Targeting of a G alpha subunit (Gi1 alpha) and c-Src tyrosine kinase to caveolae membranes: clarifying the role of N-myristoylation.
Many signaling molecules contain the consensus protein sequence Met-Gly at their N-termini that specifies N-myristoylation. Additionally, some of these proteins contain a cysteine at position-3 (Met-Gly-Cys) that can undergo palmitoylation. As many acylated proteins [G-protein subunits (alpha and beta gamma); c-Src and Src-family tyrosine kinases; H-Ras and Ras-related GTPases; endothelial nitric oxide synthase] are known to be targeted to caveolae membranes, it has been suggested that acylation is required or greatly facilitates this targeting event. However, it remains unclear whether myristoylation of Src-family kinases is necessary or sufficient for caveolar targeting. Our current study aims at clarifying the role of myristoylation in caveolar targeting using well-characterized acylation mutants of two model proteins, namely Gi1 alpha and c-Src. Here, we have used: i) detergent-free subcellular fractionation and ii) acylation mutants of Gi1 alpha and c-Src to systematically evaluate the relative contribution of myristoylation and palmitoylation to their caveolar targeting. Myristoylation (G2A) and palmitoytation (C3S) mutants of Gi1 alpha were poorly targeted to caveolae-enriched membrane fractions, while approximately 35% of total wild-type Gi1 alpha co-fractionated with caveolin, a caveolar marker protein. Similarly, a myristoylation minus mutant of c-Src was quantitatively excluded from caveolae. In contrast to a previous study, we conclude that myristoylation of Gi1 alpha and c-Src proteins is required for their correct caveolar targeting. However, the caveolar targeting of Gi1 alpha is dramatically augmented approximately 4-fold by palmitoylation. Our current studies are directly supported by the earlier in vivo observation that N-terminal myristoylation of v-Src is required for v-Src to phosphorylate caveolin on tyrosine residues in intact cells.
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Whitehead Institute for Biomedical Research, Cambridge, MA 02142-1479, USA.
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Galbiati
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D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D000081247:CSK Tyrosine-Protein Kinase; D051242:Caveolin 1; D022461:Caveolins; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D016384:Consensus Sequence; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009227:Myristic Acids / Q000378:metabolism; D010169:Palmitic Acids / Q000378:metabolism; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D019061:src-Family Kinases
D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D009227:Myristic Acids; D010169:Palmitic Acids; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D019204:GTP-Binding Proteins