Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E-cyclin-dependent kinase 2 complex in vitro.
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The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem. 269, 18299-18302]. Here we have investigated the phosphorylation of MARCKS by various cyclin-dependent kinases (Cdks) in vitro. We established that Cdk2, Cdk4 and, to a smaller extent, Cdk1 that have been immunoprecipitated from cellular extracts phosphorylate MARCKS. Comparison of MARCKS phosphorylation by protein kinase C (PKC) and by the purified cyclin E-Cdk2 complex suggested that two residues were phosphorylated by Cdk2 under these conditions. To identify these sites, Cdk2-phosphorylated MARCKS was digested with lysyl endoprotease and analysed by electrospray MS. Comparison with the digests obtained from the unphosphorylated protein demonstrated that two peptides, Gly12-Lys30 and Ala138-Lys152, were phosphorylated by cyclin E-Cdk2. The identity of these peptides was confirmed by automatic Edman degradation. On the basis of the consensus phosphorylation sequence described for Cdk2, and on MS/MS analysis of the Ala138-Lys152 peptide, we concluded that Ser27, one of the phosphorylation sites identified in vivo, and Thr150 were the Cdk2 targets in vitro. None of the other sites described in vivo were phosphorylated in these conditions. Interestingly, a preliminary phosphorylation of MARCKS by PKC improved the initial rate of phosphorylation by Cdk2 without modifying the number of sites concerned. In contrast, phosphorylation of MARCKS by Cdk2 did not significantly affect further phosphorylation by PKC. (+info)
Molecular mechanisms underlying interferon-alpha-induced G0/G1 arrest: CKI-mediated regulation of G1 Cdk-complexes and activation of pocket proteins.
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One prominent effect of IFNs is their cell growth-inhibitory activity. The mechanism behind this inhibition of proliferation is still not fully understood. In this study, the effect of IFN-alpha treatment on cell cycle progression has been analysed in three lymphoid cell lines, Daudi, U-266 and H9. Examination of the growth-arrested cell populations shows that Daudi cells accumulate in a G0-like state, whereas U-266 cells arrest later in G1. H9 cells are completely resistant to IFN-alpha's cell growth-inhibitory effects. The G0/G1-phase arrest is preceded by a rapid induction of the cyclin-dependent kinase inhibitors (CKIs), p21 and p15. In parallel, the activities of the G1 Cdks are significantly reduced. In addition to p21/p15 induction, IFN-alpha regulates the expression of another CKI, p27, presumably by a post-transcriptional mechanism. In the G1 Cdk-complexes, there is first an increased binding of p21 and p15 to their respective kinases. At longer exposure times, when Cdk-bound p15 and p21 decline, p27 starts to accumulate. Furthermore, we found that IFN-alpha not only suppresses the phosphorylation of pRb, but also alters the phosphorylation and expression of the other pocket proteins p130 and p107. These data suggest that induction of p21/p15 is involved in the primary IFN-alpha response inhibiting G1 Cdk activity, whereas increased p27 expression is part of a second set of events which keep these Cdks in their inactive form. Moreover, elevated levels of p27 correlated with a dissociation of cyclin E/Cdk2-p130 or p107 complexes to yield cyclin E/Cdk2-p27 complexes. In resistant H9 cells, which possess a homozygous deletion of the p15/p16 genes and lack p21 protein expression, IFN-alpha causes no detectable changes in p27 expression and, furthermore, no effects are observed on either pocket proteins in this cell line. Taken together, these data suggest that the early decline in G1 Cdk activity, subsequent changes in phosphorylation of pocket proteins, and G1/G0 arrest following IFN-alpha treatment, is not primarily due to loss of the G1 kinase components, but result from the inhibitory action of CKIs on these complexes. (+info)
Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest.
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p27[KIP1] (p27) is a cyclin dependent kinase inhibitor, involved in the negative regulation of G1 progression in response to a number of anti-proliferative signals. In this study we show, in growing mouse hybridoma (7TD1) and human myeloma (U266) cell lines, that p27 is highly expressed but slightly upregulated when cells are arrested, regardless to the phases of the cell cycle. In contrast, the specific blockade of these cells in early G1 phase reveals the induction of a protein of 23 kDa (p23) specifically recognized by polyclonal anti-p27 antibodies raised against the NH2 terminal part of p27 but not by anti-p21[CIP1] antibodies. Experiments using caspase inhibitors strongly suggest that p23 results from the proteolysis of p27 by a 'caspase-3-like' protease. This cleavage leads to the cytosolic sequestration of p23 but does not alter its binding properties to CDK2 and CDK4 kinases. Indeed, p23 associated in vivo with high molecular weight complexes and coprecipitated with CDK2 and CDK4. We demonstrate by transfection experiments in SaOS-2 cells that p23 induces a G1 phase growth arrest by inhibition of cyclin/CDK2 activity. In summary we describe here a caspase-cleaved form of p27, induced in absence of detectable apoptosis and likely involved in cell cycle regulation. (+info)
Transduced p16INK4a peptides inhibit hypophosphorylation of the retinoblastoma protein and cell cycle progression prior to activation of Cdk2 complexes in late G1.
