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 Ser(8) and Thr(138) as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr(138) was dephosphorylated in adult rat, although both Ser(8) and Thr(138) were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser(8) p35 to dual phospho-Ser(8)/Thr(138) p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr(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.
Highlights
Because aberrant Cyclin-dependent kinase 5 (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
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]
Aberrant Cdk5 activity may contribute to neuronal cell death via phosphorylation of other survival factors such as the tumor suppressor protein p53 [17] and retinoblastoma protein [14]
Summary
Chemicals and Antibodies—cDNAs encoding human Cdk and p35 within pCMV vectors were provided by Dr L.-H. Expression and Metabolic Phosphorylation of p35 in HEK293 Cells—HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 100 units/ml of penicillin, and 0.1 mg/ml streptomycin. Expression of Cdk5-p35 in COS-7 Cells, Cleavage of p35 to p25 by Calpain, and Dephosphorylation of p35—COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 0.1 mg/ml of streptomycin. Dephosphorylation of p35 expressed in HEK293 cells was performed in the cell extract (50 mM Tris-HCl, pH 8.5, 0.15 M NaCl, and 1% Nonidet P-40) by incubation with bacterial alkaline phosphatase (20 unit/ml, Wako, Osaka, Japan) at 37 °C for 1 h. All experiments in this study were performed more than three times with similar results and representative results are shown in the figures
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