Ac. Guappone et al., FORMATION OF A STABLE SRC-AFAP-110 COMPLEX THROUGH EITHER AN AMINO-TERMINAL OR A CARBOXY-TERMINAL SH2-BINDING MOTIF, Molecular carcinogenesis, 22(2), 1998, pp. 110-119
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.