Interaction with receptor for activated C-kinase 1 (RACK1) sensitizes the phosphodiesterase PDE4D5 towards hydrolysis of cAMP and activation by protein kinase C.
Interaction with receptor for activated C-kinase 1 (RACK1) sensitizes the phosphodiesterase PDE4D5 towards hydrolysis of cAMP and activation by protein kinase C.
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DOI：
10.1042/bj20101010
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发表时间：
2010-11-15
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影响因子：
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通讯作者：
Yarwood SJ
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作者：
Bird RJ;Baillie GS;Yarwood SJ
我们以前已经确定了PKC（蛋白激酶C）锚定蛋白RACK 1（活化C-激酶1的受体），作为cAMP特异性磷酸二酯酶PDE 4D 5的特异性结合伴侣，这表明PKC和cAMP信号通路之间的潜在串扰位点。在本研究中，我们发现用β2-肾上腺素受体激动剂异丙肾上腺素（异丙肾上腺素）升高细胞内cAMP，导致HEK（人胚肾）-293细胞颗粒和可溶性组分中PDE 4酶的活化。相反，PDE 4D 5与异丙肾上腺素和PKC激活剂PMA的激活仅限于颗粒部分，其中它与RACK 1相互作用;然而，RACK 1不适于将PDE 4D 5锚定到颗粒部分。动力学研究表明，RACK 1改变了颗粒相关的PDE 4D 5的构象，使其更容易与其底物cAMP和rolipram相互作用，rolipram是一种特异性靶向酶活性位点的PDE 4抑制剂。与RACK 1的相互作用对于PKC依赖性和ERK（细胞外信号调节激酶）非依赖性磷酸化（在Ser 126上）以及响应PMA和异丙肾上腺素的PDE 4D 5活化也是必不可少的，这两者都触发了PKCα向RACK 1的募集。这些结果共同揭示了新的信号传导串扰，其中RACK 1介导响应于细胞内cAMP升高的HEK-293细胞颗粒部分中的PDE 4D 5的PKC依赖性活化。
We have previously identified the PKC (protein kinase C)-anchoring protein RACK1 (receptor for activated C-kinase 1), as a specific binding partner for the cAMP-specific phosphodiesterase PDE4D5, suggesting a potential site for cross-talk between the PKC and cAMP signalling pathways. In the present study we found that elevation of intracellular cAMP, with the β2-adrenoceptor agonist isoproterenol (isoprenaline), led to activation of PDE4 enzymes in the particulate and soluble fractions of HEK (human embryonic kidney)-293 cells. In contrast activation of PDE4D5, with isoproterenol and the PKC activator PMA, was restricted to the particulate fraction, where it interacts with RACK1; however, RACK1 is dispensable for anchoring PDE4D5 to the particulate fraction. Kinetic studies demonstrated that RACK1 alters the conformation of particulate-associated PDE4D5 so that it more readily interacts with its substrate cAMP and with rolipram, a PDE4 inhibitor that specifically targets the active site of the enzyme. Interaction with RACK1 was also essential for PKC-dependent and ERK (extracellular-signal-regulated kinase)-independent phosphorylation (on Ser126), and activation of PDE4D5 in response to PMA and isoproterenol, both of which trigger the recruitment of PKCα to RACK1. Together these results reveal novel signalling cross-talk, whereby RACK1 mediates PKC-dependent activation of PDE4D5 in the particulate fraction of HEK-293 cells in response to elevations in intracellular cAMP.