Formation of a stable src-AFAP-110 complex through either an amino-terminal or a carboxy-terminal SH2-binding motif
Abstract
The actin-filament-associated protein (AFAP-1 10) forms a stable complex with activated variants of the Pp60c-src (Src) non-receptor tyrosine kinase through SH2 and SH3 interactions. In this report, site-directed mutagenesis and a transient expression system that permits co-expression of activated pp60c-src (Src527F) and AFAP-110 in Cos-1 cells were used to identify the SH2-binding motif in AFAP-110. Four tyrosine residues, two in the amino terminus (Y93 and Y94) and two in the carboxy terminus (Y451 and Y453), were mutated to phenylalanine, significantly reducing overall steady-state levels of tyrosine phosphorylation and preventing Src527F from forming a stable complex with AFAP-110. These data indicate that the major sites for tyrosine phosphorylation are among these four tyrosine residues and that one or more of these tyrosines may function as an SH2-binding motif. Mutagenesis of just two tyrosines in either the amino terminus (Y93/Y94) or in the carboxy terminus (Y451/Y453) 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 Src527F can form a stable complex with AFAP-110 through either of two indep...Continue Reading
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