Qian Yong, Baisden Joseph M, Cherezova Lidia, Summy Justin M, Guappone-Koay Anne, Shi Xianglin, Mast Tom, Pustula Jennifer, Zot Henry G, Mazloum Nayef, Lee Marietta Y, Flynn Daniel C
The Mary Babb Randolph Cancer Center and the Department of Microbiology and Immunology, West Virginia University, Morgantown, West Virginia 26506-9300, USA.
Mol Biol Cell. 2002 Jul;13(7):2311-22. doi: 10.1091/mbc.e01-12-0148.
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.
肌动蛋白丝相关蛋白与Src结合伴侣AFAP-110是一种衔接蛋白，可将信号分子与肌动蛋白丝相连。AFAP-110直接结合肌动蛋白丝，并通过亮氨酸拉链基序形成多聚体。Src(527F)下游的细胞信号可调节多聚化。在此，我们通过横向缔合协同测定了重组AFAP-110（rAFAP-110）结合的肌动蛋白丝。我们证明rAFAP-110具有交联肌动蛋白丝的能力，且这种能力取决于羧基末端肌动蛋白结合域的完整性。亮氨酸拉链基序的缺失或PKC磷酸化会影响AFAP-110的构象，这与多聚化的变化相关，并增加了rAFAP-110交联肌动蛋白丝的能力。AFAP-110既是PKC的底物，也是其结合伴侣。PKC激活后，应力纤维组织消失，运动结构形成，且AFAP-110与运动结构强烈共定位。无法结合PKC的AFAP-110缺失突变体的表达阻断了佛波酯对肌动蛋白丝的作用。我们推测，PKC激活后，AFAP-110可协同募集到新形成的肌动蛋白丝上，就像细胞运动结构中存在的那些肌动蛋白丝一样，并且PKC磷酸化会引起构象变化，这可能使AFAP-110能够促进细胞膜处的肌动蛋白丝交联。
