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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 CaMKIIinvolved 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/ GTP␥S-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/GTP␥S. Finally, we confirmed by immunocytochemical analysis that the transfection of constitutively active rat ␣PAK (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 ␣PAK 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.

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Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in Ca2+-induced insulin secretion. We have previously reported that treatment of insulinoma MIN6 cells with secretagogues activated CaM kinase II and increased the phosphorylation of synapsin I, followed by insulin secretion. Here, we identified isoforms of CaM kinase II in MIN6 cells and rat islets. Immunoblot analysis suggested that the major isoforms of CaM kinase II were beta'e and delta2 at the protein level in MIN6 cells. Only the beta'e isoform was detected in rat islets by both RT-PCR and immunoblot analysis. We transiently overexpressed beta'e and delta2 isoforms in MIN6 cells and confirmed that treatment of cells with tolbutamide and glucose activated the isoforms. Immunoblot analysis with an antibody against synapsin I phosphorylated by CaM kinase II demonstrated that treatment with tolbutamide and glucose rapidly increased phosphorylation of synapsin I and that phosphorylation was potentiated by overexpression of the isoforms. The secretagogue-induced insulin secretion was potentiated by overexpression of the isoforms. Our results further support our conclusion that activation of CaM kinase II and the concomitant phosphorylation of synapsin I contribute to insulin secretion from pancreatic beta-cells.