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The cyclin-dependent kinase 5 (CDK-5) activating protein, p35, is important for acute herpes simplex virus 1 (HSV-1) replication in mice. This report shows that HSV-1 increases p35 levels, changes the primary localization of CDK-5 from the nucleus to the cytoplasm, and enhances CDK-5 activity during lytic or acute infection. Infected neurons also stained positive for the DNA damage response (DDR) marker γH2AX. We propose that CDK-5 is activated by the DDR to protect infected neurons from apoptosis.

To investigate the intrinsic activities of the epidermal growth factor receptor and the role of its kinase domain in these functions within a cellular environment lacking endogenous ErbB protein expression, wild-type EGF receptor (WT-EGFR) and two kinase-impaired mutants, D813A and K721R, were expressed in 32D murine hematopoietic cells, a line which is normally dependent on interleukin 3 (IL3) for growth and survival. Addition of EGF in the absence of IL3 stimulates receptor autophosphorylation and, in the presence of serum, mitosis in cells expressing WT-EGFR, but not in cells expressing D813A or K721R. Unexpectedly, cells expressing WT-EGFR or K721R exhibited IL3-independent survival in the presence of fetal bovine serum; parental 32D cells and cells expressing D813A did not survive, apparently undergoing apoptosis in the absence of IL3, whether or not serum was present. Addition of EGF did not prevent the apoptosis of WT-EGFR or K721R cells in serum-free medium. Activation of Akt was not necessary to mediate the prosurvival activity of EGF receptor expression. These results suggest that the EGF receptor can mediate the prevention of apoptosis independently of both receptor–ligand binding and receptor kinase activity, and this activity is disrupted by the D813A mutation.

The p21(WAF1/cIP1) cyclin-dependent kinase (cdk) inhibitor is a regulator of the G(1)-S cell cycle checkpoint. Despite the importance of p21 in cell cycle inhibition, its role as a tumor suppressor is uncertain. p21 mutations are infrequent in human tumors, and p21 null mice exhibit no increased tumor incidence. To ascertain whether p21 could influence tumor formation or progression in the context of other oncogenic stimuli, we crossed p21-deficient mice with mammary tumor susceptible Wnt-1 transgenic mice. The p21+/+, p21+/-, and p21-/- Wnt-1 transgenic female offspring were monitored for mammary tumor incidence and growth rates. p21 status had no effect on the age at which mammary tumors formed. However, p21+/- mammary tumors grew significantly faster than p21+/+ and p21-/- mammary tumors. The increased growth rates were confirmed by mitotic index counts and by BrdUrd labelling assays, indicating that a significantly higher percentage of p21+/- tumor cells were in S phase and mitosis than their p21+/+ and p21-/- counterparts. Moreover, cyclin D1-associated phosphorylation of retinoblastoma protein was significantly increased in p21+/- tumor lysates compared with p21+/+ and p21-/- lysates. These results are consistent with data indicating that reduced levels of p21 can facilitate cyclin/cdk complex formation while enhancing cdk activity. Thus, a reduction of p21 dosage may promote tumor progression in the presence of other oncogenic initiators. The dependence of p21 on prior oncogenic stimuli for its tumor-promoting activities suggests that it may behave as a tumor modifier gene rather than as a tumor suppressor gene.

The mechanisms of neuronal damage in hypoxic cerebral cortex are complicated. Recent studies indicated that deregulation of Cdk5 was involved in neuronal death induced by hypoxia (1% O2). However, the pathological effect of Cdk5 is not fully elucidated. Therefore, in order to decipher the effect of Cdk5 on cellular death in hypoxic condition, the Cdk5 and its activator p35/p25 were investigated in cortical neurons at 10 DIV (Days In Vitro). Upon exposure to hypoxia, the cortical neurons showed a time-dependent increase of neuronal death compared to normoxia-treated control neurons. In correlation to the increase of neuronal death under hypoxia, the level of p25, a truncated form of p35, also increased in a time-dependent manner. Importantly, inhibition of Cdk5 kinase activity by roscovitine protected neurons from death under hypoxic stress. In contrast, ectopic upregulation of Cdk5 kinase activity in neurons expressing p25 led to an increase of neuronal death in comparison to control neurons expressing GFP. It suggests that ectopic increase of Cdk5 kinase activity through conversion of p35 to p25 is involved in the process of neuronal death induced by hypoxia.

