Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27.

The p27(Kip1) protein associates with G1-specific cyclin-CDK complexes and inhibits their catalytic activity. p27(Kip1) is regulated at various levels, including translation, degradation by the ubiquitin/proteasome pathway and non-covalent sequestration. Here, we describe point mutants of p27 deficient in their interaction with either cyclins (p27(c-)), CDKs (p27(k-)) or both (p27(ck-)), and demonstrate that each contact is critical for kinase inhibition and induction of G1 arrest. Through its intact cyclin contact, p27(k-) associated with active cyclin E-CDK2 and, unlike wild type p27, p27(c-) or p27(ck-), was efficiently phosphorylated by CDK2 on a conserved C-terminal CDK target site (TPKK). Retrovirally expressed p27(k-) was rapidly degraded through the proteasome in Rat1 cells, but was stabilized by secondary mutation of the TPKK site to VPKK. In this experimental setting, exogenous wild-type p27 formed inactive ternary complexes with cellular cyclin E-CDK2, was not degraded through the proteasome, and was not further stabilized by the VPKK mutation. p27(ck-), which was not recruited to cyclin E-CDK2, also remained stable in vivo. Thus, selective degradation of p27(k-) depended upon association with active cyclin E-CDK2 and subsequent phosphorylation. Altogether, these data show that p27 must be phosphorylated by CDK2 on the TPKK site in order to be degraded by the proteasome. We propose that cellular p27 must also exist transiently in a cyclin-bound non-inhibitory conformation in vivo.     

Expression of the cyclin-dependent kinase inhibitor p27Kip1 by developing retinal pigment epithelium

The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 contributes to the timing of cell cycle withdrawal during development and, consequently, in organogenesis. Within the retina, this effector protein is up-regulated during the birth of neuronal and glial cells [Dev. Biol. (2000) 299]. However, its expression within the retinal pigment epithelium (RPE), a supporting cell layer that is essential for neural retina development and function, has not previously been reported. We show that p27Kip1 protein expression in the RPE occurs in two phases: an up-regulation during mid-to late embryonic stages and a down-regulation during the subsequent postnatal period. In the early phase of up-regulation, an inverse relationship is seen between expression of p27Kip1 and PCNA, an indicator of cycling cells. During both up-and down-regulation, the change in spatial pattern of expression proceeds in a central to peripheral manner, with p27Kip1 up-regulation paralleling retinal maturation. These data suggest that this cell cycle regulator may be an important factor controlling the timing of RPE cell cycle withdrawal.     

Regulation of nuclear transport and degradation of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1

The regulation of the vertebrate cell cycle is controlled by the function of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. The Xenopus laevis kinase inhibitor, p27(Xic1) (Xic1) is a member of the p21(Cip1)/p27(Kip1)/p57(Kip2) CDK inhibitor family and inhibits CDK2-cyclin E in vitro as well as DNA replication in Xenopus egg extracts. Xic1 is targeted for degradation in interphase extracts in a manner dependent on both the ubiquitin conjugating enzyme, Cdc34, and nuclei. Here we show that ubiquitination of Xic1 occurs exclusively in the nucleus and that nuclear localization of Xic1 is necessary for its degradation. We find that Xic1 nuclear localization is independently mediated by binding to CDK2-cyclin E and by nuclear localization sequences within the C terminus of Xic1. Our results also indicate that binding of Xic1 to CDK2-cyclin E is dispensable for Xic1 ubiquitination and degradation. Moreover, we show that amino acids 180-183 of Xic1 are critical determinants of Xic1 degradation. This region of Xic1 may define a motif of Xic1 essential for recognition by the ubiquitin conjugation machinery or for binding an alternate protein required for degradation.     

Mutational analysis of the human cyclin-dependent kinase inhibitor p27kip1 in primary breast carcinomas

The human p27^kip1 gene encodes a cyclin-dependent kinase inhibitor implicated in the negative regulation of the cell cycle. In order to elucidate the possible role of p27 mutations in the development or progression of human breast cancer, we have studied the occurrence of genetic abnormalities in this gene in a series of 30 primary breast carcinomas. Direct sequence analysis of the polymerase chain reaction amplified human p27 gene revealed the occurrence of two sequence variations with respect to the reported sequence; both variants were also present in the lymphocyte DNA from the same patients. First, a silent G to A change at codon 142 (Thr) was detected in a single case. Second, a T to G transversion at codon 109, resulting in a Val to Gly change, was observed in eight tumour DNA samples (26%) and in 31 out of 80 unrelated normal individuals (39%). This latter change creates a BglI restriction site that might be useful for genetic analysis of human tumours. Despite the occurrence of these polymorphisms, we did not however find any evidence of somatic mutations in the coding region of the p27 gene. On the basis of these results, we suggest that alterations in the integrity of the human p27 gene are not common events in human breast carcinomas.     

