Synapsin I Is Phosphorylated at Ser603 by p21-activated Kinases (PAKs) in Vitro and in PC12 Cells Stimulated with Bradykinin

Abstract

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.

Cav1.2 and Cav1.3 Are Differentially Coupled to Glucagon-Like Peptide-1 Potentiation of Glucose-Stimulated Insulin Secretion in the Pancreatic β-Cell Line INS-1

“…Our results are consistent with a report that GSIS in MIN6 cells is markedly inhibited by both PD98059 and siRNA-mediated knockdown of ERK1 and ERK2 (Longuet et al, 2005). Furthermore, Longuet et al found that ERK stimulation of insulin secretion is mediated by a mechanism that involves phosphorylation of synapsin I. Synapsin I is also phosphorylated by p21-activated kinases in a CDC42-dependent manner in response to bradykinin receptor stimulation (Sakurada et al, 2002). CDC42 is a small Rho family GTPase that is required for second-phase GSIS (Wang et al, 2007).…”

Phosphorylation by cAMP-dependent protein kinase is essential for synapsin-induced enhancement of neurotransmitter release in invertebrate neurons

“…These data indicate that phosphorylation of synapsin domain A is essential for the synapsin-induced enhancement of neurotransmitter release and suggest that endogenous kinases phosphorylating this domain play a central role in the regulation of the efficiency of the exocytotic machinery. al., 1993;Jovanovic et al, 2000;Sakurada et al, 2002), but their presence varies in the different isoforms and orthologues.…”

Phosphorylation-regulated Inhibition of the Gz GTPase-activating Protein Activity of RGS Proteins by Synapsin I

“…9C after prolonged exposure. ) The protein kinases PAK, PKA, and CaM kinase II all phosphorylate Ser 9 in vivo in response to multiple regulatory inputs (23,24). The data of Fig.…”

“…9B). Phosphorylation at Ser 603 , reported by Sakurada et al (24), was not detected. Phosphorylation by PKA inhibited the regulatory activity of synapsin by 76% and phosphorylation by PAK1 inhibited by 66% (Fig.…”

“…Both CaM kinase II and p21-regulated kinase (PAK) catalyze phosphorylation of two serine residues, and phosphorylation at these sites results in the inability of synapsin to bind to actin (23)(24)(25). One of these sites, serine 9, is also phosphorylated by cyclic AMP-dependent protein kinase (PKA) (23).…”