Schwartz D A, Rubin C S
J Biol Chem. 1985 May 25;260(10):6296-303.
The concentration of regulatory subunits (R) of type II cAMP-dependent protein kinase increased 4- to 5-fold when Friend erythroleukemic cells were either grown in medium containing 0.5 mM 8-bromo-cAMP and 0.2 mM methylisobutylxanthine or stimulated to differentiate. Two species of RII with apparent Mr values of 54,000 (RII-54) and 52,000 (RII-52) are expressed in Friend cells. Both forms of RII were (a) covalently labeled with 8-N3-[32P]cAMP, (b) phosphorylated by the catalytic subunit of protein kinase II, and (c) complexed by polyclonal anti-RII IgGs. RII-52 and RII-54 were not interconverted by phosphorylation or dephosphorylation. A monoclonal antibody that recognizes an internal site in RII resolved the two cAMP-binding proteins by preferentially binding RII-54. The structural diversity suggested by the monoclonal antibody experiment was further examined by comparing two-dimensional maps of tryptic peptides obtained from metabolically labeled [( 35S]met) RII-52 and RII-54. Groups of 35S-labeled peptides that were either uniquely derived from RII-54 or obtained only from RII-52 were readily distinguished, thereby demonstrating that Friend cells produce two separate and distinct forms of type II cAMP-binding subunits. The relative rate of synthesis of RII-52 increased 12- to 14-fold during erythroid differentiation and treatment with 8-bromo-cAMP, while the rate of RII-54 synthesis either declined slowly or was unchanged. Thus, two homologous forms of RII are subject to different modes of physiological (differentiation) and pharmacological (chronic 8-Br-cAMP) regulation, and the accumulation of total RII observed in the present and previous (Schwartz, D. A., and Rubin, C. S. (1983) J. Biol. Chem. 258, 777-784) studies results from a selective increase in the rate of biosynthesis of RII-52.
当弗氏红白血病细胞在含有0.5 mM 8-溴-cAMP和0.2 mM甲基异丁基黄嘌呤的培养基中生长或被刺激分化时，II型cAMP依赖性蛋白激酶调节亚基（R）的浓度增加了4至5倍。在弗氏细胞中表达了两种表观分子量分别为54,000（RII-54）和52,000（RII-52）的RII。两种形式的RII均：（a）被8-N3-[32P]cAMP共价标记；（b）被蛋白激酶II的催化亚基磷酸化；（c）与多克隆抗RII IgG形成复合物。RII-52和RII-54不会通过磷酸化或去磷酸化相互转化。一种识别RII内部位点的单克隆抗体通过优先结合RII-54来分辨这两种cAMP结合蛋白。通过比较从代谢标记的[（35S]甲硫氨酸）RII-52和RII-54获得的胰蛋白酶肽的二维图谱，进一步研究了单克隆抗体实验所提示的结构多样性。很容易区分出仅源自RII-54或仅从RII-52获得的35S标记肽组，从而证明弗氏细胞产生两种单独且不同的II型cAMP结合亚基形式。在红细胞分化和用8-溴-cAMP处理期间，RII-52的相对合成速率增加了12至14倍，而RII-54的合成速率要么缓慢下降，要么保持不变。因此，两种同源形式的RII受到不同的生理（分化）和药理（慢性8-溴-cAMP）调节模式，并且在本研究及先前（施瓦茨，D.A.，和鲁宾，C.S.（1983年）《生物化学杂志》258，777 - 784）研究中观察到的总RII积累是由于RII-52生物合成速率的选择性增加所致。
