Analysis of the role of the leucine zipper motif in regulating the ability of AFAP-110 to alter actin filament integrity.

AFAP-110 has an intrinsic ability to alter actin filament integrity as an actin filament crosslinking protein. This capability is regulated by a carboxy terminal leucine zipper (Lzip) motif. The Lzip motif facilitates self-association stabilizing the AFAP-110 multimers. Deletion of the Lzip motif (AFAP-110(Deltalzip)) reduces the stability of the AFAP-110 multimer and concomitantly increases its ability to crosslink actin filaments, in vitro, and to activate cSrc and alter actin filament integrity, in vivo. We sought to determine how the Lzip motif regulates AFAP-110 function. Substitution of the c-Fos Lzip motif in place of the AFAP-110 Lzip motif (AFAP-110(fos)) was predicted to preserve the alpha-helical structure while changing the sequence. To alter the structure of the alpha-helix, a leucine to proline mutation was generated in the AFAP-110 alpha-helical Lzip motif (AFAP-110(581P)), which largely preserved the sequence. The helix mutants, AFAP-110(Deltalzip), AFAP-110(fos), and AFAP-110(581P), demonstrated reduced multimer stability with an increased capacity to crosslink actin filaments, in vitro, relative to AFAP-110. An analysis of opposing binding sites indicated that the carboxy terminus/Lzip motif can contact sequences within the amino terminal pleckstrin homology (PH1) domain indicating an auto-inhibitory mechanism for regulating multimer stability and actin filament crosslinking. In vivo, only AFAP-110(Deltalzip) and AFAP-110(581P) were to activate cSrc and to alter cellular actin filament integrity. These data indicate that the intrinsic ability of AFAP-110 to crosslink actin filaments is dependent upon both the sequence and structure of the Lzip motif, while the ability of the Lzip motif to regulate AFAP-110-directed activation of cSrc and changes in actin filament integrity in vivo is dependent upon the structure or presence of the Lzip motif. We hypothesize that the intrinsic ability of AFAP-110 to crosslink actin filaments or activate cSrc are distinct functions.