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 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

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

14-3-3 sigma, implicated in cell cycle arrest by p53, was cloned by expression cloning through cyclin-dependent kinase 2 (CDK2) association. 14-3-3 sigma shares cyclin-CDK2 binding motifs with different cell cycle regulators, including p107, p130, p21(CIP1), p27(KIP1), and p57(KIP2), and is associated with cyclin.CDK complexes in vitro and in vivo. Overexpression of 14-3-3 sigma obstructs cell cycle entry by inhibiting cyclin-CDK activity in many breast cancer cell lines. Overexpression of 14-3-3 sigma can also inhibit cell proliferation and prevent anchorage-independent growth of these cell lines. These findings define 14-3-3 sigma as a negative regulator of the cell cycle progression and suggest that it has an important function in preventing breast tumor cell growth.
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PMID:Association of the cyclin-dependent kinases and 14-3-3 sigma negatively regulates cell cycle progression. 1076 98

Neuronal death evoked by DNA damage requires cyclin-dependent kinase 4 (Cdk4) and 6 activity and is accompanied by elevation of cyclin D1-associated kinase activity. Because Cdk4/6 phosphorylates retinoblastoma protein (pRb) family members that then modulate the transcriptional activity of E2F/DP1 complexes, we examined the involvement of these components in DNA damage-evoked neuronal death. Camptothecin induced rapid pRb and p107 phosphorylation at a Cdk4/6 phosphorylation site followed by selective loss of Rb and p107. The CDK inhibitor flavopiridol suppressed pRb and p107 phosphorylation and loss, implicating CDK activity in these events. Moreover, the loss of pRb and p107 appeared to be mediated by caspases because it was blocked by general caspase inhibitors. The role of phosphorylation and pRb and p107 loss in the death pathway was indicated by observations that virally mediated expression of pRb mutated at sites of phosphorylation, including the Cdk4/6 site, inhibited death. Finally, expression of dominant-negative versions of DP1, known to compromise E2F transcriptional activity, protects cortical neurons from death induced by camptothecin and sympathetic neurons from death evoked by UV treatment. Taken together, these results implicate the CDK-pRb/E2F/DP pathway as a required element in the neuronal death evoked by DNA damage.
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PMID:Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage. 1077 74

Ectopic expression of the CDK inhibitors (CKIs) p16INK4a and p27Kip1 in Rat1 fibroblasts induces dephosphorylation and activation of Retinoblastoma-family proteins (pRb, p107 and p130), their association with E2F proteins, and cell cycle arrest in G1. The growth-inhibitory action of p16, in particular, is believed to be mediated essentially via pRb activation. The 12S E1A protein of human Adenovirus 5 associates with pRb-family proteins via residues in its Conserved Regions (CR) 1 and 2, in particular through the motif LXCXE in CR2. These interactions are required for E1A to prevent G1 arrest upon co-expression of CKIs. We show here that mutating either of two conserved motifs adjacent to LXCXE in CR2, GFP and SDDEDEE, also impairs the ability of E1A to overcome G1 arrest by p16 or p27. Strikingly, however, these mutations affect neither the association of E1A with pRb, p07 and p130, nor its ability to derepress E2F-1 transcriptional activity in transient transfection assays. One of the EIA mutants, however, is defective in derepressing several endogenous E2F target genes in the presence of p16 or p27. Thus, CR2 possesses an essential function besides pRb-binding. We speculate that this function might be required for the full derepression of E2F-regulated genes in their natural chromatin context.
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PMID:Conserved region 2 of adenovirus E1A has a function distinct from pRb binding required to prevent cell cycle arrest by p16INK4a or p27Kip1. 1080 68

The retinoblastoma family of proteins, pRb/p105, p107, and pRb2/ p130, cooperate to regulate cell cycle progression through the G1 phase of the cell cycle. Each of the family members realize their common goal of G1-S checkpoint regulation through overlapping and unique growth regulatory pathways. We took advantage of a tetracycline-regulated gene expression system to control the expression of RB2/p130 in JC virus-induced hamster brain tumor cells to study in vivo the molecular mechanisms used by pRb2/p130 to elicit its growth-suppressive function. We have previously used this system to demonstrate that induction of pRb/ p130 expression suppresses tumor growth in vivo by overcoming neoplastic transformation mediated by the large T-antigen oncoprotein of JCV (JCV TAg). Here we found that induction of pRb2/p130 in vivo specifically inhibits cyclin A- and cyclin E-associated kinase activity and by doing so induces p27Kip1 levels presumably by inhibiting p27Kip1-targeted proteolysis by cyclin E-Cdk2 phosphorylation of p27Kip1. RB2/p130 induction also decreased cyclin A and the transcription factor E2F-1 while increasing cyclin E at both the transcriptional and protein levels of expression. The growth inhibitory activity of pRb2/p130 also correlated with its E2F-binding capacity. Furthermore, p27Kip1 and pRb2/p130 were found to be targets of the JCV TAg oncoprotein and to interact in vivo with each other independently from the presence of TAg. Interestingly, pRb2/p130 expression negatively modulated the binding of p27Kip1 to JCV TAg. These data suggest that pRb2/p130 and p27Kip1 may cooperate in regulating cellular proliferation, and both may be involved in a negative feedback regulatory loop with cyclin E.
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PMID:Inducible pRb2/p130 expression and growth-suppressive mechanisms: evidence of a pRb2/p130, p27Kip1, and cyclin E negative feedback regulatory loop. 1082 49

TGFbeta1 is a potent growth inhibitor of both primitive and more differentiated human myeloid leukemic cells. The extent of the growth inhibitory response to TGFbeta varies with cell type, and is not linked to stages of differentiation of cell lines. Downregulation of multiple cell cycle-regulatory molecules is a dominant event in TGFbeta1-mediated growth inhibition of human MV4-11 myeloid leukemia cells. Both G1-phase and G2-phase cyclins and cdks participate in the regulation of TGFbeta1-mediated growth inhibition of MV4-11 cells. By both depressing cdk2 synthesis and up-regulating cyclin E-associated p27, TGFbeta1 may magnify its inhibitory efficiency. TGFbeta1 also rapidly inhibits phosphorylation of pRb at several serine and threonine residues. The underphosphorylated pRb associates with E2F-4 in G1 phase, whereas the phosphorylated pRb mainly binds to E2F-1 and E2F-3 in proliferating MV4-11 cells. Since TGFbeta1 upregulates p130/E2F-4 complex formation and downregulates p107/E2F-4 complex formation, with E2F-4 levels remaining constant, our results suggest that E2F-4 is switched from p107 to pRb and p130 when cells exit from the cell cycle and arrest in G1 by TGFbeta1. In summary, TGFbeta1 inhibits growth of human myeloid leukemic cells through multiple pathways, whereas the "cdk inhibitor" p27 is both a positive and negative regulator.
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PMID:Cell cycle and transcriptional control of human myeloid leukemic cells by transforming growth factor beta. 1083 Jul 31


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