PHORBOL ESTER-INDUCED STIMULATION AND PHOSPHORYLATION OF ADENYLYL-CYCLASE-2

被引:148
作者
JACOBOWITZ, O
IYENGAR, R
机构
[1] Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York
关键词
EPITOPE TAG; RECOMBINANT ENZYME; S19; CELLS;
D O I
10.1073/pnas.91.22.10630
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Adenylyl cyclase 2 was expressed in Sf9 cells by recombinant baculovirus infection. Phorbol 12-myristate 13-acetate (PMA) treatment of cells expressing adenylyl cyclase 2 (AC2) increased basal activity. This increase was blocked by staurosporine, a protein kinase C inhibitor. PMA treatment increased V-max without affecting K-m. Greatest increase in basal activity was seen at physiologically relevant Mg2+ concentrations. PMA treatment did not alter sensitivity to guanine nucleotide stimulatory factor (G(s)) but enhanced stimulation at all concentrations of activated G(s) alpha subunit tested. AC2 was tagged at the N terminus with an 8-amino acid epitope. Epitope-tagged AC2 was purified to apparent homogeneity in a single step by using an antiepitope antibody-affinity column. The eluate was resolved by SDS/PAGE. Silver staining of the gel showed a 106-kDa band. The purified protein was recognized by antipeptide antibody against a region common to all mammalian adenylyl cyclases. The epitope-tagged enzyme expressed in Sf9 cells was also stimulated by PMA. When cells were labeled with P-32 and treated with PMA, a 3-fold increase in P-32 incorporation of purified epitope-tagged AC2 was observed. We conclude that activation of protein kinase C results in phosphorylation and stimulation of AC2, a cell-surface G protein effector enzyme. Thus, covalent modification of cell-surface effecters may provide an independent mode for signal transmission through G protein pathways.
引用
收藏
页码:10630 / 10634
页数:5
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