Thr-370 is responsible for CDK11(p58) autophosphorylation, dimerization, and kinase activity

CDK11(p58), a member of the p34(cdc2)-related kinase family, is associated with cell cycle progression, tumorigenesis, and proapoptotic signaling. It is also required for the maintenance of chromosome cohesion, the maturation of centrosome, the formation of bipolar spindle, and the completion of mitosis. Here we identified that CDK11(p58) interacted with itself to form homodimers in cells, whereas D224N, the kinase-dead mutant, failed to form homodimers. CDK11(p58) was autophosphorylated, and the main functions of CDK11(p58), such as kinase activity, transactivation of nuclear receptors, and proapoptotic signal transduction, were dependent on its autophosphorylation. Furthermore, the in vitro kinase assay indicated that CDK11(p58) was autophosphorylated at Thr-370. By mutagenesis, we created CDK11(p58) T370A and CDK11(p58) T370D, which mimic the dephosphorylated and phosphorylated forms of CDK11(p58), respectively. The T370A mutant could not form dimers and be phosphorylated by the wild type CDK11(p58) and finally lost the kinase activity. Further functional research revealed that T370A failed to repress the transactivation of androgen receptor and enhance the cell apoptosis. Overall, our data indicated that Thr-370 is responsible for the autophosphorylation, dimerization, and kinase activity of CDK11(p58). Moreover, Thr-370 mutants might affect CDK11(p58)-mediated signaling pathways.     

Increased expression of CDK11p58 and cyclin D3 following spinal cord injury in rats

Protein kinases are critical signalling molecules for normal cell growth and development. CDK11^p58 is a p34^cdc2-related protein kinase, and plays an important role in normal cell cycle progression. However its distribution and function in the central nervous system (CNS) lesion remain unclear. In this study, we mainly investigated the protein expression and cellular localization of CDK11 during spinal cord injury (SCI). Western blot analysis revealed that CDK11^p58 was not detected in normal spinal cord. It gradually increased, reached a peak at 3 day after SCI, and then decreased. The protein expression of CDK11^p58 was further analyzed by immunohistochemistry. The variable immunostaining patterns of CDK11^p58 were visualized at different periods of injury. Double immunofluorescence staining showed that CDK11 was co-expressed with NeuN, CNPase and GFAP. Co-localization of CDK11/active caspase-3 and CDK11/proliferating cell nuclear antigen (PCNA) were detected in some cells. Cyclin D3, which was associated with CDK11^p58 and could enhance kinase activity, was detected in the normal and injured spinal cord. The cyclin D3 protein underwent a similar pattern with CDK11^p58 during SCI. Double immunofluorescence staining indicated that CDK11 co-expressed with cyclin D3 in neurons and glial cells. Coimmunoprecipitation further showed that CDK11^p58 and cyclin D3 interacted with each other in the damaged spinal cord. Thus, it is likely CDK11^p58 and cyclin D3 could interact with each other after acute SCI. Another partner of CDK11^p58 was β-1,4-galactosyltransferase 1 (β-1,4-GT 1). The co-localization of CDK11/β-1,4-GT 1 in the damaged spinal cord was revealed by immunofluorescence analysis. The cyclin D3-CDK4 complexes were also present by coimmunoprecipitation analysis. Taken together, these data suggested that both CDK11 and cyclin D3 may play important roles in spinal cord pathophysiology. The authors Yuhong Ji and Feng Xiao contributed equally to this work.     

Different effects of p58PITSLRE on the apoptosis induced by etoposide,cycloheximide and serum-withdrawal in human hepatocarcinoma cells

Minimal overexpression of the p58^PITSLRE protein kinase in Chinese hamster ovary cells induces telephase delay, abnormal cytokinesis, retarded cell growth and apoptosis. Fas mediated T cell death is correlated with p58^PITSLRE proteolysis and an increase in its histone H1 kinase activity. In this study, it was found that p58^PITSLRE had different effects on the apoptosis induced by etoposide, cycloheximide and serum-withdrawal in human hepatocarcinoma cells. The ectopic expression of p58^PITSLRE in human hepatocarcinoma cells suppressed apoptosis induced by etoposide, while enhancing the apoptosis induced by cycloheximide and serum-withdrawal respectively. Elevated expression of p58^PITSLRE was found during the apoptosis induced by etoposide, whereas most of p58^PITSLRE was proteolytically processed during apoptosis induced by cycloheximide and serum-withdrawal. Furthermore, transient transfection of p50^PITSLRE resembling the proteolytic form of p58^PITSLRE enhanced the 7721 cells susceptibility to apoptosis induced by all the three stimuli. These findings suggest that the full-length p58^PITSLRE might protect the cells from the apoptosis induced by etoposide and its proteolysis might contribute to and enhance the apoptosis induced by cycloheximide and serum-withdrawal respectively.     

