Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src.
pp60v-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 pp60v-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 pp60v-src. To verify this possibility, we investigated the direct interaction between pp60v-src and Ca2+/CaM using a myristoylated peptide corresponding to the N-terminal region of pp60v-src. The binding assay indicated that only the myristoylated peptide binds to Ca2+/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 (KD) of 2.07 x 10(-9)M (KD1) and 3.93 x 10(-6)M (KD2), 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 2-3A smaller than that of the known Ca2+/CaM-target molecule complexes. These results demonstrate clearly the direct interaction between pp60v-src and Ca2+/CaM in a novel manner different from that of known Ca2+/CaM, the target molecules, interactions.
Hayashi
Nobuhiro
N
Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan. nhayashi@fujita-hu.ac.jp
Nakagawa
Chisako
C
Ito
Yutaka
Y
Takasaki
Akihiko
A
Jinbo
Yuji
Y
Yamakawa
Yoshinori
Y
Titani
Koiti
K
Hashimoto
Keiichiro
K
Izumi
Yoshinobu
Y
Matsushima
Norio
N
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D019814:Myristic Acid / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D015688:Oncogene Protein pp60(v-src) / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000494:pharmacology; D014961:X-Ray Diffraction
D002147:Calmodulin; D010446:Peptide Fragments; D019814:Myristic Acid; D015688:Oncogene Protein pp60(v-src); D011493:Protein Kinase C; D002118:Calcium