Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
J Mol Biol. 2011 Aug 26;411(4):823-36. doi: 10.1016/j.jmb.2011.06.034. Epub 2011 Jun 29.
The cAMP-dependent protein kinase [protein kinase A (PKA)] mediates a myriad of cellular signaling events, and its activity is tightly regulated in both space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that, in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted byeither interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of the myristoylated C-subunit of PKA (PKA-C) steers the enzyme toward lipids independent of its regulatory subunit or an A-kinase anchoring protein, providing an additional mechanism to localize the enzyme near membrane-bound substrates.
环腺苷酸依赖的蛋白激酶[蛋白激酶 A (PKA)]介导了无数的细胞信号事件，其活性在空间和时间上受到严格调控。在这些调控机制中，N-豆蔻酰化作用一直难以捉摸。我们使用热力学、动力学和光谱学方法的组合，分析了 N-豆蔻酰化和丝氨酸 10 磷酸化对 PKA 与模型膜相互作用的影响。我们发现，在没有脂质的情况下，豆蔻酰基被塞进酶的疏水性结合口袋中（myr-in 状态）。与脂质双层结合后，豆蔻酰基被挤出并插入脂质双层的烃区域（myr-out 状态）。NMR 数据表明，酶在 myr-in 和 myr-out 状态之间发生构象平衡，这种平衡可以通过与膜相互作用和/或丝氨酸 10 的磷酸化来改变。我们的结果提供了证据，表明 PKA 的豆蔻酰化 C 亚基（PKA-C）的膜结合基序引导酶独立于其调节亚基或 A-激酶锚定蛋白向脂质移动，为酶在膜结合底物附近的定位提供了另一种机制。
