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

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（actin filament）结合蛋白及Src（Src）结合伴侣AFAP-110是一类衔接蛋白，可将信号分子与肌动蛋白丝相连。AFAP-110可直接结合肌动蛋白丝，并通过亮氨酸拉链基序（leucine zipper motif）发生多聚化。Src(527F)下游的细胞信号可调控AFAP-110的多聚化过程。本研究通过侧向缔合的协同作用，对重组AFAP-110（rAFAP-110）结合的肌动蛋白丝进行了表征。我们证实，rAFAP-110具备交联肌动蛋白丝的能力，且该功能依赖于羧基末端肌动蛋白结合结构域的完整性。亮氨酸拉链基序的缺失或蛋白激酶C（PKC）的磷酸化修饰会改变AFAP-110的构象，这与多聚化状态的改变相关，同时增强了rAFAP-110交联肌动蛋白丝的能力。AFAP-110同时是PKC的底物及结合伴侣。当PKC被激活时，应力纤维（stress filament）的组装结构会消失，细胞运动相关结构开始形成，且AFAP-110会与运动结构发生强共定位。表达无法结合PKC的AFAP-110缺失突变体，可阻断佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）对肌动蛋白丝的作用。我们提出如下假说：当PKC被激活时，AFAP-110可被协同招募至新形成的肌动蛋白丝（如细胞运动结构中的肌动蛋白丝），且PKC的磷酸化会诱导构象改变，从而使AFAP-110能够在细胞膜处促进肌动蛋白丝的交联。