THE INTEGRITY OF THE SH3 BINDING MOTIF OF AFAP-110 IS REQUIRED TO FACILITATE TYROSINE PHOSPHORYLATION BY, AND STABLE COMPLEX-FORMATION WITH, SRC

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).