Ac. Guappone et Dc. Flynn, THE INTEGRITY OF THE SH3 BINDING MOTIF OF AFAP-110 IS REQUIRED TO FACILITATE TYROSINE PHOSPHORYLATION BY, AND STABLE COMPLEX-FORMATION WITH, SRC, Molecular and cellular biochemistry, 175(1-2), 1997, pp. 243-252
The actin filament-associated protein AFAP-110 forms a stable complex
with activated variants of Src in chick embryo fibroblast cells. Stabl
e complex formation requires the integrity of the Src SH2 and SH3 doma
ins. In addition, AFAP-110 encodes two adjacent SH3 binding motifs and
six candidate SH2 binding motifs. These data indicate that both SH2 a
nd SH3 domains may work cooperatively to facilitate Src/AFAP-110 stabl
e complex formation. As a test for this hypothesis, we sought to under
stand whether one or both SH3 binding motifs in AFAP-110 modulate inte
ractions with the Src SH3 domain and if this interaction was required
to present AFAP-110 for tyrosine phosphorylation by, and stable comple
x formation with, Src. A proline to alanine site-directed mutation in
the amino terminal SH3 binding motif (SH3bm I) was sufficient to abrog
ate absorption of AFAP-110 with GST-SH3(src). Go-expression of activat
ed Src (pp60(527F)) With AFAP-110 in Cos-l cells permit tyrosine phosp
horylation of AFAP-110 and stable complex formation with pp60(527F). H
owever, co-expression of the SH3 null-binding mutant (AFAP(71A)) with
pp60(527F) revealed a 2.7 fold decrease in steady-state levels of tyro
sine phosphorylation, compared to AFAP-110. Although a lower but detec
table level of AFAP(71A) was phosphorylated on tyrosine, AFAP(71A) cou
ld not be detected in stable complex with pp60(527F), unlike AFAP-110.
These data indicate that SH3 interactions facilitate presentation of
AFAP-110 for tyrosine phosphorylation and are also required for stable
complex formation with pp60(527F).