Palmitylation of neuromodulin (GAP-43) is not required for phosphorylation by protein kinase C.
Neuromodulin (also designated GAP-43, B-50, and F-1) is a prominent protein kinase C substrate attached to the membranes of neuronal growth cones during development and to presynaptic membranes in discrete subsets of adult synapses. In this study, we have examined the relationship between the attachment of neuromodulin to membranes and its phosphorylation by protein kinase C. To address this issue, we have compared wild-type and mutant neuromodulins expressed in cells that normally lack the protein. Wild-type neuromodulin expressed in Chinese hamster ovary cells was associated with membranes, incorporated [3H]palmitic acid, and was phosphorylated in response to phorbol ester treatment. Substitution of serine 41, the in vitro protein kinase C site, abolished the phorbol ester response, indicating that serine 41 serves as the sole protein kinase C phosphorylation site in vivo. Substitution of the putative fatty acylation sites, cysteines 3 and 4, abolished membrane association as well as [3H]palmitic acid labeling of neuromodulin. Fatty acylation therefore appears to serve as the mechanism for anchoring neuromodulin to membranes. Surprisingly, the soluble cysteine substitution mutant was phosphorylated by protein kinase C at a rate indistinguishable from that of the wild-type protein. Therefore, membrane association may not be required for the phosphorylation of neuromodulin by protein kinase C.
Chapman
E R
ER
Department of Pharmacology, University of Washington, Seattle 98195.
Estep
R P
RP
Storm
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine; D019922:GAP-43 Protein; D005822:Genetic Vectors; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D009838:Oligodeoxyribonucleotides; D010169:Palmitic Acids; D019308:Palmitic Acid; D011493:Protein Kinase C; D003545:Cysteine