Inhibition of Lck: evidence for a novel natural Src family kinase inhibitor.

Src family kinase (SFK) is a family of protein tyrosine kinases that play important roles in the development of various cancers. Here, we showed that a naturally occurring inhibitory factor of SFK can be extracted from the rat brain. This inhibitor strongly suppressed the activity of SFKs including Lck and Fyn. It did not inhibit other protein tyrosine kinases including Wee1 or serine/threonine kinases Mst2, Cdk5/p25, Cdk5/p35, and Cdk2/cyclin A. The inhibitor was not an ATPase, a phosphatase that dephosphorylates substrates of the SFK reaction, or a protease that degrades SFKs. Activity of mutant Lck with C-terminal tyrosine substituted with phenylalanine was also suppressed by the inhibitor to a similar extent of wild-type Lck, indicating that the inhibitor was not CSK. Gel filtration chromatography indicated that the molecular size of the prevalent form of this inhibitor was approximately 44 kDa.

Tyk2/STAT3 signaling mediates beta-amyloid-induced neuronal cell death: implications in Alzheimer's disease.

One of the pathological hallmarks of Alzheimer's disease (AD) is deposition of extracellular amyloid-beta (Abeta) peptide, which is generated from the cleavage of amyloid precursor protein (APP). Accumulation of Abeta is thought to associate with the progressive neuronal death observed in AD. However, the precise signaling mechanisms underlying the action of Abeta in AD pathophysiology are not completely understood. Here, we report the involvement of the transcription factor signal transducer and activator of transcription 3 (STAT3) in mediating Abeta-induced neuronal death. We find that tyrosine phosphorylation of STAT3 is elevated in the cortex and hippocampus of APP/PS1 transgenic mice. Treatment of cultured rat neurons with Abeta or intrahippocampal injection of mice with Abeta both induces tyrosine phosphorylation of STAT3 in neurons. Importantly, reduction of either the expression or activation of STAT3 markedly attenuates Abeta-induced neuronal apoptosis, suggesting that STAT3 activation contributes to neuronal death after Abeta exposure. We further identify Tyk2 as the tyrosine kinase that acts upstream of STAT3, as Abeta-induced activation of STAT3 and caspase-3-dependent neuronal death can be inhibited in tyk2(-/-) neurons. Finally, increased tyrosine phosphorylation of STAT3 is also observed in postmortem brains of AD patients. Our observations collectively reveal a novel role of STAT3 in Abeta-induced neuronal death and suggest the potential involvement of Tyk2/STAT3 signaling in AD pathophysiology.

Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors.

CA1 pyramidal neurons degenerate after transient forebrain ischemia, whereas neurons in other regions of the hippocampus remain intact. Here we show that in rat hippocampal CA1 neurons, forebrain ischemia induces the phosphorylation of the N-methyl-D-aspartate (NMDA) receptor 2A subunit at Ser1232 (phospho-Ser1232). Ser1232 phosphorylation is catalyzed by cyclin-dependent kinase 5 (Cdk5). Inhibiting endogenous Cdk5, or perturbing interactions between Cdk5 and NR2A subunits, abolished NR2A phosphorylation at Ser1232 and protected CA1 pyramidal neurons from ischemic insult. Thus, we conclude that modulation of NMDA receptors by Cdk5 is the primary intracellular event underlying the ischemic injury of CA1 pyramidal neurons.

Identification of a novel Wee1 isoform.

We have identified a novel isoform of Wee1 kinase (Wee1i), which uses Met215 of Wee1 as its initiation codon. RT-PCR, Western blot, and in situ hybridization verified wee1i expression in mammalian cells, rat brain, and rat thymus. Recombinant and partially purified Wee1i from rat thymus displayed kinase activity comparable to or higher than Wee1. The N-terminal 214 residues of Wee1 facilitate its ubiquitin-dependent degradation to trigger mitotic entry. Since Wee1i, lack of these 214 residues, it may evade this degradation and thus provide constitutive Wee1-like kinase activity to inhibit mitotic cell proliferation. Thus, Wee1i may play an important role in differentiation and in tumor suppression.

