Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization.
The Src family consists of nine related tyrosine protein kinases with a common domain structure, including a myristylated N-terminal glycine residue. In this report, we identify cysteine residues within the N-terminal region of the Src family member Fyn which serve as sites for palmitylation. To facilitate detection of protein fatty acylation, p59fyn was overexpressed in COS cells and incubated with radioiodinated fatty acid analogs of myristate (IC13) or palmitate (IC16). Incorporation of both fatty acids into p59fyn was readily observed. Acylation with the palmitate analog was prevented when Gly-2 was mutated to alanine, implying that N-myristylation is required for palmitylation, and when either Cys-3 or Cys-6 was mutated to serine. Palmitylation was shown to alter the distribution of p59fyn between membrane-bound and soluble fractions. In contrast, no incorporation of the palmitate analog into pp60v-src, which lacks N-terminal cysteine residues, was observed. Mutation of Ser-3 of Src to cysteine, but not Ser-6, resulted in incorporation of the palmitate analog. These results serve to delineate sequence elements important for dual acylation of proteins, and further illustrate the utility of radioiodinated fatty acid analogs for studies of protein fatty acid acylation.
Alland
L
L
Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Peseckis
S M
SM
Atherton
R E
RE
Berthiaume
L
L
Resh
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D016391:Genes, src; D006801:Humans; D007457:Iodine Radioisotopes; D007668:Kidney; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D051076:Proto-Oncogene Proteins c-fyn; D015183:Restriction Mapping; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection; D014316:Tritium
D007457:Iodine Radioisotopes; D009227:Myristic Acids; D010169:Palmitic Acids; D011518:Proto-Oncogene Proteins; D019814:Myristic Acid; D014316:Tritium; D019308:Palmitic Acid; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn