Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60^v-src

pp60^v-src tyrosine protein kinase was suggested to interact with Ca²⁺-bound calmodulin (Ca²⁺/CaM) through the N-terminal region based on its structural similarities to CAP-23/NAP-22, a myristoylated neuron-specific protein, whose myristoyl group is essential for interaction with Ca²⁺/CaM; (1) the N terminus of pp60 ^v-src is myristoylated like CAP-23/NAP-22; (2) both lysine residues are required for the myristoylation-dependent interaction and serine residues that are thought to regulate the interaction through the phosphorylations located in the N-terminal region of pp60^v-src. To verify this possibility, we investigated the direct interaction between pp60 ^v-src and Ca²⁺/CaM using a myristoylated peptide corresponding to the N-terminal region of pp60^v-src. The binding assay indicated that only the myristoylated peptide binds to Ca ²⁺/CaM, and the non-myristoylated peptide is not able to bind to Ca²⁺/CaM. Analyses of the binding kinetics revealed two independent reactions with the dissociation constants (K_D) of 2.07×10 ^-9M (K_D1) and 3.93×10^-6M (K _D2), respectively. Two serine residues near the myristoyl moiety of the peptide (Ser2, Ser11) were phosphorylated by protein kinase C in vitro, and the phosphorylation drastically reduced the interaction. NMR experiments indicated that two molecules of the myristoylated peptide were bound around the hydrophobic clefts of a Ca²⁺/CaM molecule. The small-angle X-ray scattering analyses showed that the size of the peptide-Ca²⁺/CaM complex is 2-3Å smaller than that of the known Ca²⁺/CaM-target molecule complexes. These results demonstrate clearly the direct interaction between pp60^v-src and Ca²⁺/CaM in a novel manner different from that of known Ca²⁺/CaM, the target molecules, interactions.