Alpha-chimaerin exists in a functional complex with the Cdk5 kinase in brain.

Cyclin-dependent kinase 5 (Cdk5) in association with its neuronal activators p35 and p39 shows a complex involvement in the control of neurocytoskeletal dynamics. Here we show that alpha-chimaerin, a GTPase-activating protein specific for Rac and Cdc42, is a p35-binding protein. The interaction domains of p35 and alpha-chimaerin were delineated. In transfected HeLa cells, p35 and alpha-chimaerin displayed an overlapping distribution pattern and they could be co-immunoprecipitated from the cell lysate. As alpha-chimaerin has a regulatory function in actin repolymerization, these results suggested that the regulation of neurocytoskeleton dynamics by Cdk5 is mediated at least in part via alpha-chimaerin.

Aberrant motor axon projection, acetylcholine receptor clustering, and neurotransmission in cyclin-dependent kinase 5 null mice.

Cyclin-dependent kinase (Cdk)5 is a key regulator of neural development. We have previously demonstrated that Cdk5/p35 are localized to the postsynaptic muscle and are implicated in the regulation of neuregulin/ErbB signaling in myotube culture. To further elucidate whether Cdk5 activity contributes to neuromuscular junction (NMJ) development in vivo, the NMJ of Cdk5-/- mice was examined. Consistent with our previous demonstration that Cdk5 phosphorylates ErbB2/3 to regulate its tyrosine phosphorylation, we report here that the phosphorylation of ErbB2 and ErbB3 and the ErbB2 kinase activity are reduced in Cdk5-deficient muscle. In addition, Cdk5-/- mice also display morphological abnormalities at the NMJ pre- and postsynaptically. Whereas the outgrowth of the main nerve trunk is grossly normal, the intramuscular nerve projections exhibit profuse and anomalous branching patterns in the Cdk5-/- embryos. The central band of acetylcholine receptor (AChR) clusters is also wider in Cdk5-/- diaphragms, together with the absence of S100 immunoreactivity along the phrenic nerve during late embryonic stages. Moreover, we unexpectedly discovered that the agrin-induced formation of large AChR clusters is significantly increased in primary muscle cultures prepared from Cdk5-null mice and in C2C12 myotubes when Cdk5 activity was suppressed. These abnormalities are accompanied by elevated frequency of miniature endplate potentials in Cdk5-null diaphragm. Taken together, our findings reveal the essential role of Cdk5 in regulating the development of motor axons and neuromuscular synapses in vivo.

Expression of Cdk5 and its activators in NT2 cells during neuronal differentiation.

We have recently developed a rapid protocol involving NT2 cell aggregation and treatment with retinoic acid (RA) to produce terminally differentiated CNS neurons. As a first step to explore the functional roles of cell-cycle regulatory proteins in the process of neuronal differentiation, the expression profiles of cyclin-dependent kinases (Cdks) and their regulators were examined in NT2 cells following treatment with RA. One of the Cdks, Cdk5, has been demonstrated to affect the process of neuronal differentiation and suggested to play an important role in development of the nervous system. We found that the expression of Cdk5 was gradually increased, while its activators (p35 and p39) as well as Cdk5 kinase activity were induced in NT2 cells during the process of neuronal differentiation. Moreover, both p35 and p39 were localized along the axons and varicosity-like structures of differentiated NT2 neurons. Taken together, our results demonstrated that NT2 cells provide a good in vitro model system to examine signaling pathways involved in the regulation of Cdk5 activators and to elucidate the functional roles of Cdk5 in neuronal differentiation.

Regulation of mammalian STE20-like kinase 2 (MST2) by protein phosphorylation/dephosphorylation and proteolysis.

