Böl G F, Hülster A, Pfeuffer T
Dept. of Physiol. Chemistry II, Heinrich-Heine University, Düsseldorf, Germany.
Biochim Biophys Acta. 1997 Oct 11;1358(3):307-13. doi: 10.1016/s0167-4889(97)00073-6.
Adenylyl cyclases of the type II family differ from other subforms in that they are conditionally stimulated via alpha(s)/betagamma subunits and regulated by PKC mediated phosphorylation. AC II, stably expressed in HEK 239 cells, was incubated with the PKC activator tetradecanoylphorbol acetate (TPA). Using cells metabolically labeled with [32P]phosphate, TPA caused concerted stimulation of basal and forskolin activated adenylyl cyclase together with incorporation of [32P]phosphate into AC II protein. Enhanced phosphorylation was also indicated by a monoclonal anti-phosphothreonine antibody. Assignment of TPA-induced [32P]phosphate-incorporation to specific sites was achieved by a combination of chemical and immunochemical methods. Three out of five [32P]labeled peptides that were generated by fragmentation with N-chlorosuccinimide were also recognized by the monoclonal antibody BBC-4 [S. Mollner, T. Pfeuffer, Eur. J. Biochem. 171 (1988) 265-271] directed against an epitope 8 kDa from the extreme C-terminus. These findings suggested Ser-871 (consensus sequence ARSLK) and Thr-1057 (CTCR) as acceptor candidates of phorbolester induced phosphoryl transfer.
II型腺苷酸环化酶与其他亚型不同，它们通过α(s)/βγ亚基受到条件性刺激，并受蛋白激酶C介导的磷酸化调节。在HEK 293细胞中稳定表达的AC II，与蛋白激酶C激活剂十四酰佛波醇乙酸酯（TPA）一起孵育。使用用[32P]磷酸盐进行代谢标记的细胞，TPA导致基础和福斯可林激活的腺苷酸环化酶协同刺激，同时[32P]磷酸盐掺入AC II蛋白中。单克隆抗磷酸苏氨酸抗体也表明磷酸化增强。通过化学和免疫化学方法的组合，将TPA诱导的[32P]磷酸盐掺入特定位点。用N-氯代琥珀酰亚胺裂解产生的五个[32P]标记肽中的三个也被针对极端C末端8 kDa表位的单克隆抗体BBC-4识别[S. Mollner, T. Pfeuffer, Eur. J. Biochem. 171 (1988) 265-271]。这些发现表明Ser-871（共有序列ARSLK）和Thr-1057（CTCR）是佛波酯诱导的磷酸转移的受体候选位点。
