Guappone A C, Weimer T, Flynn D C
Mary Babb Randolph Cancer Center and Department of Microbiology and Immunology, West Virginia University, Morgantown 26506-9300, USA.
Mol Carcinog. 1998 Jun;22(2):110-9. doi: 10.1002/(sici)1098-2744(199806)22:2<110::aid-mc6>3.0.co;2-q.
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 independently functional SH2-binding motifs. Triple-tyrosine mutation demonstrated that Y93 was not significantly phosphorylated on tyrosine and would not facilitate stable complex formation, whereas Y94, Y451, and Y453 could be phosphorylated on tyrosine and would facilitate stable-complex formation. We hypothesize that Src527F and AFAP-110 interact through a multistep binding mechanism that may either extend interactions between Src527F and actin filaments or permit reorientation of Src527F on AFAP-110, which could facilitate the presentation of Src527F toward other signaling molecules.
肌动蛋白丝相关蛋白（AFAP-1 10）通过SH2和SH3相互作用与Pp60c-src（Src）非受体酪氨酸激酶的活化变体形成稳定复合物。在本报告中，利用定点诱变和一个允许在Cos-1细胞中共表达活化的pp60c-src（Src527F）和AFAP-110的瞬时表达系统来鉴定AFAP-110中的SH2结合基序。四个酪氨酸残基，两个在氨基末端（Y93和Y94），两个在羧基末端（Y451和Y453），被突变为苯丙氨酸，显著降低了酪氨酸磷酸化的总体稳态水平，并阻止Src527F与AFAP-110形成稳定复合物。这些数据表明，酪氨酸磷酸化的主要位点在这四个酪氨酸残基之中，并且这些酪氨酸中的一个或多个可能作为SH2结合基序发挥作用。仅将氨基末端（Y93/Y94）或羧基末端（Y451/Y453）中的两个酪氨酸突变为苯丙氨酸，对酪氨酸磷酸化的稳态水平只有适度影响，并且不足以消除稳定复合物的形成。这些数据表明，Src527F可以通过两个独立发挥功能的SH2结合基序中的任何一个与AFAP-110形成稳定复合物。三酪氨酸突变表明，Y93的酪氨酸没有明显磷酸化，也不会促进稳定复合物的形成，而Y94、Y451和Y453的酪氨酸可以被磷酸化，并会促进稳定复合物的形成。我们推测，Src527F和AFAP-110通过多步结合机制相互作用，这可能会扩展Src527F与肌动蛋白丝之间的相互作用，或者允许Src527F在AFAP-110上重新定向，这可能有助于将Src527F呈递给其他信号分子。
