Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
J Mol Biol. 2011 Sep 16;412(2):155-64. doi: 10.1016/j.jmb.2011.06.041. Epub 2011 Jul 2.
Phosphorylation of membrane proteins is a central regulatory and signaling mechanism across cell compartments. However, the recognition process and phosphorylation mechanism of membrane-bound substrates by kinases are virtually unknown. cAMP-dependent protein kinase A (PKA) is a ubiquitous enzyme that phosphorylates several soluble and membrane-bound substrates. In cardiomyocytes, PKA targets phospholamban (PLN), a membrane protein that inhibits the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA). In the unphosphorylated state, PLN binds SERCA, reducing the calcium uptake and generating muscle contraction. PKA phosphorylation of PLN at S16 in the cytoplasmic helix relieves SERCA inhibition, initiating muscle relaxation. Using steady-state kinetic assays, NMR spectroscopy, and molecular modeling, we show that PKA recognizes and phosphorylates the excited, membrane-detached R-state of PLN. By promoting PLN from a ground state to an excited state, we obtained a linear relationship between rate of phosphorylation and population of the excited state of PLN. The conformational equilibrium of PLN is crucial to regulate the extent of PLN phosphorylation and SERCA inhibition.
磷酸化是横跨细胞区室的一种核心调节和信号机制。然而，激酶对膜结合底物的识别过程和磷酸化机制实际上还是未知的。环腺苷酸依赖性蛋白激酶 A（PKA）是一种普遍存在的酶，它可以磷酸化几种可溶性和膜结合的底物。在心肌细胞中，PKA 的靶标是磷蛋白（PLN），一种抑制肌浆网 Ca2+-ATP 酶（SERCA）的膜蛋白。在未磷酸化状态下，PLN 与 SERCA 结合，减少钙的摄取并产生肌肉收缩。PLN 在细胞质螺旋中的 S16 处的 PKA 磷酸化可解除 SERCA 的抑制作用，从而引发肌肉松弛。我们通过稳态动力学测定、NMR 光谱和分子建模，证明 PKA 识别并磷酸化了处于兴奋状态、与膜分离的 PLN 的 R 态。通过将 PLN 从基态促进到激发态，我们获得了磷酸化速率与 PLN 激发态的种群之间的线性关系。PLN 的构象平衡对于调节 PLN 磷酸化和 SERCA 抑制的程度至关重要。
