Specific proteolysis of neuronal protein GAP-43 by calpain: characterization, regulation, and physiological role.
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 micro-calpain (PD151746), but it is completely blocked by micro- 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 micro-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.
Zakharov
V V
VV
Molecular and Radiation Biophysics Division, Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Gatchina, Leningrad Region, 188300, Russia. v.zakharov@vz5518.spb.edu
Bogdanova
M N
MN
Mosevitsky
M I
MI
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein / Q000378:metabolism* / Q000502:physiology; D020439:Growth Cones / Q000378:metabolism / Q000502:physiology; D006868:Hydrolysis / Q000187:drug effects; D066298:In Vitro Techniques; D008954:Models, Biological; D010766:Phosphorylation; D017729:Presynaptic Terminals / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D002147:Calmodulin; D019922:GAP-43 Protein; D011493:Protein Kinase C; D002154:Calpain; D002118:Calcium