Accumulation of the cyclin-dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation.

Many types of vertebrate precursor cells divide a limited number of times before they stop and terminally differentiate. In no case is it known what causes them to stop dividing. We have been studying this problem in the proliferating precursor cells that give rise to postmitotic oligodendrocytes, the cells that make myelin in the central nervous system. We show here that two components of the cell cycle control system, cyclin D1 and the Cdc2 kinase, are present in the proliferating precursor cells but not in differentiated oligodendrocytes, suggesting that the control system is dismantled in the oligodendrocytes. More importantly, we show that the cyclin-dependent kinase (Cdk) inhibitor p27 progressively accumulates in the precursor cells as they proliferate and is present at high levels in oligodendrocytes. Our findings are consistent with the possibility that the accumulation of p27 is part of both the intrinsic counting mechanism that determines when precursor cell proliferation stops and differentiation begins and the effector mechanism that arrests the cell cycle when the counting mechanism indicates it is time. The recent findings of others that p27-deficient mice have an increased number of cells in all of the organs examined suggest that this function of p27 is not restricted to the oligodendrocyte cell lineage.     

The cyclin-dependent kinase inhibitor p27kip1 is required for transplantation tolerance induced by costimulatory blockade

The cyclin-dependent kinase (CDK) inhibitor p27kip1 is an important negative regulator of the cell cycle that sets a threshold for mitogenic signals in T lymphocytes, and is required for T cell anergy in vitro. To determine whether p27(kip1) is required for tolerance in vivo, we performed cardiac allograft transplantation under conditions of combined CD28/CD40L costimulatory blockade. Although this treatment induced long-term allograft survival in wild-type recipients, costimulatory blockade was no longer sufficient to induce tolerance in mice lacking p27kip1. Rejected allografts from p27kip1-/- mice contained more CD4+ T lymphocytes and exhibited more tissue damage than allografts from tolerant, wild-type mice. Infiltrating p27kip1-deficient T cells, but not wild-type T cells, exhibited nuclear expression of cyclins E and A, indicating uncontrolled T cell cycle progression in the graft. The failure of tolerance in p27kip1-/- mice was also accompanied by markedly increased numbers of allospecific, IFN-gamma-producing cells in the periphery, and occurred despite apparently normal regulatory T cell activity. These data demonstrate that the CDK inhibitor p27kip1 enforces the costimulatory requirement for the expansion and differentiation of alloimmune effector T lymphocytes in vivo, and point to CDKs as novel targets for immunosuppressive or tolerance-inducing therapies.     

Purification and crystallization of cyclin-dependent kinase inhibitor p21.

p21, a universal inhibitor of mammalian cyclin-dependent kinases (CDK), regulates cell cycle progression by forming various distinct protein complexes with cyclins, CDKs, and the proliferating cell nuclear antigen. We have overexpressed recombinant human p21 in E. coli and purified active p21 to near homogeneity on a large scale. Crystals of recombinant p21 have been grown in the space group P2(1) a = 157.4, b = 152.7, c = 90.6 A, and beta = 92.7 degrees. The diffraction data of the recombinant p21 have been collected to 2.5 and 3.5 A resolution for the native crystal and two heavy atom derivatives of mercury and iridium.     

Minimal requirements for the nuclear localization of p27(Kip1), a cyclin-dependent kinase inhibitor

p27(Kip1) is a cyclin-dependent kinase inhibitor, and its nuclear localization is a prerequisite for it to function as a cell cycle regulator. In the present study, the minimal requirement for the nuclear localization signal (NLS) of p27(Kip1) was determined by analyzing the localization of various mutants of p27(Kip1) tagged with green fluorescent protein (GFP) in HeLa cells and porcine aortic endothelial cells. Wild-type p27(Kip1) exclusively localized into nucleus, while GFP alone localized in both cytosol and nucleus. A comparison of various truncation mutants revealed residues 153-166 to be the minimal region necessary for nuclear localization. However, a fusion of this region to GFP showed cytoplasmic retention in addition to nuclear localization, thus suggesting that some extension flanking this region is required to achieve a full function of NLS. The site-directed mutation of the full-length p27(Kip1) therefore showed that four basic residues (K153, R154, K165, R166), especially R166, play a critical role in the nuclear localization of p27(Kip1).     

