Phosphorylation of specific residues in the activation loops of AGC family protein kinases is required for activity of most of these kinases, including the catalytic subunit of protein kinase A (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it "constitutively" phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FxxF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in both cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FxxF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FxxW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.

中文翻译：

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三方协作机制赋予蛋白激酶A催化亚基抗去磷酸化作用。

AGC家族蛋白激酶激活环中特定残基的磷酸化是大多数这些激酶（包括蛋白激酶A（PKAc）的催化亚基）的活性所必需的。尽管许多磷酸化的AGC激酶对磷酸酶介导的去磷酸化敏感，但PKAc激活环独特地抵抗了去磷酸化，使其在细胞中“组成性”地被磷酸化。先前的生物物理实验和结构建模表明，PKAc中的N末端肉豆蔻酰化信号和C末端的FxxF基序调节其热稳定性和催化作用。在这里，使用定点诱变，分子建模以及在无细胞和基于细胞的系统中，我们证明，PKAc肉豆蔻酰化信号或FxxF基序的取代只能适度地降低磷酸化，并且不能影响细胞中的PKAc功能。但是，我们观察到这两个位点与N末端FxxW基序协作，共同建立PKAc的磷酸酶抗性，同时不影响激活环的激酶依赖性磷酸化。我们注意到，磷酸酶抗性的这种三方协作机制在功能上是相关的，这通过在这三个位点上被含有氨基酸取代的PKAc变体转染的人气道平滑肌细胞的形态，粘附和迁移所证实。这些发现确定了位于PKAc N和C末端的三个变构位点可协调调节该酶的磷酸酶敏感性。