cells in vitro. Induction of C/EBPalpha in stable 3T3-L1 clones revealed that C/EBPalpha causes disruption of p107/E2F complexes in these cells. In contrast, E2F/p130 complexes are induced in cells expressing C/EBPalpha. Our data suggest that induction of p130/E2F complexes by C/EBPalpha occurs via up-regulation of p21, which, in turn, leads to association with and inhibition of, cdk2 kinase activity. The reduction in cdk2 kinase activity correlates with alterations of p130 phosphorylation and with induction of p130/E2F complexes in 3T3-L1 stable clones. Our data suggest two pathways of C/EBPalpha-dependent regulation of E2F/Rb family complexes: disruption of S phase-specific E2F/p107 complexes and induction of E2F/p130 complexes.
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PMID:E2F/p107 and E2F/p130 complexes are regulated by C/EBPalpha in 3T3-L1 adipocytes. 1044 55

Recent studies suggest that cyclin D1 mediates progression of hepatocytes through G(1) phase of the cell cycle. The present study further examines the regulation of cyclin D1-dependent kinase activity and the interplay between cyclin D1 and other G(1) phase regulatory proteins during liver regeneration. After 70% partial hepatectomy in rats, there was upregulation of kinase activity associated with cyclins (A, D1, D3, and E), cyclin-dependent kinases (Cdk2 and Cdk4), and Cdk-inhibitory proteins (p27, p107, and p130). Although cyclin D1/Cdk4 complexes were more abundant in the cytoplasmic fraction after partial hepatectomy, kinase activity was detected primarily in the nuclear fraction. Cytoplasmic cyclin D1/Cdk4 complexes were activated by recombinant cyclin H/Cdk7. Because endogenous Cdk7 activity was found in the nucleus, this suggests that activation of cyclin D1/Cdk4 requires nuclear importation and subsequent phosphorylation by cyclin H/Cdk7. Recombinant cyclin E/Cdk2 was inhibited by extracts from quiescent liver, and cyclin D1 could titrate out this inhibitory activity. Induction of cyclin D1 was accompanied by increased abundance of cyclin D1/p27 complexes, and most p27 was sequestered by cyclin D1 after partial hepatectomy. Thus cyclin D1 appears to play two roles during G(1) phase progression in the regenerating liver: it forms a nuclear kinase complex, and it promotes activation of Cdk2 by sequestering inhibitory proteins such as p27. These experiments underscore the complexity of cyclin/Cdk regulatory networks in the regenerating liver.
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PMID:Regulation of G(1) cyclin-dependent kinases in the liver: role of nuclear localization and p27 sequestration. 1060 Aug 18

The tumour suppressor PTEN, also named MMAC1 or TEP1, is associated with a number of malignancies in human populations. This protein has a dual protein phosphatase activity, being also capable to dephosphorylate phosphatidylinositol 3,4,5 triphosphate. We have studied the mechanism of growth suppression attributable to PTEN. We observed that PTEN overexpression inhibits cell growth in a variety of normal and transformed, human and murine cells. Bromodeoxyuridine (BrdU) incorporation and TUNEL labelling experiments in transiently transfected cells demonstrate that this inhibition is due to a cell cycle arrest rather than induction of apoptosis. Given that PTEN is unable to cause cell growth arrest in retinoblastoma (Rb)-deficient cell lines, we have explored the possible requirement for pRb in the PTEN-induced inhibition of cell proliferation. We found that the co-expression of SV40 antigen, but not a mutant form (which binds exclusively to p53), and cyclin D1/cdk4 are able to overcome the PTEN-mediated growth suppression. In addition, the reintroduction of a functional pRb, but not its relatives p107 or p130, in Rb-deficient cells restores the sensitivity to PTEN-induced arrest. Finally, the hyperphosphorylation of transfected pRb is inhibited by PTEN co-expression and restored by PI-3K co-expression. Accordingly, PTEN gene is mostly expressed, in parallel to Akt, in mid-late G1 phase during cell cycle progression prior to pRb hyperphosphorylation. Finally, we have studied the signal transduction pathways modulated by PTEN expression. We found that PTEN-induced growth arrest can be rescued by the co-expression of active PI-3K and downstream effectors such as Akt or PDK1, and also certain small GTPases such as Rac1 and Cdc42, but not by active Ha-ras, raf or RhoA. Collectively, our data link the tumour suppressor activities of PTEN to the machinery controlling cell cycle through the modulation of signalling molecules whose final target is the functional inactivation of the retinoblastoma gene product.
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PMID:PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein. 1060 5

