Ki-Young Lee

Elevated neuronal Cdc2-like kinase activity in the Alzheimer disease brain

Neurofibrillary tangles (NFT) in Alzheimers disease (AD) consist largely of hyperphosphorylated tau protein. Many of the phosphorylation sites on tau are serine/threonine-proline sequences, several of which are phosphorylated in vitro by neuronal Cdc2-like kinase (Nclk), a kinase composed of Cdk5 and its activator(s). Thus, tau hyperphosphorylation in AD may result in part from deregulation of Nclk. To test this hypothesis, we examined Nclk activity in prefrontal and cerebellar cortex from 15 postmortem AD and 16 age-matched control subjects, and corrected either for Cdk5 level or for neuronal loss. The ratio of Nclk activity in prefrontal versus cerebellar cortex was then compared. When corrections were made for neuronal loss, the ratios of kinase activity in prefrontal versus cerebellar cortex were significantly higher in AD (6.45+/-0.86) than the controls (3.13+/-0.46; P = 0.003). This finding is consistent with a role for Nclk in the pathogenesis of NFT in AD.

Interaction of Cyclin-dependent Kinase 5 (Cdk5) and Neuronal Cdk5 Activator in Bovine Brain

Neuronal cdc2-like kinase (Nclk) purified from bovine brain is a heterodimer of Cdk5 and an essential 25-kDa regulatory subunit (Lew, J., and Wang, J. H.(1995) Trends Biochem. Sci. 20, 33-37). The regulatory subunit is an N-terminal truncated derivative of a 35-kDa protein expressed specifically in brain, hence the name neuronal Cdk5 activator, p25/p35. In this study, we probe the relationship between the two different forms of Nck5a and their interaction with and activation of Cdk5 in bovine brain extract. Using protein fractionation procedures in combination with Western blot analysis and protein kinase assay, three forms of Cdk5 have been detected in bovine brain: a monomeric Cdk5 that can be activated by bacterially expressed GST-p21, a heterodimer of Cdk5 and p25 that displays high kinase activity, and a Cdk5•p35 complex that is inactive and refractory to GST-p21 activation. Analysis of the Cdk5•p35 complex by gel filtration chromatography indicated that the complex was part of a macromolecular structure with a molecular mass of 670 kDa. When the macromolecular complex was subjected to gel filtration chromatography in the presence of 10% ethylene glycol, the fractions containing both p35 and Cdk5, although eluting at the same position as control, displayed high kinase activity. The result is compatible with the suggestion that the macromolecular complex contained a kinase inhibitory factor that dissociated from the complex in 10% ethylene glycol.

ASK1–p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide‐induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen‐activated protein kinase (MAPK), c‐Jun N‐terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn‐JNK, JNK1(A‐F) or SAPKβ(K‐R), blocked anandamide‐induced cell death, whereas a specific inhibitor of MEK‐1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide‐induced cell death. An important role for apoptosis signal‐regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn‐ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1ΔN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide‐induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide‐induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.

Cdk5/p25nck5a interaction with synaptic proteins in bovine brain

Cyclin‐dependent kinase 5 (Cdk5) exists in large multimeric complexes, but its function and binding partners in these complexes are unclear. We explored these issues by chromatographic and immunochemical analyses of Cdk5 and p25nck5a (a neuronal Cdk5 activator) and their associated proteins from bovine brain. Mono‐S column enzyme eluates were divided into three fractions and analyzed by gel filtration. The majority of p25nck5a from Mono‐S fractions I, II, and III eluted from the gel filtration column at ∼60, 200, and 400 kDa, respectively, and Cdk5 was abundant in fractions >400 kDa. We characterized these macromolecular structures by immunoprecipitating p25nck5a, followed by a second immunoprecipitation of remaining unbound proteins using a Cdk5 antibody. The p25nck5a immunoprecipitates showed association with Cdk5. Amphiphysin was detected in the 400‐kDa complex and synapsin I in the >400 kDa structure. The Cdk5 immunoprecipitates, however, revealed abundant retained Cdk5 but no remaining p25nck5a, indicating that Cdk5 in macromolecular structures is mostly unassociated with p25nck5a. Thus, we demonstrate: an amphiphysin‐associated 400‐kDa Cdk5/p25nck5a complex, a synapsin I‐associated >400‐kDa Cdk5/p25nck5a complex, and nck5a‐free Cdk5 complexes (200 to >400 kDa). Amphiphysin acts as a Cdk5/p25nck5a substrate in the 400‐kDa complex and we speculate that Cdk5/p25nck5a participates in amphiphysin‐mediated endocytosis. J. Cell. Biochem. 78:151–159, 2000.

Ebselen inhibits NO-induced apoptosis of differentiated PC12 cells via inhibition of ASK1-p38 MAPK-p53 and JNK signaling and activation of p44/42 MAPK and Bcl-2

Ebselen, a selenium‐containing heterocyclic compound, prevents ischemia‐induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO‐induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO‐induced phosphatidyl Serine exposure, cytochrome c release, and caspase‐3 activation by ebselen. Analysis of key apoptotic regulators during NO‐induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling‐regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen‐activated protein kinase (MAPK) and c‐jun N‐terminal protein kinase (JNK). Moreover, ebselen inhibits NO‐induced p53 phosphorylation at Ser15 and c‐Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol‐redox‐dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl‐2 in SNP‐treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.

L6 myoblast differentiation is modulated by Cdk5 via the PI3K-AKT-p70S6K signaling pathway.

