Braun R K, Vulliet P R, Carbonaro-Hall D A, Hall F L
Department of Veterinary Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis 95616.
Arch Biochem Biophys. 1991 Aug 15;289(1):187-91. doi: 10.1016/0003-9861(91)90460-z.
Previous studies identified proline-directed protein kinase (PDPK) as a growth factor-sensitive serine/threonine protein kinase that is active in the cytosol of proliferative cells and tissues during interphase. In this communication, we report that the regulatory subunit (RII) of bovine cardiac muscle cAMP-dependent protein kinase (PKA) is a putative substrate for the multifunctional PDPK. Purified RII is readily phosphorylated by PDPK in vitro in a time-dependent, enzyme-dependent manner to a stoichiometry approaching 0.7 mol phosphate/mol RII subunit protein. The major RII phosphorylation site is identified as a threonine residue located within a large hydrophobic tryptic peptide that is predicted to contain the cAMP binding domains. In contrast to the reported effects of RII autophosphorylation, kinetic analysis of RII function following phosphorylation by PDPK indicates that the inhibitory potency of RII toward the catalytic subunit of PKA in a reassociation assay is increased in proportion to the degree of phosphorylation. Further studies indicate that the cAMP-dependent activation of the RII2C2 holoenzyme is inhibited by PDPK phosphorylation. Taken together, the results of these studies indicate that phosphorylation of RII by PDPK attenuates the activity of PKA. This antagonistic interaction suggests a biochemical mechanism by which a growth factor-activated signaling system may function to modulate cAMP-dependent cellular responses.
先前的研究将脯氨酸定向蛋白激酶（PDPK）鉴定为一种对生长因子敏感的丝氨酸/苏氨酸蛋白激酶，在间期增殖细胞和组织的细胞质中具有活性。在本通讯中，我们报道牛心肌cAMP依赖性蛋白激酶（PKA）的调节亚基（RII）是多功能PDPK的假定底物。纯化的RII在体外很容易被PDPK以时间依赖性、酶依赖性的方式磷酸化，化学计量比接近0.7摩尔磷酸/摩尔RII亚基蛋白。主要的RII磷酸化位点被鉴定为位于一个大的疏水胰蛋白酶肽内的苏氨酸残基，该肽预计包含cAMP结合结构域。与报道的RII自磷酸化的作用相反，PDPK磷酸化后对RII功能的动力学分析表明，在重新结合试验中，RII对PKA催化亚基的抑制效力与磷酸化程度成比例增加。进一步的研究表明，PDPK磷酸化抑制了RII2C2全酶的cAMP依赖性激活。综上所述，这些研究结果表明PDPK对RII的磷酸化减弱了PKA的活性。这种拮抗相互作用提示了一种生化机制，通过该机制生长因子激活的信号系统可能发挥作用来调节cAMP依赖性细胞反应。