The specific mechanisms controlling intestinal cell differentiation remain largely undefined. The retinoblastoma (Rb) proteins (pRb, p130, and p107) appear crucial to the terminal differentiation process of certain cells through their association and repression of E2F transcription factors. We have examined the expression of pRb-related proteins p130 and p107 as well as the regulation of E2F during spontaneous differentiation of the Caco-2 intestinal cell line. Nuclear protein levels of p130 and p107 were increased with Caco-2 differentiation. Induction of a slower-migrating E2F complex was noted in postconfluent (i.e., differentiated) Caco-2 cells; p130 protein was the predominant component of this E2F complex with a minor contribution from cyclin-dependent kinase-2. A small component of p107 binding was identified by deoxycholate release gel shift assays. In contrast, no pRb binding to E2F was noted in Caco-2 cells. In addition to increased association with p130, E2F-4 phosphorylation was markedly decreased in differentiated Caco-2 cells, whereas E2F protein levels remained unchanged. Taken together, our findings suggest that the regulation of E2F function may be an important contributing factor in the cell cycle block and spontaneous differentiation of Caco-2 cells. This regulation of E2F occurs most likely through its increased association with p130 as well as decreased phosphorylation.
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PMID:Characterization and regulation of E2F activity during Caco-2 cell differentiation. 1064 18

Cdx1 is a homeodomain transcription factor that regulates intestine-specific gene expression. Experimental evidence suggests that Cdx1 may be involved in cell cycle regulation, but its role is ill defined and the mechanisms have not been explored. We used stable transfection of inducible constructs and transient expression with a replication-deficient adenovirus to induce Cdx1 expression in rat IEC6 cells, a non-transformed intestinal epithelial cell line that does not express Cdx1 protein. Expression of Cdx1 markedly reduced proliferation of IEC6 cells with accumulation of cells in the G(0)/G(1) phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the hypophosphorylated forms of the retinoblastoma protein (pRb) and the pRb-related p130 protein. Protein levels of multiple cyclin-dependent kinase inhibitors were either unchanged (p16, p18, p21, p27, and p57) or were not detected (p15 and p19). Most significantly, levels of cyclins D1 and D2 were markedly diminished with Cdx1 expression, but not cyclins D3, E, or the G(1) kinases. Additionally, cyclin-dependent kinase-4 activity was decreased in association with decreased cyclin D protein. We conclude that Cdx1 regulates intestinal epithelial cell proliferation by inhibiting progression through G(0)/G(1), most likely via modulation of cyclin D1 and D2 protein levels.
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PMID:The caudal-related homeodomain protein Cdx1 inhibits proliferation of intestinal epithelial cells by down-regulation of D-type cyclins. 1066 Jun 24

The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G(1)/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of "free" hyperphosphorylated p130, which could act as a CDK inhibitor.
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PMID:E1A blocks hyperphosphorylation of p130 and p107 without affecting the phosphorylation status of the retinoblastoma protein. 1070 33

Dog thyroid epithelial cells in primary culture constitute a physiologically relevant model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (thyroid-stimulating hormone [TSH]). As previously shown in this system, the cAMP-dependent mitogenic pathway differs from growth factor cascades as it stimulates the accumulation of p27(kip1) but not cyclins D. Nevertheless, TSH induces the nuclear translocations and assembly of cyclin D3 and cdk4, which are essential in cAMP-dependent mitogenesis. Here we demonstrate that transforming growth factor beta(1) (TGFbeta(1)) selectively inhibits the cAMP-dependent cell cycle in mid-G1 and various cell cycle regulatory events, but it weakly affects the stimulation of DNA synthesis by epidermal growth factor (EGF), hepatocyte growth factor, serum, and phorbol esters. EGF+serum and TSH did not interfere importantly with TGFbeta receptor signaling, because they did not affect the TGFbeta-induced nuclear translocation of Smad 2 and 3. TGFbeta inhibited the phosphorylation of Rb, p107, and p130 induced by TSH, but it weakly affected the phosphorylation state of Rb-related proteins in EGF+serum-treated cells. TGFbeta did not inhibit c-myc expression. In TSH-stimulated cells, TGFbeta did not affect the expression of cyclin D3, cdk4, and p27(kip1), nor the induced formation of cyclin D3-cdk4 complexes, but it prevented the TSH-induced relocalization of p27(kip1) from cdk2 to cyclin D3-cdk4. It prevented the nuclear translocations of cdk4 and cyclin D3 without altering the assembly of cyclin D3-cdk4 complexes probably formed in the cytoplasm, where they were prevented from sequestering nuclear p27(kip1) away from cdk2. This study dissociates the assembly of cyclin D3-cdk4 complexes from their nuclear localization and association with p27(kip1). It provides a new mechanism of regulation of proliferation by TGFbeta, which points out the subcellular location of cyclin D-cdk4 complexes as a crucial factor integrating mitogenic and antimitogenic regulations in an epithelial cell in primary culture.
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PMID:Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1). 1071 20