Mammalian STE20-like kinase 2 (MST2), a member of the STE20-like kinase family, has been shown in previous studies to undergo proteolytic activation by caspase-3 during cell apoptosis. A few studies have also implicated protein phosphorylation reactions in MST2 regulation. In this study, we examined the mechanism of MST2 regulation with an emphasis on the relationship between caspase-3 cleavage and protein phosphorylation. Both the full-length MST2 and the caspase-3-truncated form of MST2 overexpressed in 293T cells exist in a phosphorylated state. On the other hand, the endogenous full-length MST2 from rat thymus or from proliferating cells is mainly unphosphorylated whereas the caspase-3-truncated endogenous MST2 from apoptotic cells is highly phosphorylated. Cell transfection studies using mutant MST2 constructs indicate that MST2 depends on the autophosphorylation of a unique threonine residue, Thr(180), for kinase activity. The autophosphorylation reaction shows strong dependence on MST2 concentration suggesting that it is an intermolecular reaction. While both the full-length MST2 and the caspase-3-truncated form of MST2 undergo autophosphorylation, the two forms of the phosphorylated MST2 display marked difference in susceptibility to protein phosphatases. The full-length phospho-MST2 is rapidly dephosphorylated by protein phosphatase 1 or protein phosphatase 2A whereas the truncated MST2 is remarkably resistant to the dephosphorylation. Based on the present results, a novel molecular mechanism for MST2 regulation in apoptotic cells is postulated. In normal cells, because of the low concentration and the ready reversal of the autophosphorylation by protein phosphatases, MST2 is present mainly in the unphosphorylated and inactive state. During cell apoptosis, MST2 is cleaved by caspase-3 and undergoes irreversible autophosphorylation, thus resulting in the accumulation of active MST2.

Specific inhibition of cyclin-dependent kinases and cell proliferation by harmine.

As key regulators of the cell proliferation cycle, cyclin-dependent kinases (CDKs) are attractive targets for the development of anti-tumor drugs. In the present study, harmine was identified from a collection of herbal compounds to be a specific inhibitor of Cdk1/cyclin B, Cdk2/cyclin A, and Cdk5/p25 with IC50 values at low micromoles. It displayed little effect on other serine/threonine and tyrosine kinases tested. The CDK inhibition by harmine is competitive with ATP-Mg2+, suggesting that it binds to the ATP-Mg2+-binding pocket of CDKs. In cytotoxicity assays, harmine exhibited a strong inhibitory effect on the growth and proliferation of carcinoma cells whereas it had no significant effect on quiescent fibroblasts. Further, harmine was found to block DNA replication in the carcinoma cells. Taken together, harmine is a selective inhibitor of CDKs and cell proliferation.

The protein SET binds the neuronal Cdk5 activator p35nck5a and modulates Cdk5/p35nck5a activity.

The neuronal Cdk5 kinase is composed of the catalytic subunit Cdk5 and the activator protein p35(nck5a) or its isoform, p39(nck5ai). To identify novel p35(nck5a)- and p39(nck5ai)-binding proteins, fragments of p35(nck5a) and p39(nck5ai) were utilized in affinity isolation of binding proteins from rat brain homogenates, and the isolated proteins were identified using mass spectrometry. With this approach, the nuclear protein SET was shown to interact with the N-terminal regions of p35(nck5a) and p39(nck5ai). Our detailed characterization showed that the SET protein formed a complex with Cdk5/p35(nck5a) through its binding to p35(nck5a). The p35(nck5a)-interacting region was mapped to a predicted alpha-helix in SET. When cotransfected into COS-7 cells, SET and p35(nck5a) displayed overlapping intracellular distribution in the nucleus. The nuclear co-localization was corroborated by immunostaining data of endogenous SET and Cdk5/p35(nck5a) from cultured cortical neurons. Finally, we demonstrated that the activity of Cdk5/p35(nck5a), but not that of Cdk5/p25(nck5a), was enhanced upon binding to the SET protein. The tail region of SET, which is rich in acidic residues, is required for the stimulatory effect on Cdk5/p35(nck5a).

