Synapsin I is phosphorylated at Ser603 by p21-activated kinases (PAKs) in vitro and in PC12 cells stimulated with bradykinin.
The function of synapsin I is regulated by phosphorylation of the molecule at multiple sites; among them, the Ser(603) residue (site 3) is considered to be a pivotal site targeted by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Although phosphorylation of the Ser(603) residue responds to several kinds of stimuli, it is unlikely that many or all of the stimuli activate the CaMKII-involved pathway. Among the several stimulants tested in PC12 cells, bradykinin evoked the phosphorylation of Ser(603) without inducing the autophosphorylation of CaMKII, which was determined using phosphorylation site-specific antibodies against phospho-Ser(603)-synapsin I (pS603-Syn I-Ab) and phospho-Thr(286/287)-CaMKII. The bradykinin-evoked phosphorylation of Ser(603) was not suppressed by the CaMKII inhibitor KN62, whereas high KCl-evoked phosphorylation was accompanied by CaMKII autophosphorylation and inhibited by KN62. Thus, we attempted to identify Ser(603) kinase(s) besides CaMKII. We consequently detected four and three fractions with Ca(2+)/calmodulin-independent Ser(603) kinase activity on the DEAE column chromatography of bovine brain homogenate and PC12 cell lysate, respectively, two of which were purified and identified by amino acid sequence of proteolytic fragments as p21-activated kinase (PAK) 1 and PAK3. The immunoprecipitants from bovine brain homogenate with anti-PAK1 and PAK3 antibodies incorporated (32)P into synapsin I in a Cdc42/GTPgammaS-dependent manner, and its phosphorylation site was confirmed as Ser(603) using pS603-Syn I-Ab. Additionally, recombinant GST-PAK2 could phosphorylate the Ser(603) residue in the presence of Cdc42/GTPgammaS. Finally, we confirmed by immunocytochemical analysis that the transfection of constitutively active rat alphaPAK (PAK1) in PC12 cells evokes the phosphorylation of Ser(603) even in the resting mutant cells and enhances it in the bradykinin-stimulated cells, whereas that of dominant-negative alphaPAK quenches the phosphorylation. These results raise the possibility that Ser(603) on synapsin I is alternatively phosphorylated by PAKs, not only by CaMKII, in neuronal cells in response to some stimulants.
Sakurada
Katsuhiko
K
Frontier 21 Project, Institute for Life Science Research, Asahi Chemical Industry Co., Ltd. 2-1 Samejima, Fuji, Shizuoka 416-8501, Japan.
Kato
Hirotsugu
H
Nagumo
Hiromitsu
H
Hiraoka
Hideji
H
Furuya
Kaoru
K
Ikuhara
Toshihiko
T
Yamakita
Yoshihiko
Y
Fukunaga
Kouji
K
Miyamoto
Eishichi
E
Matsumura
Fumio
F
Matsuo
Yuri-Ikeda
YI
Naito
Yasuhito
Y
Sasaki
Yasuharu
Y
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001920:Bradykinin / Q000378:metabolism*; D001923:Brain Chemistry; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002417:Cattle; D004791:Enzyme Inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism*; D016704:Synapsins / Q000235:genetics / Q000378:metabolism*; D054462:p21-Activated Kinases
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D016704:Synapsins; D012694:Serine; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C515906:Pak1 protein, rat; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D001920:Bradykinin; D002118:Calcium