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.

[1]  S. Eguchi,et al.  Identification of an Essential Signaling Cascade for Mitogen-activated Protein Kinase Activation by Angiotensin II in Cultured Rat Vascular Smooth Muscle Cells , 1996, The Journal of Biological Chemistry.

[2]  H. Vogel,et al.  Calcium-Calmodulin-induced Dimerization of the Carboxyl-terminal Domain from Petunia Glutamate Decarboxylase , 1998, The Journal of Biological Chemistry.

[3]  H. Hanafusa,et al.  NH2-terminal sequences of two src proteins that cause aberrant transformation. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Blumenthal,et al.  Changes in the structure of calmodulin induced by a peptide based on the calmodulin-binding domain of myosin light chain kinase. , 1989, Biochemistry.

[5]  H. Hanafusa,et al.  Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. , 1985, Science.

[6]  N. Hayashi,et al.  An expression system of rat calmodulin using T7 phage promoter in Escherichia coli. , 1998, Protein expression and purification.

[7]  A. Rhoads,et al.  Sequence motifs for calmodulin recognition , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[8]  S. Lev,et al.  A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation , 1996, Nature.

[9]  F. Cross,et al.  A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation , 1984, Molecular and cellular biology.

[10]  Y. Izumi,et al.  Binding of both Ca2+ and mastoparan to calmodulin induces a large change in the tertiary structure. , 1989, Journal of biochemistry.

[11]  T. David‐Pfeuty,et al.  Differential localization patterns of myristoylated and nonmyristoylated c-Src proteins in interphase and mitotic c-Src overexpresser cells. , 1993, Journal of cell science.

[12]  M. Resh.  Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. , 1999, Biochimica et biophysica acta.

[13]  T. Roberts,et al.  Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase. , 1992, The Biochemical journal.

[14]  J. Cox,et al.  Inhibition of rhodopsin phosphorylation by non-myristoylated recombinant recoverin. , 1994, Biochemical and biophysical research communications.

[15]  R. Lefkowitz,et al.  Ras-dependent Mitogen-activated Protein Kinase Activation by G Protein-coupled Receptors , 1997, The Journal of Biological Chemistry.

[16]  N. Hayashi,et al.  The binding of myristoylated N‐terminal nonapeptide from neuron‐specific protein CAP‐23/NAP‐22 to calmodulin does not induce the globular structure observed for the calmodulin—nonmyristoylated peptide complex , 2000, Protein science : a publication of the Protein Society.

[17]  N. Hayashi,et al.  Nef of HIV‐1 interacts directly with calcium‐bound calmodulin , 2002, Protein science : a publication of the Protein Society.

[18]  M Ikura,et al.  Molecular and structural basis of target recognition by calmodulin. , 1995, Annual review of biophysics and biomolecular structure.

[19]  M. Resh.  Regulation of cellular signalling by fatty acid acylation and prenylation of signal transduction proteins. , 1996, Cellular signalling.

[20]  D. Filman,et al.  Myristylation of picornavirus capsid protein VP4 and its structural significance , 1987, Nature.

[21]  J. Gordon,et al.  The biology and enzymology of eukaryotic protein acylation. , 1988, Annual review of biochemistry.

[22]  A. Gronenborn,et al.  Solution structure of a calmodulin-target peptide complex by multidimensional NMR. , 1994, Science.

[23]  N. Hayashi,et al.  Identification of the Calmodulin-binding Domain of Neuron-specific Protein Kinase C Substrate Protein CAP-22/NAP-22 , 1999, The Journal of Biological Chemistry.

[24]  V. Lipkin,et al.  N‐Myristoylation of recoverin enhances its efficiency as an inhibitor of rhodopsin kinase , 1995, FEBS letters.

[25]  C. Klee,et al.  Identification of the NH2‐terminal blocking group of calcineurin B as myristic acid , 1982, FEBS letters.

[26]  S. Carr,et al.  n-Tetradecanoyl is the NH2-terminal blocking group of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[27]  M Ikura,et al.  Evidence for calmodulin inter‐domain compaction in solution induced by W‐7 binding , 1999, FEBS letters.

[28]  R. Jove,et al.  Cell transformation by the viral src oncogene. , 1987, Annual review of cell biology.

[29]  B. Sefton,et al.  Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[30]  M. Lisanti,et al.  Targeting of a G alpha subunit (Gi1 alpha) and c-Src tyrosine kinase to caveolae membranes: clarifying the role of N-myristoylation. , 1997, Cellular and molecular biology.

[31]  Masaya Orita,et al.  A novel target recognition revealed by calmodulin in complex with Ca2+-calmodulin-dependent kinase kinase , 1999, Nature Structural Biology.

[32]  M. Resh,et al.  Myristylation and palmitylation of Src family members: The fats of the matter , 1994, Cell.

[33]  K L Gould,et al.  Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements. , 1986, European journal of biochemistry.

[34]  N. Hayashi,et al.  Circular Dichroism and 1H Nuclear Magnetic Resonance Studies on the Solution and Membrane Structures of GAP-43 Calmodulin-binding Domain* , 1997, The Journal of Biological Chemistry.

[35]  S. Grzesiek,et al.  The Importance of Not Saturating H2o in Protein NMR : application to Sensitivity Enhancement and Noe Measurements , 1993 .