Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the β-adrenergic receptor (β-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes β-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the β-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.

中文翻译：

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Raf 激酶抑制蛋白通过正反馈环调节 cAMP 依赖性蛋白激酶信号通路。


Raf 激酶抑制蛋白 (RKIP) 通过调节 MAP 激酶和蛋白激酶 A (PKA) 等关键激酶级联来维持细胞稳健性并防止癌症和心脏病等疾病的进展。蛋白激酶 C (PKC) 将 RKIP 在 S153 处磷酸化，触发从抑制 Raf 到抑制 G 蛋白偶联受体激酶 2 (GRK2) 的转变，从而增强激活 PKA 的 β-肾上腺素受体 (β-AR) 的信号传导。在此，我们报道 PKA 磷酸化 RKIP 促进 β-AR 激活的 PKA 信号传导。利用生化、遗传和生物物理方法，我们发现 PKA 在 S51 处磷酸化 RKIP，从而增加 PKC 对 S153 的磷酸化，从而触发 PKA 的反馈激活。 S51V 突变阻断了 RKIP 激活前列腺癌细胞中的 PKA 并诱导原代心肌细胞响应 β-AR 激活剂异丙肾上腺素收缩的能力，说明了该正反馈回路的功能重要性。正如之前针对其他激酶所显示的，当 P74L 突变导致 RKIP 不稳定时，PKA 对 RKIP 在 S51 处的磷酸化会增强。这些结果表明，S51 处的 PKA 磷酸化可能会导致与高能 RKIP 状态相关的变构变化，从而增强其他关键位点上 RKIP 的磷酸化。这种变构调节机制可能具有在疾病状态下调节 PKA 信号传导的治疗潜力。