Department of Molecular Physiology & Biophysics, Vanderbilt University School of Medicine, Nashville, TN, 37232-0615, USA.
Department of Molecular Physiology & Biophysics, Vanderbilt University School of Medicine, Nashville, TN, 37232-0615, USA; Vanderbilt Brain Institute, Vanderbilt University School of Medicine, Nashville, TN, 37232-0615, USA; Vanderbilt-Kennedy Center for Research on Human Development, Vanderbilt University School of Medicine, Nashville, TN, 37232-0615, USA.
Biochem Biophys Res Commun. 2020 Mar 26;524(1):262-267. doi: 10.1016/j.bbrc.2020.01.089. Epub 2020 Jan 23.
Protein-protein interactions can be modulated by phosphorylation of either binding partner, thereby altering subcellular localization and/or physiological function. Shank3, a master postsynaptic scaffolding protein that controls the developmental maturation of excitatory synapses, was recently shown to be phosphorylated by Protein Kinase A (PKA) at Ser685 in vivo. Mutation of Shank3 Ser685 was shown to modulate the binding of Abelson interactor 1 (ABI1), a component of the WAVE regulatory complex for actin remodeling, but a direct effect of Ser685 phosphorylation on ABI1 binding was not investigated. Here, we demonstrate that Ca/calmodulin-dependent protein kinase II alpha (CaMKIIα) also phosphorylates Shank3 at Ser685. Mutation of Ser685 to phospho-null alanine (S685A) prevented both CaMKIIα and PKA phosphorylation of a GST-Shank3 fusion protein. The co-immunoprecipitation of ABI1 with Shank3 from HEK293 cell extracts is reduced by mutation of Ser685 to either Ala or Asp. However, pre-phosphorylation of GST-Shank3 by purified CaMKIIα significantly increased binding of ABI1, and this effect was abrogated by Ser685 to Ala mutation in GST-Shank3. Taken together, our data suggest that neuronal ABI1-Shank3 interactions may be convergently regulated by Shank3 Ser685 phosphorylation in response to both Ca and cAMP signaling, potentially modulating dendritic spine morphology.
蛋白质-蛋白质相互作用可以通过结合伙伴的磷酸化来调节，从而改变亚细胞定位和/或生理功能。Shank3 是一种主要的突触后支架蛋白，可控制兴奋性突触的发育成熟，最近被发现可在体内被蛋白激酶 A（PKA）磷酸化于 Ser685。Shank3 Ser685 的突变被证明可以调节 Abelson 相互作用蛋白 1（ABI1）的结合，ABI1 是肌动蛋白重塑的 WAVE 调节复合物的一个组成部分，但 Ser685 磷酸化对 ABI1 结合的直接影响尚未被研究。在这里，我们证明钙/钙调蛋白依赖性蛋白激酶 IIα（CaMKIIα）也可磷酸化 Shank3 于 Ser685。Ser685 突变为磷酸化缺失的丙氨酸（S685A）可阻止 GST-Shank3 融合蛋白被 CaMKIIα 和 PKA 磷酸化。将 Ser685 突变为 Ala 或 Asp 均可减少从 HEK293 细胞提取物中免疫共沉淀的 ABI1 与 Shank3 的相互作用。然而，纯化的 CaMKIIα 对 GST-Shank3 的预磷酸化显著增加了 ABI1 的结合，而 GST-Shank3 中 Ser685 突变为 Ala 则消除了这种作用。综上所述，我们的数据表明神经元 ABI1-Shank3 相互作用可能通过 Shank3 Ser685 磷酸化被钙和 cAMP 信号共同调节，可能调节树突棘形态。
