Wyatt T A, Naftilan A J, Francis S H, Corbin J D
Department of Molecular Physiology and Biophysics and of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
Am J Physiol. 1998 Feb;274(2):H448-55. doi: 10.1152/ajpheart.1998.274.2.H448.
Guanosine 3',5'-cyclic monophosphate (cGMP)-binding, cGMP-specific phosphodiesterase (PDE5) is abundant in vascular smooth muscle, and this enzyme is a potent substrate for cGMP-dependent protein kinase (PKG) in vitro. Binding of cGMP to the allosteric sites of PDE5 is required for this phosphorylation to occur. Vascular smooth muscle cells (VSMC) were used to determine if PDE5 is phosphorylated in intact cells when cGMP is increased. With the use of anti-PDE5 antibodies, a phosphorylated 93-kDa protein band was immunoprecipitated from early passaged primary cultures of VSMC that had been preincubated with 32(Pi) to label cellular ATP and then treated with atrial natriuretic factor (ANF). In the absence of ANF, there was no detectable incorporation of radiolabeled phosphate into this band. Phosphorylation of the 93-kDa protein was augmented by pretreating cells with 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) to activate PKG before addition of ANF. 8-BrcGMP, which interacts poorly with the allosteric sites of PDE5, had no effect on PDE5 phosphorylation in the absence of ANF. Phosphorylation of PDE5 in response to treatment of cells with ANF was associated with a two- to fourfold increase in PDE activity in immunoprecipitates. Multiple-passaged VSMC, which are deficient in PKG but retain PDE5, demonstrated no ANF-dependent increase in phosphorylation or catalytic activity of PDE5. However, incubation of immunoprecipitated PDE5 from these cells with purified PKG, cGMP, and a phosphorylation mixture containing [gamma-32P]ATP resulted in 32(Pi) incorporation into PDE5 that was correlated with increased catalytic activity. These studies are the first to demonstrate phosphorylation of PDE5 in intact cells, thus suggesting a physiological role for this enzyme in smooth muscle regulation.
3',5'-环磷酸鸟苷（cGMP）结合的、cGMP特异性磷酸二酯酶（PDE5）在血管平滑肌中含量丰富，并且该酶在体外是cGMP依赖性蛋白激酶（PKG）的有效底物。cGMP与PDE5的变构位点结合是这种磷酸化发生所必需的。使用血管平滑肌细胞（VSMC）来确定当cGMP增加时PDE5在完整细胞中是否被磷酸化。利用抗PDE5抗体，从早期传代的原代VSMC培养物中免疫沉淀出一条93 kDa的磷酸化蛋白带，这些细胞预先用32(Pi)孵育以标记细胞ATP，然后用心房利钠因子（ANF）处理。在没有ANF的情况下，未检测到放射性标记的磷酸盐掺入该条带。在用8-溴-3',5'-环磷酸鸟苷（8-BrcGMP）预处理细胞以激活PKG后再添加ANF，93 kDa蛋白的磷酸化增强。8-BrcGMP与PDE5的变构位点相互作用较差，在没有ANF的情况下对PDE5磷酸化没有影响。用ANF处理细胞后PDE5的磷酸化与免疫沉淀物中PDE活性增加两到四倍相关。多次传代的VSMC缺乏PKG但保留PDE5，未显示出ANF依赖性的PDE5磷酸化或催化活性增加。然而，将这些细胞免疫沉淀的PDE5与纯化的PKG、cGMP和含有[γ-32P]ATP的磷酸化混合物一起孵育，导致32(Pi)掺入PDE5，这与催化活性增加相关。这些研究首次证明了PDE5在完整细胞中的磷酸化，从而表明该酶在平滑肌调节中具有生理作用。