Pctaire1 interacts with p35 and is a novel substrate for Cdk5/p35.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase that plays important roles during central nervous system development. Cdk5 kinase activity depends on its regulatory partners, p35 or p39, which are prominently expressed in the central nervous system. We have previously demonstrated the involvement of Cdk5 in the regulation of acetylcholine receptor expression at the neuromuscular junction, suggesting a novel functional role of Cdk5 at the synapse. Here we report the identification of Pctaire1, a member of the Cdk-related kinase family, as a p35-interacting protein in muscle. Binding of Pctaire1 to p35 can be demonstrated by in vitro binding assay and co-immunoprecipitation experiments. Pctaire1 is associated with p35 in cultured myotubes and skeletal muscle, and is concentrated at the neuromuscular junction. Furthermore, Pctaire1 can be phosphorylated by the Cdk5/p25 complex, and serine 95 is the major phosphorylation site. In brain and muscle of Cdk5 null mice, Pctaire1 activity is significantly reduced. Moreover, Pctaire1 activity is increased following preincubation with brain extracts and phosphorylation by the Cdk5/p25 complex. Taken together, our findings demonstrate that Pctaire1 interacts with p35, both in vitro and in vivo, and that phosphorylation of Pctaire1 by Cdk5 enhances its kinase activity.

Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor.

Neuronal Cdc2-like kinase (Nclk) plays an important role in a variety of cellular processes, including neuronal cell differentiation, apoptosis, neuron migration, and formation of neuromuscular junction. The active kinase consists of a catalytic subunit, Cdk5, and an essential regulatory subunit, neuronal Cdk5 activator (p35(nck5a) or p25(nck5a)), which is expressed primarily in neurons of central nervous tissue. In our previous study using the yeast two-hybrid screening method, three novel p35(nck5a)-associated proteins were isolated. Here we show that one of these proteins, called C42, specifically inhibits the activation of Cdk5 by Nck5a. Co-immunoprecipitation data suggested that C42 and p35(nck5a) could form a complex within cultured mammalian cells. Deletion analysis has mapped the inhibitory domain of C42 to a region of 135 amino acids, which is conserved in Pho81, a yeast protein that inhibits the yeast cyclin-dependent protein kinase Pho85. The Pho85.Pho80 kinase complex has been shown to be the yeast functional homologue of the mammalian Cdk5/p35(nck5a) kinase.

Cyclin-dependent kinase 5 phosphorylates signal transducer and activator of transcription 3 and regulates its transcriptional activity.

The activity of cyclin-dependent kinase 5 (Cdk5) depends on the association with one of its activators, p35 and p39, which are prominently expressed in the nervous system. Studies on the repertoire of protein substrates for Cdk5 have implicated the involvement of Cdk5 in neuronal migration and synaptic plasticity. Our recent analysis of the sequence of signal transducer and activator of transcription (STAT)3, a key transcription factor, reveals the presence of potential Cdk5 phosphorylation site. We report here that the Cdk5/p35 complex associates with STAT3 and phosphorylates STAT3 on the Ser-727 residue in vitro and in vivo. Intriguingly, whereas the Ser phosphorylation of STAT3 can be detected in embryonic and postnatal brain and muscle of wild-type mice, it is essentially absent from those of Cdk5-deficient embryos. In addition, treatment of cultured myotubes with neuregulin enhances the Ser phosphorylation of STAT3 and transcription of STAT3 target genes, such as c-fos and junB, in a Cdk5-dependent manner. Both the DNA-binding activity of STAT3 and the transcription of specific target genes, such as fibronectin, are reduced in Cdk5-deficient muscle. Taken together, these results reveal a physiological role of Cdk5 in regulating STAT3 phosphorylation and modulating its transcriptional activity.

Beta-carbolines as specific inhibitors of cyclin-dependent kinases.

Harmine (3), 7-fluoro-1-methyl beta-carboline (35) and 1-(5-methyl-imidazol-4-yl) beta-carboline (41) were potent and specific inhibitors of cyclin-dependent kinases. The degree of aromaticity of the tricyclic ring and the positioning of substituents are important for inhibitory activity. While most beta-carbolines inhibited CDK2 and CDK5 to the same extent, selective inhibition against CDK2 was observed in 1-(2-chlorophenyl)- (12), 1-(2-fluorophenyl)- (15), and 1-(2-chloro-5-nitrophenyl)- (28) beta-carbolines.