Oncogenic signals of HER-2/neu in regulating the stability of the cyclin-dependent kinase inhibitor p27

Overexpression and activation of HER-2/neu, a proto-oncogene, play a pivotal role in cancer formation. Strong expression of HER-2/neu in cancers has been associated with poor prognosis. Reduced expression of p27(Kip1), a cyclin-dependent kinase inhibitor, correlates with poor clinical outcome in many types of carcinomas. Because many cancers with the overexpression of HER-2/neu overlap with those affected by reduced p27 expression, we studied the link between HER-2/neu oncogenic signals and p27 regulation. We found that down-regulation of p27 correlates with HER-2/neu overexpression. To address the molecular mechanism of this inverse correlation, we found that reduction of p27 is caused by enhanced ubiquitin-mediated degradation, and the HER-2/Grb2/MAPK pathway is involved in the decrease of p27 stability. Also, HER-2/neu activity causes mislocation of p27 and Jun activation domain-binding protein 1 (JAB1), an exporter of p27, into the cytoplasm, thereby facilitating p27 degradation. These results reveal that HER-2/neu signals reduce p27 stability and thus present potential points for therapeutic intervention in HER-2/neu-associated cancers.     

The cyclin-dependent kinase inhibitors p57 and p27 regulate neuronal migration in the developing mouse neocortex

Neuronal precursors remain in the proliferative zone of the developing mammalian neocortex until after they have undergone neuronal differentiation and cell cycle arrest. The newborn neurons then migrate away from the proliferative zone and enter the cortical plate. The molecules that coordinate migration with neuronal differentiation have been unclear. We have proposed in this study that the cdk inhibitors p57 and p27 play a role in this coordination. We have found that p57 and p27 mRNA increase upon neuronal differentiation of neocortical neuroepithelial cells. Knockdown of p57 by RNA interference resulted in a significant delay in the migration of neurons that entered the cortical plate but did not affect neuronal differentiation. Knockdown of p27 also inhibits neuronal migration in the intermediate zone as well as in the cortical plate, as reported by others. We have also found that knockdown of p27 increases p57 mRNA levels. These results suggest that both p57 and p27 play essential roles in neuronal migration and may, in concert, coordinate the timing of neuronal differentiation, migration, and possibly cell cycle arrest in neocortical development.     

Direct binding of the signal-transducing adaptor Grb2 facilitates down-regulation of the cyclin-dependent kinase inhibitor p27Kip1

Ectopic expression of Jab1/CSN5 induces specific down-regulation of the cyclin-dependent kinase (Cdk) inhibitor p27 (p27(Kip1)) in a manner dependent upon transportation from the nucleus to the cytoplasm. Here we show that Grb2 and Grb3-3, the molecules functioning as an adaptor in the signal transduction pathway, specifically and directly bind to p27 in the cytoplasm and participate in the regulation of p27. The interaction requires the C-terminal SH3-domain of Grb2/3-3 and the proline-rich sequence contained in p27 immediately downstream of the Cdk binding domain. In living cells, enforcement of the cytoplasmic localization of p27, either by artificial manipulation of the nuclear/cytoplasmic transport signal sequence or by coexpression of ectopic Jab1/CSN5, markedly enhances the stable interaction between p27 and Grb2. Overexpression of Grb2 accelerates Jab1/CSN5-mediated degradation of p27, while Grb3-3 expression suppresses it. A p27 mutant unable to bind to Grb2 is transported into the cytoplasm in cells ectopically expressing Jab1/CSN5 but is refractory to the subsequent degradation. These findings indicate that Grb2 participates in a negative regulation of p27 and may directly link the signal transduction pathway with the cell cycle regulatory machinery.     

The cyclin-dependent kinase inhibitor p27Kip1 is stabilized in G(0) by Mirk/dyrk1B kinase

Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G(0)/G(1) until mitogenic signals activate G(1) cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. We now demonstrate that the arginine-directed kinase, Mirk/dyrk1B, is maximally active in G(0) in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10. The phospho-mimetic mutant p27-S10D was more stable, and the non-phosphorylatable mutant p27-S10A was less stable than wild-type when expressed in G(0)-arrested cells. Following phosphorylation by Mirk, p27 remains a functional CDK inhibitor, capable of binding to CDK2. Mirk did not induce the translocation of p27 from the nucleus in G(0), but instead co-localized with nuclear p27. Depletion of Mirk by RNA interference decreased the phosphorylation of p27 at Ser-10 and the stability of endogenous p27. RNA(i) to Mirk increased cell entry from G(0) into G(1) as shown by increased expression of proliferating cell nuclear antigen and decreased expression of p27. These data suggest a model in which Mirk increases the amount of nuclear p27 by stabilizing it during G(0) when Mirk is most abundant. Mitogen stimulation then causes cells to enter G(1), reduces Mirk levels (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354), and initiates the translocation of p27 to the cytoplasm. In addition, depletion of Mirk by RNA(i) in postmitotic C2C12 myoblasts decreased protein but not mRNA levels of p27, suggesting that stabilization of p27 by Mirk also occurs during differentiation.     

