Bonini N M, Nelson D L
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
J Cell Sci. 1990 Feb;95 ( Pt 2):219-30. doi: 10.1242/jcs.95.2.219.
Permeabilized, MgATP-reactivated cells of Paramecium (models) respond to cyclic AMP and cyclic GMP by increasing forward swimming speed. In association with the motile response, cyclic AMP and 8-bromo-cyclic GMP (8-Br-cyclic GMP) stimulated protein phosphorylation. Cyclic AMP addition to permeabilized cells reproducibly stimulated the phosphorylation of 10 proteins, ranging in molecular weight from 15 to 110K (K = 10(3) Mr). 8-Br-cyclic GMP, which selectively activates the cyclic GMP-dependent protein kinase of Paramecium, stimulated the phosphorylation of a subset of the proteins phosphorylated by cyclic AMP. Ca2+ addition caused backward swimming and stimulated the phosphorylation of four substrates, including a 25K target that may also be phosphorylated in response to cyclic nucleotide addition. Ba2+ and Sr2+ also induced backward swimming, but did not cause detectable phosphorylation. To identify ciliary targets of cyclic nucleotide-dependent protein kinase activity, permeabilized cells were deciliated following reactivation of motility with Mg-[gamma-32P]ATP in the presence or absence of cyclic nucleotide. Soluble proteins of the deciliation supernatant were enriched in 15 cyclic AMP-stimulated phosphoproteins, ranging in molecular weight from 15 to 95K. Most of the ciliary substrates were axonemal and could be released by high salt solution. A 29K protein that copurified in sucrose gradients with the 22S dynein, and a high molecular weight protein (greater than 300K) in the 19 S region were phosphorylated when cyclic AMP was added to permeabilized, motile cells. These data suggest that regulation of ciliary motility by cyclic AMP may include phosphorylation of dynein-associated proteins.
经通透处理并用MgATP重新激活的草履虫（模型）细胞，通过提高向前游动速度来响应环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）。与运动反应相关的是，cAMP和8-溴环磷酸鸟苷（8-Br-cGMP）刺激了蛋白质磷酸化。向通透处理的细胞中添加cAMP可重复性地刺激10种蛋白质的磷酸化，其分子量范围为15至110K（K = 10³ Mr）。8-Br-cGMP可选择性激活草履虫的环磷酸鸟苷依赖性蛋白激酶，刺激了cAMP磷酸化的一部分蛋白质的磷酸化。添加Ca²⁺导致向后游动，并刺激了四种底物的磷酸化，包括一种25K的靶标，其也可能响应环核苷酸添加而被磷酸化。Ba²⁺和Sr²⁺也诱导向后游动，但未引起可检测到的磷酸化。为了鉴定环核苷酸依赖性蛋白激酶活性的纤毛靶标，在用Mg-[γ-³²P]ATP在有或无环核苷酸存在的情况下重新激活运动性后，对通透处理的细胞进行去纤毛处理。去纤毛上清液中的可溶性蛋白质富含15种cAMP刺激的磷蛋白，分子量范围为15至95K。大多数纤毛底物是轴丝的，并且可以通过高盐溶液释放。当向通透处理的运动细胞中添加cAMP时，可以观察到一种在蔗糖梯度中与22S动力蛋白共纯化的29K蛋白质以及19S区域中的一种高分子量蛋白质（大于300K）发生磷酸化。这些数据表明，cAMP对纤毛运动的调节可能包括动力蛋白相关蛋白的磷酸化。
