Complex Mechanisms Underlying Impaired Activation of Cdk4 and Cdk2 in Replicative Senescence: Roles of p16, p21, and Cyclin D1

Morisaki, Hirobumi; Andõ, Akira; Nagata, Yoshiho; Pereira‐Smith, Olivia M.; Smith, James R.; Ikeda, Kyoji; Nakanishi, Makoto

doi:10.1006/excr.1999.4698

Cited by 40 publications

(34 citation statements)

References 33 publications

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“…It is likely to be totally inactive (5,40). At variance with cyclin E-bound CDK2, CDK2 associated with cyclin D1 and p21 was only faintly phosphorylated at Thr-160, even in serum-stimulated cells (not shown), in agreement with previous reports indicating that cyclin D1 is an inhibitor for CDK2 that prevents its phosphorylation by CAK (45,61,62).…”

Section: Table I Summary Of the Main Observationssupporting

confidence: 92%

Phosphorylations of Cyclin-dependent Kinase 2 Revisited Using Two-dimensional Gel Electrophoresis

Coulonval

Bockstaele²,

Paternot³

et al. 2003

Journal of Biological Chemistry

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To control the G 1 /S transition and the progression through the S phase, the activation of the cyclin-dependent kinase (CDK) 2 involves the binding of cyclin E then cyclin A, the activating Thr-160 phosphorylation within the T-loop by CDK-activating kinase (CAK), inhibitory phosphorylations within the ATP binding region at Tyr-15 and Thr-14, dephosphorylation of these sites by cdc25A, and release from Cip/Kip family (p27 kip1 and p21 cip1 ) CDK inhibitors. To re-assess the precise relationship between the different phosphorylations of CDK2, and the influence of cyclins and CDK inhibitors upon them, we introduce here the use of the high resolution power of two-dimensional gel electrophoresis, combined to Tyr-15-or Thr-160-phosphospecific antibodies. The relative proportions of the potentially active forms of CDK2 (phosphorylated at Thr-160 but not Tyr-15) and inactive forms (non-phosphorylated, phosphorylated only at Tyr-15, or at both Tyr-15 and Thr-160), and their respective association with cyclin E, cyclin A, p21, and p27, were demonstrated during the mitogenic stimulation of normal human fibroblasts. Novel observations modify the current model of the sequential CDK2 activation process: (i) Tyr-15 phosphorylation induced by serum was not restricted to cyclin-bound CDK2; (ii) Thr-160 phosphorylation engaged the entirety of Tyr-15-phosphorylated CDK2 associated not only with a cyclin but also with p27 and p21, suggesting that Cip/ Kip proteins do not prevent CDK2 activity by impairing its phosphorylation by CAK; (iii) the potentially active CDK2 phosphorylated at Thr-160 but not Tyr-15 represented a tiny fraction of total CDK2 and a minor fraction of cyclin A-bound CDK2, underscoring the rate-limiting role of Tyr-15 dephosphorylation by cdc25A.The major events of the eukaryotic cell cycle depend on the sequential and ordered formation, activation (by phosphorylation and/or dephosphorylation) and then inactivation of different complexes of cyclin-dependent kinases (CDKs). 1 CDK2 plays an essential role in controlling the G 1 /S transition and the progression through the S phase: it participates in the inactivating phosphorylations of pRb and related p130 (1-4), and it phosphorylates other key substrates whose activities are necessary to trigger and organize the DNA synthesis phase while preventing DNA re-replication (5-9). Because the critical roles of CDK2, both in positive and negative mitogenic controls of cell cycle and as the end point target of DNA damage checkpoint mechanisms, have been well established (10 -13), understanding the details of CDK2 regulation is of fundamental importance.Based on the cdc2/CDK1 activation model (14 -16) and on structural (17) and enzymatic studies (18 -24), the consensual framework of CDK2 activation is the following. The binding of a cyclin partner (cyclin E at the G 1 /S transition, cyclin A during the S phase) confers a low basal activity to the cyclin⅐CDK2 complex and enables subsequent phosphorylation of CDK2 by the CDK-activating kinase (CAK/cyclin H-CDK7) on a conser...

