FORMATION OF A STABLE SRC-AFAP-110 COMPLEX THROUGH EITHER AN AMINO-TERMINAL OR A CARBOXY-TERMINAL SH2-BINDING MOTIF

The actin-filament-associated protein (AFAP-110) forms a stable comple x with activated variants of the Pp60(c-src) (Src) non-receptor tyrosi ne kinase through SH2 and SH3 interactions. In this report, site-direc ted mutagenesis and a transient expression system that permits co-expr ession of activated pp60(c-src) (Src(527F)) and AFAP-110 in Cos-1 cell s were used to identify the SH2-binding motif in AFAP-110. Four tyrosi ne residues, two in the amino terminus (Y-93 and Y-94) and two in the carboxy terminus (Y-451 and Y-453), were mutated to phenylalanine, sig nificantly reducing overall steady-state levels of tyrosine phosphoryl ation and preventing SrC527F from forming a stable complex with AFAP-1 10. These data indicate that the major sites for tyrosine phosphorylat ion are among these four tyrosine residues and that one or more of the se tyrosines may function as an SH2-binding motif. Mutagenesis of just two tyrosines in either the amino terminus (y93N94) Or in, the carbox y terminus (Y-451/Y-453) to phenylalanine had only a modest effect on steady-state levels of tyrosine phosphorylation and was not sufficient to abrogate stable-complex formation. These data suggest that Src(527 F) can form a stable complex with AFAP-110 through either of two indep endently functional SH2-binding motifs. Triple-tyrosine mutation demon strated that Y93 was not significantly phosphorylated on tyrosine and would not facilitate stable complex formation, whereas Y-94, Y-451, an d Y-453 could be phosphorylated on tyrosine and would facilitate stabl e-complex formation. We hypothesize that Src(527F) and AFAP-110 intera ct through a multistep binding mechanism that may either extend intera ctions between Src(527F) and actin filaments or permit reorientation o f Src(527F) on AFAP-110, which could facilitate the presentation of Sr c(527F) toward other signaling molecules. Mol. Carcinog. 22:110-119, 1 998. (C) 1998 Wiley-Liss, Inc.