title: The residue at position 5 of the N-terminal region of Src and Fyn modulates their myristoylation, palmitoylation, and membrane interactions.
abstract: The interactions of Src family kinases (SFKs) with the plasma membrane are crucial for their activity. They depend on their fatty-acylated N-termini, containing N-myristate and either a polybasic cluster (in Src) or palmitoylation sites (e.g., Fyn). To investigate the roles of these moieties in SFK membrane association, we used fluorescence recovery after photobleaching beam-size analysis to study the membrane interactions of c-Src-GFP (green fluorescent protein) or Fyn-GFP fatty-acylation mutants. Our studies showed for the first time that the membrane association of Fyn is more stable than that of Src, an effect lost in a Fyn mutant lacking the palmitoylation sites. Unexpectedly, Src-S3C/S6C (containing cysteines at positions 3/6, which are palmitoylated in Fyn) exhibited fast cytoplasmic diffusion insensitive to palmitoylation inhibitors, suggesting defective fatty acylation. Further replacement of the charged Lys-5 by neutral Gln to resemble Fyn (Src-S3C/S6C/K5Q) restored Fyn-like membrane interactions, indicating that Lys-5 in the context of Src-S3C/S6C interferes with its myristoylation/palmitoylation. This was validated by direct myristoylation and palmitoylation studies, which indicated that the residue at position 5 regulates the membrane interactions of Src versus Fyn. Moreover, the palmitoylation levels correlated with targeting to detergent-resistant membranes (rafts) and to caveolin-1. Palmitoylation-dependent preferential containment of Fyn in rafts may contribute to its lower transformation potential.
authors.affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Efrat
authors.initials: E
authors.lastname: Gottlieb-Abraham
authors.affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Orit
authors.initials: O
authors.lastname: Gutman
authors.affiliation: Institute of Molecular Cell Biology, Center for Molecular Biomedicine, University Hospital, Jena 07745, Germany.
authors.forename: Govind M
authors.initials: GM
authors.lastname: Pai
authors.affiliation: Institute of Molecular Cell Biology, Center for Molecular Biomedicine, University Hospital, Jena 07745, Germany.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Rubio
authors.affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel henis@post.tau.ac.il.
authors.forename: Yoav I
authors.initials: YI
authors.lastname: Henis
mesh_terms: D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D000081247:CSK Tyrosine-Protein Kinase; D051242:Caveolin 1 / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000378:metabolism; D016391:Genes, src / Q000235:genetics* / Q000502:physiology*; D049452:Green Fluorescent Proteins; D054878:Lipoylation; D008565:Membrane Proteins; D008566:Membranes / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: C495540:CAV1 protein, human; D051242:Caveolin 1; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D049452:Green Fluorescent Proteins; D000081247:CSK Tyrosine-Protein Kinase; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; C000631346:CSK protein, human; D003545:Cysteine