Characterization of the calmodulin binding domain of neuromodulin. Functional significance of serine 41 and phenylalanine 42.
Neuromodulin (also designated P-57, GAP-43, B-50) is a major presynaptic substrate for protein kinase C. Phosphorylation of neuromodulin decreases its affinity for calmodulin, suggesting that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons, releasing calmodulin locally in response to phosphorylation by protein kinase C (Alexander, K. A., Cimler, B. M., Meier, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). In the present study, we have constructed and characterized several mutant neuromodulins to demonstrate that the amino acid sequence 39-56 is required for calmodulin binding, and that this domain contains the sole in vitro protein kinase C phosphorylation site at serine 41. We also demonstrate that the adjacent phenylalanine 42, interacts hydrophobically with calmodulin. These hydrophobic interactions may be disrupted by the introduction of negative charge at serine 41, and thereby regulate the neuromodulin/calmodulin binding interactions. The sensitivity of the neuromodulin/calmodulin binding interaction to negative charge at serine 41 was determined by substitution of serine 41 with an aspartate or an asparagine residue. The asparagine mutant retained its affinity for calmodulin-Sepharose while the aspartate mutant did not adsorb to calmodulin-Sepharose. We conclude that protein kinase C phosphorylation of neuromodulin abolishes calmodulin binding by introducing negative charges within the calmodulin binding domain at a position adjacent to the phenylalanine.
Chapman
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ER
Department of Pharmacology, University of Washington, Seattle 98195.
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Alexander
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Nicolson
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Storm
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D019922:GAP-43 Protein; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D015345:Oligonucleotide Probes; D010455:Peptides / Q000138:chemical synthesis; D010649:Phenylalanine; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D014158:Transcription, Genetic
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D010455:Peptides; D011994:Recombinant Proteins; D012694:Serine; D010649:Phenylalanine; D011493:Protein Kinase C