The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity

Abstract

We have constructed two point mutants of Rous sarcoma virus in which the amino-terminal glycine residue of the transforming protein, p6Osrc, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60`sc proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p6oSrc proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60`rc proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60`rc was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p6Osrc proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p6tVrc apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur.

Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae.

“…Myristoylation of the Gly 2 residue of Src family PTKs is necessary for membrane binding (6,9). It is possible that the .…”

“…Association of DAF with PTK depends principally on Cys 3 of PTK. (,4) HeLa cells were transiently transfected with p59 fy~ mutants with substitution of cysteine to serine at position or position 6 (lanes[4][5][6]. Cell lysates were analyzed by immtmoprecipitation with anti-DAF (lanes 2 and 5), antii)59 fyn (lanes 3 and 6), or a nonspecific control antibody (lanes 1 and 4), followed by an in vitro kinase assay, SDS-PAGE, and autoradiography.…”

Dual acylation and lipid raft association of Src-family protein tyrosine kinases are required for SDF-1/CXCL12-mediated chemotaxis in the Jurkat human T cell lymphoma cell line

“…Membrane association, although not required for its enzymatic activity, is critical for the biological activity of the SFK [15][16][17]. Association with cellular membranes is a function of the extreme N terminus of all SFK, which are myristoylated on an invariant amino-terminal glycine residue via an amide bond [18].…”

A novel c‐Src recruitment pathway from the cytosol to focal adhesions

“…Binding between Src and its substrates through the SH2 and/or SH3 domains then triggers Src to serve as a kinase. Although the majority of activated Src molecules are localized at focal adhesions [8], little is known about how activated Src molecules are recruited.Translocation of Src to the cell membrane is achieved by myristoylation of the Src N-terminal domain [9]. Membrane targeting of Src is independent of its kinase activity, although the kinase activation is Abbreviations ECM, extracellular matrix; FRAP, fluorescence recovery after photobleaching; SH2, Src-homology 2; SH3, Src-homology 3; TIRF, total internal reflection fluorescence.…”

“…Translocation of Src to the cell membrane is achieved by myristoylation of the Src N-terminal domain [9]. Membrane targeting of Src is independent of its kinase activity, although the kinase activation is Abbreviations ECM, extracellular matrix; FRAP, fluorescence recovery after photobleaching; SH2, Src-homology 2; SH3, Src-homology 3; TIRF, total internal reflection fluorescence.…”