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Progression of cells through the G1 phase of the cell cycle requires cyclin D:Cdk4/6 and cyclin E:Cdk2 complexes; however, the duration and ordering of these complexes remain unclear. To address this, we synthesized a peptidyl mimetic of the Cdk4/6 inhibitor, p16INK4a that contained an NH2-terminal TAT protein transduction domain. Transduction of TAT-p16 wild-type peptides into cells resulted in the loss of active, hypophosphorylated pRb and elicited an early G1 cell cycle arrest, provided cyclin E:Cdk2 complexes were inactive. We conclude that cyclin D:Cdk4/6 activity is required for early G1 phase cell cycle progression up to, but not beyond, activation of cyclin E:Cdk2 complexes at the restriction point and is thus nonredundant with cyclin E:Cdk2 in late G1. (+info)
c-Myc regulates cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle progression at multiple independent points.
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c-myc is a cellular proto-oncogene associated with a variety of human cancers and is strongly implicated in the control of cellular proliferation, programmed cell death, and differentiation. We have previously reported the first isolation of a c-myc-null cell line. Loss of c-Myc causes a profound growth defect manifested by the lengthening of both the G1 and G2 phases of the cell cycle. To gain a clearer understanding of the role of c-Myc in cellular proliferation, we have performed a comprehensive analysis of the components that regulate cell cycle progression. The largest defect observed in c-myc-/- cells is a 12-fold reduction in the activity of cyclin D1-Cdk4 and -Cdk6 complexes during the G0-to-S transition. Downstream events, such as activation of cyclin E-Cdk2 and cyclin A-Cdk2 complexes, are delayed and reduced in magnitude. However, it is clear that c-Myc affects the cell cycle at multiple independent points, because restoration of the Cdk4 and -6 defect does not significantly increase growth rate. In exponentially cycling cells the absence of c-Myc reduces coordinately the activities of all cyclin-cyclin-dependent kinase complexes. An analysis of cyclin-dependent kinase complex regulators revealed increased expression of p27(KIP1) and decreased expression of Cdk7 in c-myc-/- cells. We propose that c-Myc functions as a crucial link in the coordinate adjustment of growth rate to environmental conditions. (+info)
Critical role played by cyclin D3 in the MyoD-mediated arrest of cell cycle during myoblast differentiation.
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During the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and with the requirement of the p300 transcriptional coactivator. In differentiated myocytes, cyclin D3 also becomes stabilized and is found nearly totally complexed with unphosphorylated pRb. The detection of complexes containing cyclin D3, cdk4, p21, and PCNA suggests that cdk4, along with PCNA, may get sequestered into high-order structures held together by pRb and cyclin D3. Cyclin D3 up-regulation and stabilization is inhibited by adenovirus E1A, and this correlates with the ability of E1A to promote pRb phosphorylation; conversely, the overexpression of cyclin D3 in differentiated myotubes counteracts the E1A-mediated reactivation of DNA synthesis. These results indicate that cyclin D3 critically contributes to the irreversible exit of differentiating myoblasts from the cell cycle. (+info)
Role for cyclin D1 in UVC-induced and p53-mediated apoptosis.
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DNA damaging agents such as ultraviolet (UV) induce cell cycle arrest followed by apoptosis in cells where irreparable damage has occurred. Here we show that during early phase G1 arrest which occurs in UV-irradiated human U343 glioblastoma cells, there are (1) decreases in cyclin D1 and cdk4 levels which parallel a loss of S-phase promoting cyclin D1/cdk4 complexes, and (2) increases in p53 and p21 protein levels. We also show that the late phase UV-induced apoptosis of U343 cells occurs after cell cycle re-entry and parallels the reappearance of cyclin D1 and cdk4 and cyclin D1/cdk4 complexes. These findings suggest that cyclin D1 can abrogate UV-induced G1 arrest and that the p53-mediated apoptosis that occurs in these cells is dependent on cyclin D1 levels. We examined these possibilities using U343 cells that ectopically express cyclin D1 and found that indeed cyclin D1 can overcome the cell cycle arrest caused by UV. Moreover, the appearance of p53 protein and the induction of apoptosis in UV-irradiated cells was found to be dependent on the level of ectopically expressed cyclin D1. These findings, therefore, indicate that expression of cyclin D1 following DNA damage is essential for cell cycle re-entry and p53-mediated apoptosis. (+info)
Involvement of cell cycle elements, cyclin-dependent kinases, pRb, and E2F x DP, in B-amyloid-induced neuronal death.
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Previous evidence by others has indicated that a variety of cell cycle-related molecules are up-regulated in brains of Alzheimer's disease patients. The significance of this increase, however, is unclear. Accordingly, we examined the obligate nature of cyclin-dependent kinases and select downstream targets of these kinases in death of neurons evoked by B-amyloid (AB) protein. We present pharmacological and molecular biological evidence that cyclin-dependent kinases, in particular Cdk4/6, are required for such neuronal death. In addition, we demonstrate that the substrate of Cdk4/6, pRb/p107, is phosphorylated during AB treatment and that one target of pRb/p107, the E2F x DP complex, is required for AB-evoked neuronal death. These results provide evidence that cell cycle elements play a required role in death of neurons evoked by AB and suggest that these elements play an integral role in Alzheimer's disease-related neuronal death. (+info)