Premium
Formation of a stable src–AFAP‐110 complex through either an amino‐terminal or a carboxy‐terminal SH2‐binding motif
Author(s) -
Guappone Anne C.,
Weimer Tracy,
Flynn Daniel C.
Publication year - 1998
Publication title -
molecular carcinogenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.254
H-Index - 97
eISSN - 1098-2744
pISSN - 0899-1987
DOI - 10.1002/(sici)1098-2744(199806)22:2<110::aid-mc6>3.0.co;2-q
Subject(s) - sh2 domain , tyrosine , immunoreceptor tyrosine based activation motif , biology , tyrosine phosphorylation , proto oncogene tyrosine protein kinase src , phosphorylation , biochemistry , amino acid , peptide sequence , receptor tyrosine kinase , microbiology and biotechnology , gene
The actin‐filament–associated protein (AFAP‐110) forms a stable complex with activated variants of the Pp60 c‐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 pp60 c‐src (Src 527F ) 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 (Y 93 and Y 94 ) and two in the carboxy terminus (Y 451 and Y 453 ), were mutated to phenylalanine, significantly reducing overall steady‐state levels of tyrosine phosphorylation and preventing Src 527F 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 (Y 93 /Y 94 ) or in the carboxy 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 527F can form a stable complex with AFAP‐110 through either of two independently functional SH2‐binding motifs. Triple‐tyrosine mutation demonstrated that Y 93 was not significantly phosphorylated on tyrosine and would not facilitate stable complex formation, whereas Y 94 , Y 451 , and Y 453 could be phosphorylated on tyrosine and would facilitate stable‐complex formation. We hypothesize that Src 527F and AFAP‐110 interact through a multistep binding mechanism that may either extend interactions between Src 527F and actin filaments or permit reorientation of Src 527F on AFAP‐110, which could facilitate the presentation of Src 527F toward other signaling molecules. Mol. Carcinog. 22:110–119, 1998. © 1998 Wiley‐Liss, Inc.