Böl G F, Gros C, Hülster A, Bösel A, Pfeuffer T
Department of Physiological Chemistry II, Heinrich-Heine University, Düsseldorf, D-40001, Germany.
Biochem Biophys Res Commun. 1997 Aug 18;237(2):251-6. doi: 10.1006/bbrc.1997.7123.
Following up the results from previous studies on chemical fragmentation of TPA-treated, [32P]phosphate labeled adenylyl cyclase type II (AC II) (Böl, G. F., Hülster, A., and Pfeuffer, T. in press) we have replaced serine 871 or threonine 1057 by alanine using site directed mutagenesis. Both mutants had unimpaired catalytic activity, however enhancement by phorbolester TPA was reduced by 60-80 % in the T1057A mutant, but not in the S871A mutant. The stimulation of adenylyl cyclase type II by betagamma subunits of heterotrimeric G-pro teins and that by PKC have been previously shown to be mutually exclusive (Zimmermann and Taussig (1996), J. Biol. Chem. 271, 27161-27166). This is in line with the present findings that AC II expressed in COS-1 cells was only barely stimulated (10%) by coexpressed betagamma-subunits in presence of TPA. Mutation of threonine 1057 to alanine however caused partial regain of betagamma-stimulation in the presence of TPA by 40%, as compared to that of WT adenylyl cyclase type II which was 70% in the absence of TPA. These data strongly implicate the importance of threonine 1057 as phosphate acceptor following PKC-mediated sensitisation of adenylyl cyclase type II.
继先前关于佛波酯（TPA）处理的、[32P]磷酸盐标记的Ⅱ型腺苷酸环化酶（ACⅡ）化学片段化的研究结果（Böl, G. F., Hülster, A., and Pfeuffer, T.即将发表）之后，我们利用定点诱变将丝氨酸871或苏氨酸1057替换为丙氨酸。两个突变体的催化活性均未受损，然而，佛波酯TPA对T1057A突变体的增强作用降低了60 - 80%，而对S871A突变体则没有影响。此前已表明，异源三聚体G蛋白的βγ亚基对Ⅱ型腺苷酸环化酶的刺激作用与蛋白激酶C（PKC）的刺激作用相互排斥（Zimmermann和Taussig（1996年），《生物化学杂志》271卷，27161 - 27166页）。这与目前的研究结果一致，即在TPA存在的情况下，共表达的βγ亚基对COS - 1细胞中表达的ACⅡ几乎没有刺激作用（10%）。然而，将苏氨酸1057突变为丙氨酸会导致在TPA存在的情况下，βγ刺激作用部分恢复40%，而野生型Ⅱ型腺苷酸环化酶在不存在TPA时的βγ刺激作用为70%。这些数据强烈表明苏氨酸1057作为PKC介导的Ⅱ型腺苷酸环化酶致敏后磷酸盐受体的重要性。
