Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization, Regulation, and Physiological Role

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V. V. Zakharov^*, M. N. Bogdanova, and M. I. Mosevitsky

Molecular and Radiation Biophysics Division, Petersburg Nuclear Physics Institute, Russian Academy of Sciences, 188300 Gatchina, Leningrad Region, Russia; fax: (7-813) 713-2303; E-mail: v.zakharov@vz5518.spb.edu

^* To whom correspondence should be addressed.

Received July 22, 2004; Revision received August 13, 2004

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The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of µ-calpain (PD151746), but it is completely blocked by µ- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by µ-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G_o; this results in growth cone turning in response to repulsive signals.

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KEY WORDS: neuronal protein GAP-43, calpain, protein kinase C, calmodulin, synaptic terminals, growth cone guidance, proteolysis

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