Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation.
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.
Kamei
Hirotsugu
H
Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachiohji, Tokyo 192-0397, Japan.
Saito
Taro
T
Ozawa
Mirai
M
Fujita
Yuichi
Y
Asada
Akiko
A
Bibb
James A
JA
Saido
Takaomi C
TC
Sorimachi
Hiroyuki
H
Hisanaga
Shin-Ichi
S
D000818:Animals; D019556:COS Cells; D002154:Calpain / Q000737:chemistry*; D002460:Cell Line; D002522:Chlorocebus aethiops; D051360:Cyclin-Dependent Kinase 5 / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology*; D019636:Neurodegenerative Diseases / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D051381:Rats
D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; C089495:neuronal Cdk5 activator (p25-p35); D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; D002154:Calpain