Protein kinase Cdelta inhibition of S-phase transition in capillary endothelial cells involves the cyclin-dependent kinase inhibitor p27(Kip1).

Distinct protein kinase C (PKC) isoforms differentially regulate cellular proliferation in rat microvascular endothelial cells (EC). Overexpression of PKCalpha has little effect on proliferation, whereas PKCdelta slows endothelial cell proliferation and induces S-phase arrest. Analyses were performed on EC overexpressing PKCalpha (PKCalphaEC) or PKCdelta (PKCdeltaEC) to determine the role of specific cell cycle regulatory proteins in the PKCdelta-induced cell cycle arrest. Serum-induced stimulation of cyclins D1, E, and A-associated kinase activity was delayed by 12 h in the PKCdeltaEC line in association with S-phase arrest. However, the protein levels for cyclins D1, E, and A were similar. Nuclear accumulation of cyclin D1 protein in response to serum was also delayed in PKCdeltaEC. In the PKCdeltaEC line, serum induced p27(Kip1) but not p16(Ink4a) or p21(Cip1). Serum did not affect p27(Kip1) levels in the control vascular endothelial cell line. Immunoprecipitation-Western blotting analysis of p27(Kip1) showed serum stimulation of the vascular endothelial cell line resulted in increased amounts of cyclin D1 bound to p27(Kip1). In the PKCdeltaEC line, serum did not increase the amount of cyclin D1 bound to p27(Kip1). Transfection of full-length p27(Kip1) antisense into the PCKdeltaEC line reversed the S-phase arrest and resulted in normal cell cycle progression, suggesting a critical role for p27(Kip1) in the PKCdelta-mediated S-phase arrest.     

Receptor-Ck regulates the expression of cyclin-dependent kinase inhibitors (p16; p27)

The present study was addressed to understand the interrelationship between Receptor-C_k induction-activation coupling; isoprenoids (derived from mevalonate pathway) and cyclin-dependent kinase inhibitors. The results reported here unambiguously reveal that isoprenoids regulate the expression of genes coding for CDK inhibited p16 and p27. Further, Receptor-C_k dependent signalling, known to control the mevalonate pathway, had a direct effect upon the p27 gene expression. Based upon these studies coupled with our earlier findings we propose that Receptor-C_k has an important role in the regulation of cell cycle machinery.     

The C-terminal domain of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1, is both necessary and sufficient for phosphorylation-independent proteolysis

Cell cycle progression is regulated by cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. In the frog, Xenopus laevis, the CDK inhibitor p27(Xic1) (Xic1) inhibits DNA synthesis by negatively regulating CDK2-cyclin E. Using the frog egg extract as a model system for the study of Xic1, studies have demonstrated that Xic1 protein levels are regulated by nuclear ubiquitination and proteolysis. To characterize the molecular mechanism that regulates Xic1 turnover, we have identified the minimal sequences of Xic1 that are necessary and sufficient for its nuclear ubiquitination and degradation. Using deletion mutagenesis, our studies indicated that the C-terminal 50 amino acids of Xic1 are critical for its proteolysis beyond a role in nuclear transport. Replacement of the Xic1 C terminus with the SV40 nuclear localization sequence resulted in the nuclear localization of Xic1 but not its ubiquitination or degradation. Our deletion studies also indicated that the CDK2-cyclin binding domain of Xic1 is important for its efficient retention in the nucleus. Further deletion analyses identified at least 3 lysine residues within the Xic1 C terminus that are targeted for specific ubiquitination. Importantly, our studies demonstrated that the Xic1 C-terminal 50 amino acids can serve as a nuclear degradation signal when fused to a stable heterologous nuclear protein. Moreover, a 30-amino-acid region within the C terminus of Xic1 can serve as a nuclear ubiquitination signal. To address the role of phosphorylation on Xic1 turnover, all the potential phosphorylation sites within the C-terminal 50 amino acids of Xic1 were mutated to alanine to prevent possible phosphorylation. This resulted in a Xic1 protein that was nevertheless degraded in a manner similar to wild-type Xic1, suggesting that phosphorylation of Xic1 is not critical for its nuclear ubiquitination or proteolysis.