The N-terminal SH4 region of the Src family kinase Fyn is modified by methylation and heterogeneous fatty acylation: role in membrane targeting, cell adhesion, and spreading.
The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function.
Liang
Xiquan
X
Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Lu
Yun
Y
Wilkes
Meredith
M
Neubert
Thomas A
TA
Resh
Marilyn D
MD
D000215:Acylation; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002448:Cell Adhesion; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D005227:Fatty Acids / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D019814:Myristic Acid / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D051076:Proto-Oncogene Proteins c-fyn; D011993:Recombinant Fusion Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
D005227:Fatty Acids; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D051076:Proto-Oncogene Proteins c-fyn; D008239:Lysine