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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study examines in vivo the role and functional interrelationships of components regulating exit from the G1 resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57(KIP2) inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57(KIP2) function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57(KIP2) to regulate the G1/S transition in a cell type highly dependent upon pRb.
Mol Cell Biol 1999 Jan
PMID:Cyclin D- and E-dependent kinases and the p57(KIP2) inhibitor: cooperative interactions in vivo. 985 59

Entry into S phase is dependent on the coordinated activation of CDK4,6 and CDK2 kinases. Once a cell commits to S phase, there must be a mechanism to ensure the irreversibility of this decision. The activity of these kinases is inhibited by their association with p27. In many cells, p27 plays a major role in the withdrawal from the cell cycle in response to environmental cues. Thus, it is likely that p27 is a target of the machinery required to ensure the irreversibility of S-phase entry. We have been interested in understanding the mechanisms regulating p27 at the G1/S transition. In this report, we define a cell-free degradation system which faithfully recapitulates the cell cycle phase-specific degradation of p27. We show that this reaction is dependent on active CDK2 activity, suggesting that CDK2 activity is directly required for p27 degradation. In addition to CDK2, other S-phase-specific factors are required for p27 degradation. At least some of these factors are ubiquitin and proteasome dependent. We discuss the relationships between CDK2 activity, ubiquitin-dependent, and possibly ubiquitin-independent proteasomal activities in S-phase extracts as related to p27.
Mol Cell Biol 1999 Feb
PMID:Cell-free degradation of p27(kip1), a G1 cyclin-dependent kinase inhibitor, is dependent on CDK2 activity and the proteasome. 989 Oct 53

The tumour suppressor p16 is a member of the INK4 family of inhibi tors of the cyclin D-dependent kinases, CDK4 and CDK6, that are involved in the key growth control pathway of the eukaryotic cell cycle. The 156 amino acid residue protein is composed of four ankyrin repeats (a helix-turn-helix motif) that stack linearly as two four-helix bundles resulting in a non-globular, elongated molecule. The thermodynamic and kinetic properties of the folding of p16 are unusual. The protein has a very low free energy of unfolding, Delta GH-2O/D-N, of 3.1 kcal mol-1 at 25 degreesC. The rate-determining transition state of folding/unfolding is very compact (89% as compact as the native state). The other unusual feature is the very rapid rate of unfolding in the absence of denaturant of 0.8 s-1 at 25 degreesC. Thus, p16 has both thermodynamic and kinetic instability. These features may be essential for the regulatory function of the INK4 proteins and of other ankyrin-repeat-containing proteins that mediate a wide range of protein-protein interactions. The mechanisms of inactivation of p16 by eight cancer-associated mutations were dissected using a systematic method designed to probe the integrity of the secondary structure and the global fold. The structure and folding of p16 appear to be highly vulnerable to single point mutations, probably as a result of the protein's low stability. This vulnerability provides one explanation for the striking frequency of p16 mutations in tumours and in immortalised cell lines.
J Mol Biol 1999 Jan 29
PMID:Stability and folding of the tumour suppressor protein p16. 991 18

The D-type cyclins and their major kinase partners CDK4 and CDK6 regulate G0-G1-S progression by contributing to the phosphorylation and inactivation of the retinoblastoma gene product, pRB. Assembly of active cyclin D-CDK complexes in response to mitogenic signals is negatively regulated by INK4 family members. Here we show that although all four INK4 proteins associate with CDK4 and CDK6 in vitro, only p16(INK4a) can form stable, binary complexes with both CDK4 and CDK6 in proliferating cells. The other INK4 family members form stable complexes with CDK6 but associate only transiently with CDK4. Conversely, CDK4 stably associates with both p21(CIP1) and p27(KIP1) in cyclin-containing complexes, suggesting that CDK4 is in equilibrium between INK4 and p21(CIP1)- or p27(KIP1)-bound states. In agreement with this hypothesis, overexpression of p21(CIP1) in 293 cells, where CDK4 is bound to p16(INK4a), stimulates the formation of ternary cyclin D-CDK4-p21(CIP1) complexes. These data suggest that members of the p21 family of proteins promote the association of D-type cyclins with CDKs by counteracting the effects of INK4 molecules.
Mol Cell Biol 1999 Mar
PMID:Cyclin D-CDK subunit arrangement is dependent on the availability of competing INK4 and p21 class inhibitors. 1002 65

