Suppression of Calpain-dependent Cleavage of the CDK5 Activator p35 to p25 by Site-specific Phosphorylation

Abstract

Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues 138 predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration. Cyclin-dependent kinase 5 (Cdk5)2 is a unique member of the Cdk family. Its activity in postmitotic neurons is completely dependent upon association with one of two neuronal specific activators, p35 or p39. Cdk5/p35 is involved in a panoply of processes critical to central nervous system function both during development and throughout maturity including neuronal migration during corticogenesis, neurite outgrowth, regulation of the synaptic vesicle cycle, neurotransmitter release, and postsynaptic neurotransmitter receptor regulation and signaling (1-3). The mechanisms by which Cdk5 activity is normally regulated remains to be fully delineated. Furthermore, because aberrant Cdk5 activity has been implicated in the etiology of neurodegenerative diseases (4, 5), identifying the biochemical mechanisms contributing to deregulation of Cdk5 is of substantial biomedical relevance.Deregulation of Cdk5 results from removal of the first 98 amino acids of p35 by the Ca 2ϩ -dependent cysteine protease, calpain, leaving Cdk5 associated with the N-terminal truncated form p25. Cleavage of p35 to p25 changes the subcellular distribution of active Cdk5 from membranes to the cytosolic fraction (6, 7), thereby altering substrate specificity. p25 accumulates in neurons undergoing various types of cell death (6 -9). Expression of Cdk5/p25 in cultured cells results in increased phospho-Tau levels in comparison to cells expressing Cdk5/ p35 (6). Furthermore, exogenous overexpression of p25 in transgenic mice results in a neurodegenerative phenotype including the formation of paired helical filaments, Tau aggregation, and neuronal loss similar to that observed in Alzheimer disease (10, 11).Cdk5/p25 has also been implicated in ischemia-induced neuronal loss in the hippocampus via increased phosphorylation of the NR2A subunit of the N-methyl-D-aspartic acid receptor (12). In addition, several recent reports indicate that Cdk5-p25 mediates cell death via translocation to the nucleus (13-15). p25 generation increases nuclear Cdk5 activity in cultured neurons, facilitating phosphorylation and inhibition of the pro-survival transcription factor MEF2 (13,15,16). Aberrant Cdk5 activity may also contribute to neuronal cell death via phosphorylation of other survival factors such as the tumor suppressor protein p53 ...

Cdk5 activity in the brain – multiple paths of regulation

“…Rapid degradation of p35 is observed in neurons that express Cdk5, which can be inhibited by expression of a dominant-negative or kinase-dead Cdk5 mutant, or by treatment with the Cdk5 inhibitor Roscovitine (Wei et al, 2005). Cdk5 primarily phosphorylates p35 at amino acid residues Ser8 and Thr138 (hereafter S8 and T138, respectively), two phosphorylation events that are differentially regulated and have different consequences (Kamei et al, 2007). The phosphorylation level of S8 remains constant during development, whereas T138 phosphorylation is highest in fetal brains and undetectable in adult brains (Fig.…”

“…2). Because both sites are phosphorylated by Cdk5, the differential phosphorylation levels are believed to be due to increased dephosphorylation of T138 in adult brain by protein phosphatase 1 (PP1) or protein phosphatase 2A (PP2A) (Kamei et al, 2007). However, further studies are needed to confirm this possible mechanism.…”

S-Nitrosylation of Cyclin-Dependent Kinase 5 (Cdk5) Regulates Its Kinase Activity and Dendrite Growth During Neuronal Development

“…Under pathological condition, p35 or p39 undergoes calpaindependent cleavage, generating a proteolytic product, p25 or p29 (Kusakawa et al, 2000;Lee et al, 2000;Patzke and Tsai, 2002), which deregulates Cdk5 activity through the mislocalization and mistargeting of Cdk5 to its substrates. On the other hand, phosphorylation of Cdk5 or its activators can also regulate its kinase activity Kamei et al, 2007). For example, phosphorylation of Cdk5 at tyrosine 15 (Tyr 15) following stimulation by extracellular cues such as ephrinA-1 has been reported to enhance its kinase activity (Dhavan and Tsai, 2001;Fu et al, 2007).…”

Cdk5 phosphorylation of its activators p35 and p39 determines subcellular location of the holokinase in a phosphorylation site-specific manner

“…Phosphorylation of p35 by Cdk5 determines its cytoplasmic localization, and Cdk5 activity is involved in p35 nuclear localization p35 is phosphorylated at Ser8 and Thr138 by Cdk5 (Kamei et al, 2007;Hosokawa et al, 2010). Therefore, there are two possibilities as to how Cdk5 activity affects subcellular localization; through phosphorylation of p35 or of other proteins.…”

“…Subcellular localization of proteins would be regulated by other factors, such as phosphorylation. p35 is phosphorylated at Ser8 and Thr138 by Cdk5 (Kamei et al, 2007;Hosokawa et al, 2010). Phosphorylation near myristoylation sites affects the interaction of myristoylated proteins to membranes (Taniguchi and Manenti, 1993;Hayashi and Titani, 2010), and Ser8 of p35 is located particularly close to the myristoylation site.…”