Although previous studies have indicated that the neuroprotective effect of N-acetylcysteine (NAC) required activation of the Ras-extracellular-signal-regulated kinase (ERK) pathway, the detailed mechanisms and signal cascades leading to activation ERK are not clear. In the present study, we investigated the effect of NAC on A beta(25-35)-induced neuronal death. Pretreatment of neurons with NAC 1 hr before application of A beta prevented A beta-mediated cell death. NAC increased cyclin-dependent kinase 5 (Cdk5) phosphorylation, an effect that was blocked by Cdk5 inhibitor. The neuroprotective effect of NAC was significantly attenuated by Cdk5 inhibitors or in neurons transfected with Cdk5 or p35 small interfering RNA (siRNA). Conversely, pretreatment of neurons with the calpain inhibitors calpeptin or MDL28170 enhanced the neuroprotective effect of NAC. A beta(25-35) caused a significant decrease in the level of p35, with a concomitant increase in p25, which was completely prevented by NAC. This effect of NAC was blocked by the Cdk5 inhibitors roscovitine and butyrolactone. In addition, NAC increased Cdk5/p35 kinase activity but reduced Cdk5 kinase activity. A beta(25-35) treatment decreased phosphorylated levels of ERK, which could be reversed by NAC. The effect of NAC was completely blocked by Cdk5 inhibitors. NAC reversed the A beta(25-35)-induced decrease in the expression of Bcl-2, which could be blocked by the MAPK kinase (MEK) inhibitor or Cdk5 inhibitors. These results suggest that NAC-mediated neuroprotection against A beta toxicity is likely mediated by the p35/Cdk5-ERKs-Bcl-2 signal pathway.

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN.

The Ret receptor tyrosine kinase mediates physiological signals of glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) and is essential for postnatal survival in mice. It is implicated in a number of human diseases and developmental abnormalities. Here, we describe our analyses of mice expressing a Ret mutant ( Ret DN) with diminished kinase activity that inhibits wild-type Ret activity, including its activation of AKT. All Ret DN/+ mice died by 1 month of age and had distal intestinal aganglionosis reminiscent of Hirschsprung disease (HSCR) in humans. The Ret DN/+ proximal small intestine also had severe hypoganglionosis and reduction in nerve fiber density, suggesting a potential mechanism for the continued gastric dysmotility in postsurgical HSCR patients. Unlike Ret -null mice, which have abnormalities in the parasympathetic and sympathetic nervous systems, the Ret DN/+ mice only had defects in the parasympathetic nervous system. A small proportion of Ret DN/+ mice had renal agenesis, and the remainder had hypoplastic kidneys and developed tubulocystic abnormalities postnatally. Postnatal analyses of the testes revealed a decreased number of germ cells, degenerating seminiferous tubules, maturation arrest and apoptosis, indicating a crucial role for Ret in early spermatogenesis.

Stimulation of cells with TNFα leads to the formation of the TNF-R1 signaling complex (TNF-RSC) to mediate downstream cellular fate decision. Activation of the TNF-RSC is modulated by different types of ubiquitination and may lead to cell death, including apoptosis and necroptosis, in both RIPK1-dependent and RIPK1-independent manners. Spata2 (spermatogenesis-associated 2) is an adaptor protein recruited into the TNF-RSC to modulate the interaction between the linear ubiquitin chain assembly complex (LUBAC) and the deubiquitinase CYLD (cylindromatosis). However, the mechanism by which Spata2 regulates the activation of RIPK1 is unclear. Here, we report that Spata2-deficient cells show resistance to RIPK1-dependent apoptosis and necroptosis and are also partially protected against RIPK1-independent apoptosis. Spata2 deficiency promotes M1 ubiquitination of RIPK1 to inhibit RIPK1 kinase activity. Furthermore, we provide biochemical evidence for the USP domain of CYLD and the PUB domain of the SPATA2 complex preferentially deubiquitinating the M1 ubiquitin chain in vitro. Spata2 deficiency also promotes the activation of MKK4 and JNK and cytokine production independently of RIPK1 kinase activity. Spata2 deficiency sensitizes mice to systemic inflammatory response syndrome (SIRS) induced by TNFα, which can be suppressed by RIPK1 inhibitor Nec-1s. Thus, Spata2 can regulate inflammatory response and cell death in both RIPK1-dependent and RIPK1-independent manners.