To investigate the mode of action of the p16(INK4a) tumor suppressor protein, we have established U2-OS cells in which the expression of p16(INK4a) can be regulated by addition or removal of isopropyl-beta-D-thiogalactopyranoside. As expected, induction of p16(INK4a) results in a G1 cell cycle arrest by inhibiting phosphorylation of the retinoblastoma protein (pRb) by the cyclin-dependent kinases CDK4 and CDK6. However, induction of p16(INK4a) also causes marked inhibition of CDK2 activity. In the case of cyclin E-CDK2, this is brought about by reassortment of cyclin, CDK, and CDK-inhibitor complexes, particularly those involving p27(KIP1). Size fractionation of the cellular lysates reveals that a substantial proportion of CDK4 participates in active kinase complexes of around 200 kDa. Upon induction of p16(INK4a), this complex is partly dissociated, and the majority of CDK4 is found in lower-molecular-weight fractions consistent with the formation of a binary complex with p16(INK4a). Sequestration of CDK4 by p16(INK4a) allows cyclin D1 to associate increasingly with CDK2, without affecting its interactions with the CIP/KIP inhibitors. Thus, upon the induction of p16(INK4a), p27(KIP1) appears to switch its allegiance from CDK4 to CDK2, and the accompanying reassortment of components leads to the inhibition of cyclin E-CDK2 by p27(KIP1) and p21(CIP1). Significantly, p16(INK4a) itself does not appear to form higher-order complexes, and the overwhelming majority remains either free or forms binary associations with CDK4 and CDK6.
Mol Cell Biol 1999 Mar
PMID:Induced expression of p16(INK4a) inhibits both CDK4- and CDK2-associated kinase activity by reassortment of cyclin-CDK-inhibitor complexes. 1002 85

The INK4 family of cyclin-dependent kinase (CDK) inhibitors includes four 15- to 19-kDa polypeptides (p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d)) that bind to CDK4 and CDK6. By disrupting cyclin D-dependent holoenzymes, INK4 proteins prevent phosphorylation of the retinoblastoma protein and block entry into the DNA-synthetic phase of the cell division cycle. The founding family member, p16(INK4a), is a potent tumor suppressor in humans, whereas involvement, if any, of other INK4 proteins in tumor surveillance is less well documented. INK4c and INK4d are expressed during mouse embryogenesis in stereotypic tissue-specific patterns and are also detected, together with INK4b, in tissues of young mice. INK4a is expressed neither before birth nor at readily appreciable levels in young animals, but its increased expression later in life suggests that it plays some checkpoint function in response to cell stress, genotoxic damage, or aging per se. We used targeted gene disruption to generate mice lacking INK4d. These animals developed into adulthood, had a normal life span, and did not spontaneously develop tumors. Tumors did not arise at increased frequency in animals neonatally exposed to ionizing radiation or the carcinogen dimethylbenzanthrene. Mouse embryo fibroblasts, bone marrow-derived macrophages, and lymphoid T and B cells isolated from these animals proliferated normally and displayed typical lineage-specific differentiation markers. Males exhibited marked testicular atrophy associated with increased apoptosis of germ cells, although they remained fertile. The absence of tumors in INK4d-deficient animals demonstrates that, unlike INK4a, INK4d is not a tumor suppressor but is instead involved in spermatogenesis.
Mol Cell Biol 2000 Jan
PMID:INK4d-deficient mice are fertile despite testicular atrophy. 1059 39

Active cyclin-dependent kinases (CDKs) are required for progression through the G1 phase of the cell cycle and entry into S phase. Activity of G1 CDKs is controlled by mechanisms including phosphorylation of Thr14 and Tyr15 residues. Removal of inhibitory phosphates on these amino acid residues is required for G1 CDK activation, and is mediated by the Cdc25A phosphatase. Regulation of active Cdc25A phosphatase levels may be important for the proliferation of hematopoietic progenitor cells, effects assessed in the human growth-factor-dependent cell line Mo7e. Constitutive Cdc25A protein levels were enhanced with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus steel factor (SF). Cdc25A is thought to exert its activity in the nucleus, and nuclear protein levels of Cdc25A were also enhanced with GM-CSF and SF. GM-CSF plus SF promote synergistic growth of Mo7e cells. Pretreatment with macrophage inflammatory protein (MIP-1alpha) inhibited GM-CSF- plus SF-induced growth and upregulation of Cdc25A protein levels. Stimulation with GM-CSF and SF also rapidly increased Cdc25A phosphatase activity, an effect suppressed by MIP-1alpha. A concomitant inhibition of increased CDK4 kinase activity correlated with increased phosphotyrosine levels on CDK4 when cells were pretreated with MIP-1alpha prior to GM-CSF and SF. These data suggest that Cdc25A expression and activity are regulated during proliferation of Mo7e cells.
Cytokines Cell Mol Ther 1999 Sep
PMID:The effects of GM-CSF, steel factor and MIP-1alpha on the expression and activation of Cdc25A phosphatase in Mo7e cells. 1064 70

The p53-inducible gene PC3 (TIS21, BTG2) is endowed with antiproliferative activity. Here we report that expression of PC3 in cycling cells induced accumulation of hypophosphorylated, growth-inhibitory forms of pRb and led to G(1) arrest. This latter was not observed in cells with genetic disruption of the Rb gene, indicating that the PC3-mediated G(1) arrest was Rb dependent. Furthermore, (i) the arrest of G(1)-S transition exerted by PC3 was completely rescued by coexpression of cyclin D1 but not by that of cyclin A or E; (ii) expression of PC3 caused a significant down-regulation of cyclin D1 protein levels, also in Rb-defective cells, accompanied by inhibition of CDK4 activity in vivo; and (iii) the removal from the PC3 molecule of residues 50 to 68, a conserved domain of the PC3/BTG/Tob gene family, which we term GR, led to a loss of the inhibition of proliferation as well as of the down-regulation of cyclin D1 levels. These data point to cyclin D1 down-regulation as the main factor responsible for the growth inhibition by PC3. Such an effect was associated with a decrease of cyclin D1 transcript and of cyclin D1 promoter activity, whereas no effect of PC3 was observed on cyclin D1 protein stability. Taken together, these findings indicate that PC3 impairs G(1)-S transition by inhibiting pRb function in consequence of a reduction of cyclin D1 levels and that PC3 acts, either directly or indirectly, as a transcriptional regulator of cyclin D1.
Mol Cell Biol 2000 Mar
PMID:Arrest of G(1)-S progression by the p53-inducible gene PC3 is Rb dependent and relies on the inhibition of cyclin D1 transcription. 1066 55

The presence of two families of seven distinct mammalian cyclin-dependent kinase (CDK) inhibitor genes is thought to mediate the complexity of connecting a variety of cellular processes to the cell cycle control pathway. The distinct pattern of tissue expression of CDK inhibitor genes suggests that they may function as tumor suppressors with different tissue specificities. To test this hypothesis, we have characterized two strains of double mutant mice lacking either p18(INK4c) and p27(KIP1) or p18(INK4c) and p21(CIP1/WAF1). Loss of both p18 and p27 function resulted in the spontaneous development by 3 months of age of at least eight different types of hyperplastic tissues and/or tumors in the pituitary, adrenals, thyroid, parathyroid, testes, pancreas, duodenum, and stomach. Six of these hyperplastic tissues and tumors were in endocrine organs, and several types of tumors routinely developed within the same animal, a phenotype reminiscent of that seen in combined human multiple endocrine neoplasia syndromes. The p18-p21 double null mice, on the other hand, developed pituitary adenomas, multifocal gastric neuroendocrine hyperplasia, and lung bronchioalveolar tumors later in life. G(1) CDK2 and CDK4 kinase activities were increased in both normal and neoplastic tissues derived from mice lacking individual CDK inhibitors and were synergistically stimulated by the simultaneous loss of two CDK inhibitors. This indicates that an increase in G(1) CDK kinase activity is a critical step during but is not sufficient for tumor growth. Our results suggest that functional collaborations between distinct CDK inhibitor genes are tissue specific and confer yet another level of regulation in cell growth control and tumor suppression.
Mol Cell Biol 2000 Aug
PMID:Functional collaboration between different cyclin-dependent kinase inhibitors suppresses tumor growth with distinct tissue specificity. 1091 96

The eukaryotic cell cycle is regulated by cyclin-dependent kinases (CDKs). CDK4 and CDK6, which are activated by D-type cyclins during the G(1) phase of the cell cycle, are thought to be responsible for phosphorylation of the retinoblastoma gene product (pRb). The tumor suppressor p16(INK4A) inhibits phosphorylation of pRb by CDK4 and CDK6 and can thereby block cell cycle progression at the G(1)/S boundary. Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II by general transcription factor TFIIH is believed to be an important regulatory event in transcription. TFIIH contains a CDK7 kinase subunit and phosphorylates the CTD. We have previously shown that p16(INK4A) inhibits phosphorylation of the CTD by TFIIH. Here we report that the ability of p16(INK4A) to inhibit CDK7-CTD kinase contributes to the capacity to induce cell cycle arrest. These results suggest that p16(INK4A) may regulate cell cycle progression by inhibiting not only CDK4-pRb kinase activity but also by modulating CDK7-CTD kinase activity. Regulation of CDK7-CTD kinase activity by p16(INK4A) thus may represent an alternative pathway for controlling cell cycle progression.
Mol Cell Biol 2000 Oct
PMID:Regulation of CDK7-carboxyl-terminal domain kinase activity by the tumor suppressor p16(INK4A) contributes to cell cycle regulation. 1100 68


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