COORDINATED REGULATION OF SYNAPSIN-I INTERACTION WITH F-ACTIN BY CA2-INHIBITION OF ACTIN-BINDING AND BUNDLING( CALMODULIN AND PHOSPHORYLATION )

The synapsins are a family of synaptic vesicle phosphoproteins whose r ole seems to be to limit the availability of small synaptic vesicles f or exocytosis by Linking them to the cytoskeleton. One member of the f amily, synapsin I, has been shown to bind calmodulin in a Ca2+-depende nt manner. In this study, we have examined whether or not calmodulin c an regulate one of the activities of synapsin I, namely, its interacti on with F-actin. Synapsin I is an actin bundling protein: this activit y is controlled by phosphorylation. Here we show that calmodulin in th e presence of Ca2+ is a competitive inhibitor of both actin binding an d bundling by synapsin I. Under the conditions of our assay (0.45 mu M synapsin I, 4 mu M F-actin), half-maximal inhibiton of actin binding and bundling by unphosphorylated synapsin I was found with 4.3 and 3.7 mu M calmodulin, respectively. The actin binding activity of synapsin I phosphorylated by cAMP-dependent protein kinase or by calmodulin-de pendent protein kinase II showed similar sensitivity to calmodulin inh ibition to unphosphorylated synapsin I. However, inhibition of bundlin g was potentiated. Half-maximal inhibition of bundling by synapsin I p hosphorylated by cAMP-dependent kinase was achieved at approximately 0 .5 mu M calmodulin. Half-maximal inhibition of bundling by synapsin I phosphorylated by calmodulin-dependent protein kinase II was achieved at less than 0.2 mu M calmodulin, although the maximum binding under t he conditions of the assay was lower. Synapsin I phosphorylated by bot h cAMP-dependent kinase and calmodulin-dependent protein kinase II was half-maximally inhibited from actin binding by less than 100 nM calmo dulin, although the binding and bundling characteristics of this form of synapsin I were weak even in the absence of calmodulin. We conclude that covalent (phosphorylation) and noncovalent (calmodulin) forms of regulation act together in regulating one of the activities of synaps in I.