Synapsin I is phosphorylated at Ser603 by p21-activated kinases (PAKs) in vitro and in PC12 cells stimulated with bradykinin

Katsuhiko Sakurada, Hirotsugu Kato, Hiromitsu Nagumo, Hideji Hiraoka, Kaoru Furuya, Toshihiko Ikuhara, Yoshihiko Yamakita, Kouji Fukunaga, Eishichi Miyamoto, Fumio Matsumura, Yuri Ikeda Matsuo, Yasuhito Naito, Yasuharu Sasaki

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26 Citations (Scopus)


The function of synapsin I is regulated by phosphorylation of the molecule at multiple sites; among them, the Ser603 residue (site 3) is considered to be a pivotal site targeted by Ca2+/calmodulin-dependent kinase II (CaMKII). Although phosphorylation of the Ser603 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 Ser603 without inducing the autophosphorylation of CaMKII, which was determined using phosphorylation site-specific antibodies against phospho-Ser603-synapsin I (pS603-Syn I-Ab) and phospho-Thr286/287 -CaMKII. The bradykinin-evoked phosphorylation of Ser603 was not suppressed by the CaMKII inhibitor KN62, whereas high KC1-evoked phosphorylation was accompanied by CaMKII autophosphorylation and inhibited by KN62. Thus, we attempted to identify Ser603 kinase(s) besides CaMKII. We consequently detected four and three fractions with Ca2+/calmodulin-independent Ser603 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 32p into synapsin I in a Cdc42/ GTPγS-dependent manner, and its phosphorylation site was confirmed as Ser603 using pS603-Syn I-Ab. Additionally, recombinant GST-PAK2 could phosphorylate the Ser6O3 residue in the presence of Cdc42/GTPγS. Finally, we confirmed by immunocytochemical analysis that the transfection of constitutively active rat αPAK (PAK1) in PC12 cells evokes the phosphorylation of Ser603 even in the resting mutant cells and enhances it in the bradykinin-stimulated cells, whereas that of dominant-negative αPAK quenches the phosphorylation. These results raise the possibility that Ser603 on synapsin I is alternatively phosphorylated by PAKs, not only by CaMKII, in neuronal cells in response to some stimulants.

Original languageEnglish
Pages (from-to)45473-45479
Number of pages7
JournalJournal of Biological Chemistry
Issue number47
Publication statusPublished - 2002 Nov 22
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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