Identification of the protein kinase C phosphorylation site in neuromodulin.
Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin [Alexander, K. A., Cimler, B. M., Meier, K. E., & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113], and we have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar Km values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [gamma-32P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32P-labeled tryptic peptide was generated from phosphorylated neuromodulin. The sequence of this peptide was IQASFR. The serine in this peptide corresponds to position 41 of the entire protein, which is adjacent to or contained within the calmodulin binding domain of neuromodulin. A synthetic peptide, QASFRGHITRKKLKGEK, corresponding to the calmodulin binding domain with a few flanking residues, including serine-41, was also phosphorylated by protein kinase C. We conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmodulin to neuromodulin.(ABSTRACT TRUNCATED AT 250 WORDS)
Apel
E D
ED
Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.
Byford
M F
MF
Au
D
D
Walsh
K A
KA
Storm
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D011493:Protein Kinase C; D014357:Trypsin