SRC CAN REGULATE CARBOXY-TERMINAL INTERACTIONS WITH AFAP-110, WHICH INFLUENCE SELF-ASSOCIATION, CELL LOCALIZATION AND ACTIN FILAMENT INTEGRITY

The SH2 and SH3 binding partner AFAP-110 is a tyrosine phosphorylated substrate of Src, AFAP-110 has been hypothesized to link Src to actin filaments, which may contribute to the effects of Src upon actin filam ent integrity, However, it has been unclear what effect activated Src (Src(527F)) has upon AFAP-110 structure or function and whether AFAP-1 10 plays a role in actin filament integrity. We report here that the c arboxy terminal 127 amino acids of AFAP-110 are comprised of an alpha- helical region that contains a leucine zipper motif, This indicated th e potential of AFAP-110 to self-associate, Expression of the carboxy t erminus as a fusion protein (GST-cterm) mill permit affinity absorptio n of cellular AFAP-110, The integrity of the alpha-helical leucine zip per motif in GST-cterm is required for affinity absorption, but bindin g is not due to a classical leucine zipper interaction. Co-expression of Src(527F), unlike cSrc, mill abrogate affinity absorption of AFAP-1 10 with GST-cterm, These data indicate that Src(527F) has affected a c hange in the carboxy terminal structure that renders AFAP-110 unavaila ble for affinity absorption. Superose chromatography demonstrate that AFAP-110 will fractionate as a monomer or multimer, indicating AFAP-11 0 can be detected in a self-associated form in cell lysates. Go-expres sion of Src(527F) resulted in AFAP-110 fractionating with a molecular weight that predicts only a multimeric population. Deletional mutagene sis also indicate a biological role for the carboxy terminus in cellul ar localization and actin filament integrity. Deletion of the entire c arboxy terminal alpha-helix (84 amino acids) will not permit AFAP-110 to efficiently colocalize with actin filaments or the cell membrane. D eletion of only the leucine zipper region of the carboxy terminal alph a-helix (44 amino acids) from AFAP-110 (AFAPA(Delta zip)) demonstrate that both AFAP(Delta lzip) and actin filaments are repositioned into r osette-like structures, similar to the effects of Src(527F), while co- expression of AFAP-110 with cSrc will not affect actin filaments. Thes e data indicate that AFAP-110 can play an important role in modulating actin filament integrity through carboxy terminal interactions that c an be affected by Sr-527F.