Cdk5 regulates myogenesis but the signaling cascade through which Cdk5 modulates this process remains to be characterized. Here, we investigated whether PI3K, Akt, p70S6K, p38 MAPK, p44/42 MAPK, and Egr-1 serve as upstream regulators of Cdk5 during L6 myoblast differentiation. Upon serum reduction, we found that besides elevated expression of Cdk5 and its activator, p35, and increased Cdk5/p35 activity, Egr-1, Akt, p70S6K, and p38 MAPK activity were upregulated in differentiating L6 cells. However, p44/42 MAPK was downregulated and SAPK/JNK was unaffected. LY294002, a PI3K inhibitor, blocked the activation of Akt and p70S6K, indicating that Akt and p70S6K activation is linked to PI3K activation. The lack of LY294002 effect on p38 MAPK suggests that p38 MAPK activation is not associated with PI3K activation. Rapamycin, a specific inhibitor of FRAP/mTOR (the upstream kinase of p70S6K), also blocked p70S6K activation, indicating the involvement of FRAP/mTOR activation. LY294002 and rapamycin also blocked the enhancement of Egr-1 level, Cdk5 activity, and myogenin expression, suggesting that upregulation of these factors is coupled to PI3K–p70S6K activation. Overexpression of dominant-negative-Akt also reduced Cdk5/p35 activity and myogenin expression, indicating that the PI3K–p70S6K–Egr-1–Cdk5 signaling cascade is linked to Akt activation. SB2023580, a p38 MAPK inhibitor, had no effect on p70S6K, Egr-1, or Cdk5 activity, suggesting that p38 MAPK activation lies in a pathway distinct from the PI3K–Akt–p70S6K–Egr-1 pathway that we identify as the upstream modulator of Cdk5 activity during L6 myoblast differentiation.

Cyclin E in breast tumors is cleaved into its low molecular weight forms by calpain

Abundant levels of the hyperactive low molecular weight (LMW) forms of cyclin E contribute to deregulation of Cdk2 in breast tumors, but the mechanism through which they arise is not fully understood. Here, we explored the hypothesis that post-translational processing by a protease generates the LMW forms of cyclin E in breast tumors. In ZR75 tumor cell lysates, calcium-induced cyclin E truncation into peptides corresponding in size with LMW forms of cyclin E in tumor tissues. Calpeptin inhibited calcium-stimulated cyclin E truncation, indicating that cleavage resulted from activity of the calcium-dependent protease, calpain. Consistently, calcium+calpain caused truncation of cyclin E immunoprecipitated from tumor cells and tissues. Calcium also caused truncation of the calpain regulatory subunit in tumor cell lysates, indicating that elevated calpain activity accompanies cyclin E truncation. Increased levels of the calpain small subunit were also observed in breast tumors, and significant amounts of its proteolyzed forms indicated increased calpain activity. While elastase also caused cyclin E truncation, the cleavage pattern was distinct from that generated by calpain, suggesting discrete mechanisms in regulating the formation of LMW cyclin E in breast tumors. Treatment of ZR75 cultures with calcium+A23187 recapitulated the formation of the calcium/calpain-induced LMW forms of cyclin E. Altered calcium homeostasis and/or inability of the endogenous calpain inhibitor to control the activity of high levels of the calpain small subunit may contribute to increased calpain activity in breast tumors, causing abundant levels of LMW cyclin E.

Neuronal Cdc2-like kinases: Neuron-specific forms of Cdk5

Neuronal Cdc2-like kinase, Nclk, is a heterodimer of a Cdk5 catalytic subunit and a 25 kDa regulatory subunit derived proteolytically from a neuron- and central nervous system-specific 35 kDa protein. The regulatory subunit is mandatory for kinase activity, hence it is designated the neuronal Cdk5 activator, p25/p35nck5a. Nclk has been suggested to play a regulatory role in neuro-cytoskeleton dynamics and in neuronal differentiation. In addition to the activation by Nck5a, Cdk5 is regulated by other mechanisms including additional activator proteins and inhibition by phosphorylation of specific amino acid residues. While Nclk shares common catalytic and regulatory properties with other members of the cdc2-like kinase family, it also displays unique characteristics that may be important for its neuronal functions.

Reduced expression and novel splice variants of ING4 in human gastric adenocarcinoma

ING4, a new member of the ING (inhibitor of growth) family of tumour suppressor genes, has been found to be deleted or down‐regulated in gliomas, breast tumours, and head and neck squamous cell carcinomas. The goal of the present study was to investigate whether the expression and alternative splicing of ING4 transcripts are involved in the initiation and progression of stomach adenocarcinoma. ING4 mRNA and protein expression was examined in gastric adenocarcinoma tissues and human gastric adenocarcinoma cell lines by RT‐PCR, real‐time RT‐PCR, tissue microarray immunohistochemistry, and western blot analysis. Alterations in ING4 transcripts were determined through sequence analysis of ING4 cDNA. Our data showed that ING4 mRNA and protein were dramatically reduced in stomach adenocarcinoma cell lines and tissues, and significantly less in female than in male patients. We also found that reduced ING4 mRNA expression correlated with the stage of the tumour. Interestingly, by sequence analysis, we discovered five novel aberrantly spliced variant forms of ING4_v1 and ING4_v2. These variants cause a codon frame‐shift and, eventually, deletion of the NLS or PHD domain contributing to the mislocalization of p53 and/or HAT/HDAC complexes and, subsequently, altered gene expression in gastric adenocarcinoma. These results suggest that attenuated and aberrant ING4 expression may be involved in the initiation and progression of stomach adenocarcinoma. Copyright

Down-regulation of ING4 is associated with initiation and progression of lung cancer.

Wang Q‐s, Li M, Zhang L‐y, Jin Y, Tong D‐d, Yu Y, Bai J, Huang Q, Liu F‐L, Liu A, Lee K‐Y & Fu S‐b
(2010) Histopathology 57, 271–281
Down‐regulation of ING4 is associated with initiation and progression of lung cancer