A pro-apoptotic effect of the CDK inhibitor p57(Kip2) on staurosporine-induced apoptosis in HeLa cells

Apoptosis, or programmed cell death, is involved in many biological events, including tumorigenesis. Recently, it has been reported that two members of the Cip/Kip family of CDK inhibitors, p21(Cip1) and p27(Kip1), are involved in the regulation of apoptosis. Here, we report that selective expression of the third member in this family, p57(Kip2), potentiated staurosporine-induced apoptosis in HeLa cells. This pro-apoptotic effect was associated with an increased caspase-3 activity. In contrast, glucocorticoid treatment, despite inducing p57(Kip2) expression in HeLa cells, was found to have an inhibitory effect on staurosporine-induced apoptosis. This anti-apoptotic effect of glucocorticoids could be explained by a concomitant increase in Bcl-x(L) expression. The results presented in this study show that p57(Kip2) has a stimulatory effect on apoptosis induced by staurosporine, suggesting a role for p57(Kip2) in the response of tumor cells to cytotoxic drugs.     

CDK11p110 plays a critical role in the tumorigenicity of esophageal squamous cell carcinoma cells and is a potential drug target

Esophageal squamous cell carcinoma (ESCC) is a serious malignancy with limited options for targeted therapy. The exploration of novel targeted therapies for combating ESCC is urgently needed. Cyclin-dependent kinases (CDKs) play important roles in the progression of cancers; however, the function of CDK11^p110 (cyclin-dependent kinase 11^p110) in ESCC is still unknown. Here, we investigated the effects and molecular mechanisms of CDK11^p110 in the proliferation and growth of ESCC by examining the expression of CDK11^p110 in ESCC tissues and by detecting phenotypic changes in ESCC cells after CDK11^p110 knockdown or overexpression in vitro and in vivo. According to the tissue microarray analysis, compared with its expression level in normal tissues, the expression level of CDK11^p110 was significantly elevated in ESCC tissues; this result was in concordance with the data in TCGA (The Cancer Genome Atlas) datasets. In addition, RNAi-mediated CDK11^p110 silencing exerted a substantial inhibitory effect on the proliferation, clonogenicity and migration ability of ESCC cells. Further study indicated that CDK11^p110 knockdown arrested ESCC cells in the G2/M phase of the cell cycle and induced cell apoptosis. Moreover, stable shRNA-mediated CDK11^p110 knockdown inhibited tumor growth in an ESCC xenograft model, and overexpression of CDK11^p110 enhanced tumor growth. In addition, the Ki67 proliferation index was closely associated with the elevation or depletion of CDK11^p110 in vivo. In summary, this study provides evidence that CDK11^p110 play a critical role in the tumorigenicity of ESCC cells, which suggests that CDK11^p110 may be a promising therapeutic target in ESCC.

Abbreviations: CDKs: cyclin-dependent kinases; CDK11: Cyclin-dependent kinase 11; CDK11^p110: Cyclin-dependent kinase 11^p110, the larger isomer of cyclin-dependent kinase 11; ESCC: esophageal squamous cell carcinoma; FACS: fluorescence-activated cell sorting; FDA: the Food and Drug Administration; TCGA: The Cancer Genome Atlas; TMA: tissue microarray.     

p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.     

p33(ING1) enhances UVB-induced apoptosis in melanoma cells

The biological functions of the tumor suppressor ING1 have been studied extensively in the past few years since it was cloned. It shares many biological functions with p53 and has been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, chemosensitivity, and DNA repair. Some of these functions, such as cell cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins. Two recent reports by Scott and colleagues demonstrate that p33(ING1) (one of the ING1 isoforms) translocates to the nucleus and binds to PCNA upon UV irradiation. Here we report that p33(ING1) mediates UV-induced cell death in melanoma cells. We found that overexpression of p33(ING1) increased while the introduction of an antisense p33(ING1) plasmid reduced the apoptosis rate in melanoma cells after UVB irradiation. We also demonstrated that enhancement of UV-induced apoptosis by p33(ING1) required the presence of p53. Moreover, we found that p33(ING1) enhanced the expression of endogenous Bax and altered the mitochondrial membrane potential. Taken together, these observations strongly suggest that p33(ING1) cooperates with p53 in UVB-induced apoptosis via the mitochondrial cell death pathway in melanoma cells.     