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Section: Table I Summary Of the Main Observationssupporting

confidence: 92%

Phosphorylations of Cyclin-dependent Kinase 2 Revisited Using Two-dimensional Gel Electrophoresis

Coulonval

Bockstaele²,

Paternot³

et al. 2003

Journal of Biological Chemistry

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“…It has long been suggested that assembly of CDK4/6 with cyclin D is regulated by factor(s), so called 'assembly factor', which is induced or activated by mitogenic stimulation in fibroblasts (Cheng et al, 1998;Matsushime et al, 1994;Sherr and Roberts, 1999;Sugimoto et al, 1999). In contrast to p27 Kip1 , p21 Cip1 expression is induced upon mitogenic stimulation in fibroblasts (Morisaki et al, 1999;Sherr and Roberts, 1999) and is able to Figure 4 Differential effect of p34 SEI-1 and p27 Kip1 on cyclin D1-CDK4. (A,B) Both types of asynchronously proliferating MEFs (passage 15) were infected with recombinant adenoviruses encoding either GFP, flag-tagged p34 SEI-1 or p27 Kip1 .…”

Section: Discussionmentioning

confidence: 99%

Activation of cyclin D1-kinase in murine fibroblasts lacking both p21Cip1 and p27Kip1

et al. 2002

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Deregulation of D-type cyclin-dependent kinases (CDK4 and 6) is widely observed in various human cancers, illustrating their importance in cell cycle control. Like other cyclin-dependent kinases (CDKs), assembly with cyclins is the most critical step for activation of CDK4/ 6. As previously reported elsewhere, we observed that the level of cyclinD1-CDK4 complex and its associated kinase activity were significantly low in asynchronously proliferating mouse embryo fibroblasts lacking both p21 Cip1 and p27 Kip1 (p21/p27-null MEFs). These evidences imply that p21 Cip1 and p27 Kip1 CDK inhibitors are 'essential activators' of cyclin D-kinases. We, however, discovered here that both the assembly and activation of cyclin D1-CDK4 complex occur when quiescent p21/p27-null MEFs were stimulated to re-enter the cell cycle. This mitogen-induced cyclin D1-kinase activity was blocked by overexpression of p16 INK4a and resulted in the inhibition of S phase entry in p21/p27-null MEFs. Furthermore, ectopic expression of p34 SEI-1 , a mitogen-induced CDK4 binding protein, increased the levels of active cyclinD1-CDK4 complex in asynchronously proliferating p21/p27-null MEFs. Together, our results suggest that there are several independent ways to stimulate the assembly of cyclin D1-CDK4 kinases. Although p21 Cip1 and p27 Kip1 play a role in this process, our results demonstrate that additional mechanisms must occur in G0 to S phase transition.

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“…The role of p21 as an assembly activator or inhibitor depends on its expression level. At low and intermediate concentrations it is an assembly factor, while at high concentrations it is an inhibitor [87] . p21 has been shown to be part of a quaternary complex that contains cyclin, Cdk and PCNA [88] .…”

Section: P21 and Differentiationmentioning

confidence: 99%

The Biology of p21Waf1/Cip1 - Review Paper

Blundell¹

2006

American J. of Biochemistry and Biotechnology

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p21WAF1/Cip1 belongs to the Cip/Kip family of cyclin kinase inhibitors (CKI) (p21 Waf1/Cip1 , p27Kip1 , p57 Kip1 ). p21 Waf1/Cip1 was first described as a potent and universal inhibitor of cyclin-dependent kinases (Cdks). Two forms of functionally active p21 has been localized and described: a nuclear and a cytoplasmic forms. In this paper the structures of p21 has been described and as well as it functional biology in terms of differentiation, proliferation and apoptotic activities. The upregulation of p21 by p53-dependent and p53-independent mechanisms is also described.

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Complex Mechanisms Underlying Impaired Activation of Cdk4 and Cdk2 in Replicative Senescence: Roles of p16, p21, and Cyclin D1

Cited by 40 publications

References 33 publications

Phosphorylations of Cyclin-dependent Kinase 2 Revisited Using Two-dimensional Gel Electrophoresis

Phosphorylations of Cyclin-dependent Kinase 2 Revisited Using Two-dimensional Gel Electrophoresis

Activation of cyclin D1-kinase in murine fibroblasts lacking both p21Cip1 and p27Kip1

The Biology of p21Waf1/Cip1 - Review Paper

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