Jesusa L. Rosales

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.

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.

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.

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

Controversies over p25 in Alzheimer's disease

Activity of the neuronal Cdc2-like kinase composed of cyclin-dependent kinase 5 (Cdk5) and its activator, p35, is critical for the normal development of the central nervous system, including cortical lamination and axonal patterning events. Conversely, altered Cdk5 activity has been associated with several neuronal defects including neurodegeneration. Indeed, an increasing line of evidence suggests that Cdk5 contributes to neurodegeneration in AD. For example, Cdk5 immunoreactivity is consistently detected in neurofibrillary tangles (NFTs) [15]; Cdk5 phosphorylates tau and promotes dimerization of tau to an AD-like state [2, 13]; sulfated glycosaminoglycans co-exist with tau and Cdk5 in NFTs and remarkably enhance tau phosphorylation by Cdk5 [5]; protein phosphatase 1 (PP1), which shows decreased activity in AD brains [4], is inhibited by Cdk5 through phosphorylation of the PP1 inhibitor protein (I-1) [6]; in primary neuronal cultures, Aβ induces activation of Cdk5 that results in tau hyperphosphorylation, cytoskeletal collapse, and cell death [1, 9]. In addition, we and others have shown elevated Cdk5 activity in mice exhibiting tau hyperphosphorylation [10], and in AD brains [7,11]. However, the molecular mechanisms whereby Cdk5 activity is upregulated in AD brain remain to be investigated. Recently, it was suggested that Cdk5 activity is enhanced by calcium through induction of p35 cleavage to p25 via calpain activation [9]. The active truncated form of p35, p25, was then shown to accumulate in

Cdk7 Functions as a Cdk5 Activating Kinase in Brain

Activity of Cdk5, which is essential for CNS development, depends on its association with p35 (or truncated p25) or p39, but the regulatory mechanisms whereby Cdk5 is activated have not yet been fully elucidated. Studies on PC12 cells showed that Cdk5/p25 activity is modulated by an unidentified kinase that phosphorylates Cdk5 at Ser159 and enhances its kinase activity (Sharma et al., 1999 PNAS USA 96,11156). In this study, we have partially purified a bovine brain enzyme that can phosphorylate the Cdk5(149-163) peptide but not the Cdk5(149-163)A159 peptide. The enzyme, which contains Cdk7, cyclin H, and Mat1, specifically phosphorylates Ser159 of Cdk5(149-163) and enhances Cdk5/p25 activity. The active Cdk7-containing enzyme also phosphorylates and activates wt Cdk5 but not mutated Cdk5(Ser159Ala). Likewise, Cdk7 or cyclin H immunoprecipitate from mouse brain specifically phosphorylates wt Cdk5 at Ser159 and enhances Cdk5/p25 activity. Conversely, blocked Cdk7 immunoprecipitate does not phosphorylate nor activate Cdk5. In addition, the Cdk7 substrate, CTD of RNAPII, causes a dose-dependent decline in Cdk5 activation by Cdk7. These findings indicate that Cdk7 functions as a Cdk5 activating kinase in brain. Indeed, elevated Cdk5 activity in the developing E18 mouse brain coincides with increased Cdk7 kinase activity.

Viewpoint: Crosstalks between neurofibrillary tangles and amyloid plaque formation

Since its discovery, the hallmarks of Alzheimers disease (AD) brain have been recognised as the formation of amyloid plaques and neurofibrillary tangles (NFTs). Mounting evidence has suggested the active interplay between the two pathways. Studies have shown that β-amyloid (Aβ) can be internalized and generated intracellularly, accelerating NFT formation. Conversely, tau elements in NFTs are observed to affect Aβ and amyloid plaque formation. Yet the precise mechanisms which link the pathologies of the two brain lesions remain elusive. In this review, we discuss recent evidence that support five putative mechanisms by which crosstalk occurs between amyloid plaque and NFT formation in AD pathogenesis. Understanding the crosstalks in the formation of AD pathologies could provide new clues for the development of novel therapeutic strategies to delay or halt the progression of AD.

GTP‐dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein

Guanosine triphosphate (GTP) has been implicated in the regulation of Ca2+‐mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found that, in the presence of GTP, 1.0 μM free Ca2+ causes maximum secretion—equivalent to that achieved with 100 μM free Ca2+—whereas GTPγS inhibits Ca2+‐stimulated secretion. Interestingly, GTP by itself stimulates secretion. These results indicate the existence of a GTP‐regulated mechanism of secretion in neutrophils that requires GTP hydrolysis to stimulate secretion in the presence and absence of Ca2+. The stimulatory effect of GTP is only observed when GTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, gives no stimulatory response, implying that the GTP‐dependent secretory apparatus requires at least one cytosolic protein. GTP‐dependent secretion can be reconstituted with crude HL‐60 and bovine liver cytosol. The reconstituting activity binds to GTP‐agarose, suggesting that the cytosolic factor is a GTP‐binding protein or forms a complex with a GTP‐binding protein. However, it is not a member of the rho or rac families of GTPases. By gel filtration chromatography, the secretion‐reconstituting activity eluted at 870 and 200 kDa, but in the presence of GTP, eluted at 120 kDa, indicating that it is part of a high‐molecular‐weight complex that dissociates in the presence of GTP. Retention of adenosine diphosphate‐ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activity to brefeldin A led to our speculation that ARF6 may be the GTPase involved in GTP‐dependent secretion, and that activity from a BFA‐insensitive ARF6 guanine nucleotide exchange factor reconstitutes secretion. J. Cell. Biochem. 80:37–45, 2000.

Cdk5 mediates vimentin Ser56 phosphorylation during GTP-induced secretion by neutrophils.

Secretion by neutrophils contributes to acute inflammation following injury or infection. Vimentin has been shown to be important for secretion by neutrophils but little is known about its dynamics during secretion, which is regulated by cyclin‐dependent kinase 5 (Cdk5). In this study, we sought to examine the vimentin dynamics and its potential regulation by Cdk5 during neutrophil secretion. We show that vimentin is a Cdk5 substrate that is specifically phosphorylated at Ser56. In response to neutrophil stimulation with GTP, vimentin Ser56 was phosphorylated and colocalized with Cdk5 in the cytoplasmic compartment. Vimentin pSer56 and Cdk5 colocalization was consistent with coimmunoprecipitation from stimulated cells. Vimentin Ser56 phosphorylation occurred immediately after stimulation, and a remarkable increase in phosphorylation was noted later in the secretory process. Decreased GTP‐induced vimentin Ser56 phosphorylation and secretion resulted from inhibition of Cdk5 activity by roscovitine or olomoucine or by depletion of Cdk5 by siRNA, suggesting that GTP‐induced Cdk5‐mediated vimentin Ser56 phosphorylation may be related to GTP‐induced Cdk5‐mediated secretion by neutrophils. Indeed, inhibition of vimentin Ser56 phosphorylation led to a corresponding inhibition of GTP‐induced secretion, indicating a link between these two events. While fMLP also induced vimentin Ser56 phosphorylation, such phosphorylation was unaffected by roscovitine, which nonetheless, inhibited secretion, suggesting that Cdk5 regulates fMLP‐induced secretion via a mechanism independent of Cdk5‐mediated vimentin Ser56 phosphorylation. These findings demonstrate the distinct involvement of Cdk5 in GTP‐ and fMLP‐induced secretion by neutrophils, and support the notion that specific targeting of Cdk5 may serve to inhibit the neutrophil secretory process. J. Cell. Physiol. 227: 739–750, 2012.