Comparison of heterotrimeric protein phosphatase 2A containing different B subunits.
Comparison of heterotrimeric protein phosphatase 2A containing different B subunits.
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发表时间：
1994-08
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通讯作者：
C. Kamibayashi;R. Estes;R. Lickteig;S. I. Yang;C. Craft;M. Mumby
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作者：
C. Kamibayashi;R. Estes;R. Lickteig;S. I. Yang;C. Craft;M. Mumby
蛋白磷酸酶 2A (PP2A) 由结构亚基 (A)、催化亚基 (C) 和调节亚基 (B) 组成。免疫学分析确定 B α/PR55 α 是大脑 PP2A 的主要调节亚基，而独特的 B' 亚基与心肌酶相关。重组 PP2A 异源三聚体从感染表达 A 和 C 的杆状病毒以及表达 B α/PR55 α、B β/PR55 β 或 SV40 小肿瘤抗原 (st) 的病毒的昆虫细胞中纯化。 rAC-B α 和 rAC-B β 的磷酸酶活性与大脑 AC-B α 的磷酸酶活性相似，而 rAC-st 的活性低 50-80%。肝素对 rAC-st 肌球蛋白轻链磷酸酶活性没有影响，而含有 B 亚基的形式则被刺激 2-3 倍。鱼精蛋白导致 AC-B α 和 rAC-st 活性增加 3-4 倍，并显着激活 rAC-B β（6 倍）和 AC-B'（10.5 倍）。当使用组蛋白 H1 作为底物时，所有异源三聚体均被肝素刺激大约 4 倍。 Mn2+ 使 AC-B' 和 rAC-B beta 的活性增加了 2 倍，而 rAC-st 则使 AC-B' 和 rAC-B beta 的活性增加了 6 倍。 AC-B α 和 AC-B β 的化学交联产生 200 kDa 的复合物，而 AC-st 则以 150 kDa 的复合物形式存在。这些结果表明，不同的调节蛋白会影响酶活性以及对体外改变 PP2A 活性的试剂的反应。不同的PP2A异源三聚体在体内可能具有不同的功能，亚基组成的变化将对信号转导途径产生重要影响。
Protein phosphatase 2A (PP2A) is composed of structural (A), catalytic (C), and regulatory subunits (B). Immunological analyses identified B alpha/PR55 alpha as the major regulatory subunit of brain PP2A while a unique B' subunit was associated with the cardiac enzyme. Recombinant PP2A heterotrimers were purified from insect cells infected with baculoviruses expressing A and C, in combination with viruses expressing B alpha/PR55 alpha, B beta/PR55 beta, or SV40 small tumor antigen (st). Phosphatase activities of rAC-B alpha and rAC-B beta were similar to those for brain AC-B alpha, while rAC-st was 50-80% less active. Heparin had no effect on rAC-st myosin light chain phosphatase activity, while the B subunit-containing forms were stimulated 2-3-fold. Protamine caused a 3-4-fold increase in AC-B alpha and rAC-st activities and a marked activation of rAC-B beta (6-fold) and AC-B' (10.5-fold). When histone H1 was used as substrate, all of the heterotrimers were stimulated approximately 4-fold by heparin. The activity of AC-B' and rAC-B beta were increased 2-fold by Mn2+, while a 6-fold stimulation was observed with rAC-st. Chemical cross-linking of AC-B alpha and AC-B beta generated 200-kDa complexes, while AC-st was present as a 150-kDa complex. These results demonstrate that different regulatory proteins affect enzyme activity and the response to agents that modify PP2A activity in vitro. Different PP2A heterotrimers are likely to have distinct functions in vivo, and changes in subunit composition will have an important impact on signal transduction pathways.