v-Jun accelerates G(1) progression and shares the capacity of the Myc, E2F, and E1A oncoproteins to sustain S-phase entry in the absence of mitogens; however, how it does so is unknown. To gain insight into the mechanism, we investigated how v-Jun affects mitogen-dependent processes which control the G(1)/S transition. We show that v-Jun enables cells to express cyclin A and cyclin A-cdk2 kinase activity in the absence of growth factors and that deregulation of cdk2 is required for S-phase entry. Cyclin A expression is repressed in quiescent cells by E2F acting in conjunction with its pocket protein partners Rb, p107, and p130; however, v-Jun overrides this control, causing phosphorylated Rb and proliferation-specific E2F-p107 complexes to persist after mitogen withdrawal. Dephosphorylation of Rb and destruction of cyclin A nevertheless occur normally at mitosis, indicating that v-Jun enables cells to rephosphorylate Rb and reaccumulate cyclin A without exogenous mitogenic stimulation each time the mitotic "clock" is reset. D-cyclin-cdk activity is required for Rb phosphorylation in v-Jun-transformed cells, since ectopic expression of the cdk4- and cdk6-specific inhibitor p16(INK4A) inhibits both DNA synthesis and cell proliferation. Despite this, v-Jun does not stimulate D-cyclin-cdk activity but does induce a marked deregulation of cyclin E-cdk2. In particular, hormonal activation of a conditional v-Jun-estrogen receptor fusion protein in quiescent, growth factor-deprived cells stimulates cyclin E-cdk2 activity and triggers Rb phosphorylation and DNA synthesis. Thus, v-Jun overrides the mitogen dependence of S-phase entry by deregulating Rb phosphorylation, E2F-pocket protein interactions, and ultimately cyclin A-cdk2 activity. This is the first report, however, that cyclin E-cdk2, rather than D-cyclin-cdk, is likely to be the critical Rb kinase target of v-Jun.
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PMID:v-Jun overrides the mitogen dependence of S-phase entry by deregulating retinoblastoma protein phosphorylation and E2F-pocket protein interactions as a consequence of enhanced cyclin E-cdk2 catalytic activity. 1071 76

Transformation of normal melanocytes to metastatic melanoma cells is characterized by loss of dependency on external growth factors required for the viability and proliferation of normal melanocytes. The molecular events that lead to melanoma cell autonomous growth are not well defined, but are likely to include sustained activity of cyclin-dependent kinases (CDK2, CDK4 and CDK6) as a result of loss of CDK inhibitors (such as p16INK4a and possibly p27KIP1), and persistent upregulation of several cyclins (cyclin D1, cyclin A and cyclin E), the positive regulators of CDKs. CDKs phosphorylate, and consequently, inactivate the retinoblastoma family of tumor suppressor proteins (pRb, p107 and p130), termed pocket proteins. The inactivation of pocket proteins liberates E2F transcription factors from suppressive complexes ('free' E2F) that, in turn, induces the continuous expression of target genes whose products promote cell cycle progression. In normal melanocytes, external growth factors suppress the activity of all three pocket proteins, allowing E2F activity to accumulate and sustain transcription of target genes required for cell proliferation. In contrast, in melanoma cells from advanced lesions, all three pocket proteins are highly phosphorylated and inactive, even in the absence of environmental mitogens, and free E2F activity is constitutively high. Manipulations of normal mouse melanocytes in vitro, and in vivo in transgenic mouse expressing ectopic genes, further support the notion that growth rate, and release from dependency on external mitogens, positively correlate with inactivation of pocket proteins. The latter has been accomplished by sustained cell surface receptor stimulation, such as constitutive high expression of a growth factor, or by sequestration with dominantly acting viral proteins. Taken together, chronic hyperphosphorlyation/inactivation of pRb, p107 and p130 is probably one of the key events in converting growth-factor dependent normal melanocytes, to autonomously growing melanoma cells. Since all pocket proteins are regulated by CDKs activity, it is likely that agents that inhibit this class of enzymes will be effective in treating melanoma patients.
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PMID:Melanoma cell autonomous growth: the Rb/E2F pathway. 1072 88

The bipartite repressor elements, termed cell cycle-dependent element (CDE)/cell cycle regulatory element (CCRE)-cell cycle homology region (CHR) control the growth-dependent transcription of the cyclin A, cdc25C, cdc2 genes. Here, we have identified a functional element displaying the signature of the CDE-CHR in the promoter of the mouse RB2 (p130) gene, encoding the retinoblastoma protein family (pRB)-related protein p130. This element locates close to the major transcription start site where it makes major groove contacts with proteins that can be detected in a cellular context using in vivo genomic footprinting techniques. Inactivation of either the CDE or CHR sequence strongly up-regulates the p130 promoter activity in exponentially growing cells, a situation where endogenous p130 gene expression is almost undetectable. Electrophoretic mobility shift assays suggest that two different protein complexes bind independently to the p130 CDE and CHR elements, and that the protein(s) bound to the CDE might be related to those bound on cyclin A and cdc2 promoters.
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PMID:A CDE/CHR-like element mediates repression of transcription of the mouse RB2 (p130) gene. 1076 May 7


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