Cancer cells exhibit de-regulation of multiple cellular signalling pathways and treatments of various types of cancers with polyphenols are promising. We recently reported the synthesis of a series of 33 novel divanillic and trivanillic polyphenols that displayed anticancer activity, at least in vitro, through inhibiting various kinases. This study revealed that minor chemical modifications of a trivanillate scaffold could convert cytotoxic compounds into cytostatic ones. Compound 13c, a tri-chloro derivative of trivanillic ester, displayed marked inhibitory activities against FGF-, VEGF-, EGF- and Src-related kinases, all of which are implicated not only in angiogenesis but also in the biological aggressiveness of various cancer types. The pan-anti-kinase activity of 13c occurs at less than one-tenth of its mean IC(50) in vitro growth inhibitory concentrations towards a panel of 12 cancer cell lines. Of the 26 kinases for which 13c inhibited their activity by75%, eight (Yes, Fyn, FGF-R1, EGFR, Btk, Mink, Ret and Itk) are implicated in control of the actin cytoskeleton organization to varying degrees. Compound 13c accordingly impaired the typical organization of the actin cytoskeleton in human U373 glioblastoma cells. The pan-anti-kinase activity and actin cytoskeleton organization impairment provoked by 13c concomitantly occurs with calcium homeostasis impairment but without provoking MDR phenotype activation. All of these anticancer properties enabled 13c to confer therapeutic benefits in vivo in a mouse melanoma pseudometastatic lung model. These data argue in favour of further chemically modifying trivanillates to produce novel and potent anticancer drugs.

To investigate whether cyclin-dependent kinase 5 and its regulatory protein p35 was involved in staurosporine-induced apoptosis of cortical neuronal cultures.Primary cerebral cortical neurons were exposed to 300 nmol/L staurosporine. After incubation for different time, morphological alterations were observed with phase-contrast microscopy, fluorescence microscopy, and transmission electron microscopy. DNA fragmentation was detected by agarose gel electrophoresis. The protein levels of Cdk4, p53, Cdk5, and its regulatory protein p35 following staurosporine treatment were measured by Western blotting. The Cdk5 activity was assayed for histone H1 kinase activity by autoradiography.The typical morphological changes of apoptosis were observed and the nuclear DNA fragmentation showed the characteristic "ladder" pattern after the cells were treated by staurosporine. The Cdk5 protein level increased markedly at 3 h and continued to 24 h. The p35 level increased at 3 h after being exposed to staurosporine, and decreased at 12 h. The cleavage of p35 to p25 was also detected at 12 h and increased at 24 h. There was no increase in Cdk5 kinase activity despite the increased cleavage of p35. The protein level of Cdk4 protein increased at 3 h and then decreased gradually from 6 h, but it was still higher than that in the vehicle cultures at 12 h. The p53 level decreased obviously at 3 h after staurosporine treatment and then seemed to increase at 12 h, but remained lower than that of vehicle cultures.Staurosporine-induced increase in Cdk5 protein levels and the cleavage of p35 to p25 may contribute to neuronal apoptosis.