Cranial neural crest-derived mesenchymal proliferation is regulated by Msx1-mediated p19(INK4d) expression during odontogenesis

Neural crest cells are multipotential progenitors that contribute to various cell and tissue types during embryogenesis. Here, we have investigated the molecular and cellular mechanism by which the fate of neural crest cell is regulated during tooth development. Using a two- component genetic system for indelibly marking the progeny of neural crest cells, we provide in vivo evidence of a deficiency of CNC-derived dental mesenchyme in Msx1 null mutant mouse embryos. The deficiency of the CNC results from an elevated CDK inhibitor p19(INK4d) activity and the disruption of cell proliferation. Interestingly, in the absence of Msx1, the CNC-derived dental mesenchyme misdifferentiates and possesses properties consistent with a neuronal fate, possibly through a default mechanism. Attenuation of p19(INK4d) in Msx1 null mutant mandibular explants restores mitotic activity in the dental mesenchyme, demonstrating the functional significance of Msx1-mediated p19(INK4d) expression in regulating CNC cell proliferation during odontogenesis. Collectively, our results demonstrate that homeobox gene Msx1 regulates the fate of CNC cells by controlling the progression of the cell cycle. Genetic mutation of Msx1 may alternatively instruct the fate of these progenitor cells during craniofacial development.     

The p33ING1b tumor suppressor cooperates with p53 to induce apoptosis in response to etoposide in human osteosarcoma cells

p33ING1b induces cell cycle arrest and stimulates DNA repair, apoptosis and chemosensitivity. The magnitude of some p33ING1b effects may be due to activation of the tumor suppressor p53. To investigate if the p33ING1b protein affected chemosensitivity of osteosarcoma cells, we overexpressed p33ING1b in p53+/+ U2OS cells or in p53-mutant MG63 cells, and then assessed for growth arrest and apoptosis after treatment with etoposide. p33ING1b increased etoposide-induced growth inhibition and apoptosis to a much greater degree in p53+/+ U2OS cells than in p53-mutant MG63 cells. Moreover, ectopic expression of p33ING1b markedly upregulated p53, p21WAF1 and bax protein levels and activated caspase-3 protein kinase in etoposide-treated U2OS cells. Together, our data indicate that p33ING1b prominently enhances etoposide-induced apoptosis through p53-dependent pathways in human osteosarcoma cells. p33ING1b may be an important marker and/or therapeutic target in the prevention and treatment of metastatic osteosarcoma.     

CDK inhibitor p57Kip2 is downregulated by Akt during HER2-mediated tumorigenicity

HER2/neu oncogene is frequently deregulated in cancers, and the (PI3K)-Akt signaling is one of the major pathways in mediating HER2/neu oncogenic signal. p57^Kip2, an inhibitor of cyclin-depependent kinases, is pivotal in regulating cell cycle progression, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to p57^Kip2 regulation, and that Akt is a negative regulator of p57^Kip2. Ectopic expression of Akt can decrease the expression of p57^Kip2, while Akt inhibition leads to p57^Kip2 stabilization. Mechanistic studies show that Akt interacts with p57^Kip2 and causes cytoplasmic localization of p57^Kip2. Akt phosphorylates p57 on Ser 282 or Thr310. Akt activity results in destabilization of p57 by accelerating turnover rate of p57 and enhancing p57 ubiquitination. Importantly, the negative impact of HER2/Akt on p57 stability contributes to HER2-mediated cell proliferation, transformational activity and tumorigenicity. p57 restoration can attenuate these defects caused by HER2. Significantly, Kaplan-Meier analysis of tumor samples demonstrate that in tumors where HER2 expression was observed, high expression levels of p57^Kip2 were associated with better overall survival. These data suggest that HER2/Akt is an important negative regulator of p57^Kip2, and that p57 restoration in HER2-overexpressing cells can reduce breast tumor growth. Our findings indicate the applicability of employing p57 regulation as a therapeutic intervention in HER2-overexpressing cancers.     

Differential regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68

Members of the gamma2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21(Cip) and p27(Kip). Furthermore, M cyclin directed cdk2 to phosphorylate p27(Kip1) on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27(Kip1) and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27(Kip1)-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21(Cip1) and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.     

CREB represses p53-dependent transactivation of MDM2 through the complex formation with p53 and contributes to p53-mediated apoptosis in response to glucose deprivation

Recently, we have described that CREB (cAMP-responsive element-binding protein) has the ability to transactivate tumor suppressor p53 gene in response to glucose deprivation. In this study, we have found that CREB forms a complex with p53 and represses p53-mediated transactivation of MDM2 but not of p21^WAF1. Immunoprecipitation analysis revealed that CREB interacts with p53 in response to glucose deprivation. Forced expression of CREB significantly attenuated the up-regulation of the endogenous MDM2 in response to p53. By contrast, the mutant form of CREB lacking DNA-binding domain (CREBΔ) had an undetectable effect on the expression level of the endogenous MDM2. During the glucose deprivation-mediated apoptosis, there existed an inverse relationship between the expression levels of MDM2 and p53/CREB. Additionally, p53/CREB complex was dissociated from MDM2 promoter in response to glucose deprivation. Collectively, our present results suggest that CREB preferentially down-regulates MDM2 and thereby contributing to p53-mediated apoptosis in response to glucose deprivation.