Mass spectrometric analysis of GAP-43/neuromodulin reveals the presence of a variety of fatty acylated species.
GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications.
Liang
Xiquan
X
Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Lu
Yun
Y
Neubert
Thomas A
TA
Resh
Marilyn D
MD
D000215:Acylation; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D005227:Fatty Acids / Q000032:analysis*; D019922:GAP-43 Protein / Q000737:chemistry*; D006801:Humans; D019811:Hydroxylamine / Q000494:pharmacology; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry; D018613:Microscopy, Confocal; D009154:Mutation; D016716:PC12 Cells; D010169:Palmitic Acids / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013228:Stearates / Q000737:chemistry; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D005227:Fatty Acids; D019922:GAP-43 Protein; D010169:Palmitic Acids; D011993:Recombinant Fusion Proteins; D013228:Stearates; D019811:Hydroxylamine; D008715:Methionine; D014357:Trypsin; D003545:Cysteine