H-ras oncogenes have been identified in greater than 50% of the most common forms of human neoplasia. Ras-related proteins have been postulated to mediated signal transduction pathways involving mitogen-activated protein (MAP) kinases and nuclear responses that may be involved in the induction of apoptosis. We examined whether expression of H-ras oncogene conferred resistance or susceptibility to the morphologic effects of the protein phosphatase inhibitor, okadaic acid, using a tumorigenic H-ras-transformed normal rat kidney epithelial cell line, NRK-H/6.1. We also examined whether okadaic acid induced apoptosis correlated with a differential effect on kinase activity in H-Ras-transformed cells as compared to the nontransformed NRK-52E cells. Treatment with various concentrations of okadaic acid produced rapid and extensive morphologic changes characteristic of apoptosis in both cell types. Equimolar okadaic acid concentrations for 2 or 4 hr resulted in cell detachment and loss of membrane integrity (as measured by propidium iodide uptake) in 74% (0.5 microM) and 78% (1.0 microM) of the H-Ras-transformed cells as compared to 8 and 25%, respectively, in the non-transformed cells. Furthermore, a higher basal level of kinase activity was observed in the H-Ras-transformed cells as compared to the nontransformed cells. Okadaic acid-induced apoptosis correlated with activation of members of the MAP kinase family, raf-1 and protein kinase C (PKC). These studies show that H-ras oncogene expression imparts selective susceptibility to cell death induced by phosphatase inhibition. The observed increase in susceptibility to okadaic acid-induced apoptosis appears to involve the modulation of raf-1, PKC, and MAP kinase activities. These findings may be significant in the elucidation of mechanisms for selective induction of cell death in tumor cells expressing H-ras oncogene.

cmi13027-sup-0001-Table_S1.xlsx cmi13027-sup-0002-Table_S2.xlsx cmi13027-sup-0003-Table_S3.docx cmi13027-sup-0004-suppl_figures.pptx; International audience; Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the etiologic agent of cecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterized. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N-terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co-immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterization confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its non-canonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.

Insulin and insulin-like growth factor 1 (IGF-1) act as anti-apoptotic hormones. We found that, unexpectedly, double knockout (DKO) cells that lacked both insulin and IGF-1 receptors (IR and IGF1R, respectively), were resistant to apoptosis induced through either the intrinsic or extrinsic pathway. This resistance to apoptosis was associated with decreased abundance of the pro-apoptotic protein Bax and increases in abundance of the anti-apoptotic proteins Bcl-2, Bcl-xL, XIAP, and Flip. These changes in protein abundance involved primarily post-transcriptional mechanisms. Restoration of the insulin or IGF-1 receptor to DKO cells also restored their sensitivity to apoptosis. Notably, expression of a catalytically inactive mutant form of the insulin receptor also restored susceptibility to apoptosis. Thus, the insulin and IGF-1 receptors have bidirectional roles in the control of cell survival and can be viewed as previously-unidentified dependence receptors. Insulin and IGF-1 binding stimulates receptor tyrosine kinase activity and blocks apoptosis, whereas unliganded insulin and IGF-1 receptors, acting through a mechanism independent of their catalytic activity, exert a permissive effect on cell death.

Despite significant advances in treatment, breast cancer is still the second leading cause of cancer-related deaths in women in the United States. Therefore, significant efforts are being given to develop novel strategies for the prevention of breast cancer in recent years. Our laboratory and others have been studying the effects of a potential chemopreventive agent, indole-3-carbinol (I3C), in breast cancer cells. We have previously shown that I3C induces apoptosis in breast cancer cells and found that the induction of apoptotic processes was partly mediated by dysregulation of anti- and pro-apoptotic molecules. However, the precise molecular mechanism(s) by which I3C induces apoptosis in breast cancer cells has not been fully elucidated. For the present study, we focused our investigation on important cell signaling molecules such as Akt and NF-kappaB during I3C-induced apoptosis in breast cancer cells. We found that I3C induces apoptotic processes in MCF10A-derived cell lines with premalignant (DCIS.com) and malignant (MCF10CA1a) phenotypes but not in nontumorigenic parental MCF10A cells. Immunoprecipitation, kinase assays, and Western blot analysis showed that I3C specifically inhibits Akt kinase activity and abrogates the EGF-induced activation of Akt in breast cancer cells. NF-kappaB DNA-binding analysis and transfection studies with Akt cDNA and NF-kappaB-Luc reporter constructs revealed that Akt gene transfection directly activates NF-kappaB, and this activation was completely abrogated by I3C treatment. In addition, I3C also abrogated the EGF-induced activation of NF-kappaB, which was mediated via the Akt signaling pathway. From these results, we conclude that there is a direct cross-talk between Akt and NF-kappaB pathways and that the inactivation of Akt and NF-kappaB activity plays important roles in mediating I3C-induced apoptosis in breast cancer cells. These results also suggest that I3C may be a potential chemopreventive agent by virtue of its selective apoptosis-inducing ability in premalignant and malignant breast epithelial cells.