Phosphorylation of spinophilin modulates its interaction with actin filaments
Phosphorylation of spinophilin modulates its interaction with actin filaments
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DOI：
10.1074/jbc.m205754200
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发表时间：
2003-01-10
影响因子：
4.8
通讯作者：
Greengard, P
中科院分区：
文献类型：
--
作者：
Hsieh-Wilson, LC;Benfenati, F;Greengard, P
Spinophilin 是一种蛋白磷酸酶 1 (PP1) 和肌动蛋白结合蛋白，可调节兴奋性突触传递和树突棘形态。我们报道了亲旋蛋白在体外被蛋白激酶 A (PKA) 磷酸化。用多巴胺 D1 受体激动剂或毛喉素处理新纹状体神经元可刺激亲旋蛋白的磷酸化，这与亲旋蛋白是完整细胞中 PKA 的底物一致。通过胰蛋白酶磷酸肽作图、定点诱变和微测序分析，我们确定了两个主要磷酸化位点 Ser-94 和 Ser-177，它们位于亲旋蛋白的肌动蛋白结合域内。 PKA 对亲旋蛋白的磷酸化调节亲旋蛋白与肌动蛋白细胞骨架之间的关联。亚细胞分级后，未磷酸化的亲旋蛋白在突触后密度中富集，而磷酸化的亲旋蛋白库则在胞质中发现。 F-肌动蛋白共沉淀和叠加分析表明，亲旋蛋白的磷酸化降低了亲旋蛋白-肌动蛋白相互作用的化学计量。相反，亲旋蛋白与 PP1 结合的能力保持不变。综上所述，我们的研究表明，PKA 对亲旋蛋白的磷酸化调节了亲旋蛋白-PP1 复合物在树突棘内的锚定，从而可能有助于突触传递的功效和可塑性。
Spinophilin is a protein phosphatase 1 (PP1)- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We report that spinophilin is phosphorylated in vitro by protein kinase A (PKA). Phosphorylation of spinophilin was stimulated by treatment of neostriatal neurons with a dopamine D1 receptor agonist or with forskolin, consistent with spinophilin being a substrate for PKA in intact cells. Using tryptic phosphopeptide mapping, site-directed mutagenesis, and microsequencing analysis, we identified two major sites of phosphorylation, Ser-94 and Ser-177, that are located within the actin-binding domain of spinophilin. Phosphorylation of spinophilin by PKA modulated the association between spinophilin and the actin cytoskeleton. Following subcellular fractionation, unphosphorylated spinophilin was enriched in the postsynaptic density, whereas a pool of phosphorylated spinophilin was found in the cytosol. F-actin co-sedimentation and overlay analysis revealed that phosphorylation of spinophilin reduced the stoichiometry of the spinophilin-actin interaction. In contrast, the ability of spinophilin to bind to PP1 remained unchanged. Taken together, our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission.