Vol. 13, Issue 7, 2311-2322, July 2002

PKC Phosphorylation Increases the Ability of AFAP-110 to Cross-link Actin Filaments

Yong Qian,^* Joseph M. Baisden,^* Lidia Cherezova,^* Justin M. Summy,^* Anne Guappone-Koay,^* Xianglin Shi, Tom Mast, Jennifer Pustula, Henry G. Zot, Nayef Mazloum,^§ Marietta Y. Lee,^§ and Daniel C. Flynn^*

 ^*The Mary Babb Randolph Cancer Center and the Department of Microbiology and Immunology, West Virginia University, Morgantown, West Virginia 26506-9300;  Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia 26506;  Department of Biology, Eastern Michigan University, Ypsilanti, Michigan 48197; and  ^§the Department of Biochemistry and Molecular biology, New York Medical College, Valhalla, New York 10595

The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src^527F can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.