J. Cell Biol., Volume 145, Number 2, April 19, 1999 377-389

Dual Fatty Acylation of p59^Fyn Is Required for Association with the T Cell Receptor  Chain through Phosphotyrosine-Src Homology Domain-2 Interactions

Woutervan't Hof, and Marilyn D. Resh

Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York 10021

The first 10 residues within the Src homology domain (SH)-4 domain of the Src family kinase Fyn are required for binding to the immune receptor tyrosine-based activation motif (ITAM) of T cell receptor (TCR) subunits. Recently, mutation of glycine 2, cysteine 3, and lysines 7 and 9 was shown to block binding of Fyn to TCR  chain ITAMs, prompting the designation of these residues as an ITAM recognition motif (Gauen, L.K.T., M.E. Linder, and A.S. Shaw. 1996. J. Cell Biol. 133:1007-1015). Here we show that these residues do not mediate direct interactions with TCR ITAMs, but rather are required for efficient myristoylation and palmitoylation of Fyn. Specifically, coexpression of a K_7,9A-Fyn mutant with N-myristoyltransferase restored myristoylation, membrane binding, and association with the cytoplasmic tail of TCR  fused to CD8. Conversely, treatment of cells with 2-hydroxymyristate, a myristoylation inhibitor, blocked association of wild-type Fyn with . The Fyn NH₂ terminus was necessary but not sufficient for interaction with  and both Fyn kinase and SH2 domains were required, directing phosphorylation of  ITAM tyrosines and binding to  ITAM phosphotyrosines. Fyn/ interaction was sensitive to octylglucoside and filipin, agents that disrupt membrane rafts. Moreover, a plasma membrane bound, farnesylated Fyn construct, G₂A,C₃S-FynKRas, was not enriched in the detergent insoluble fraction and did not associate with . We conclude that the Fyn SH4 domain provides the signals for fatty acylation and specific plasma membrane localization, stabilizing the interactions between the Fyn SH2 domain and phosphotyrosines in TCR  chain ITAMs.

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