Westphal R S, Coffee R L, Marotta A, Pelech S L, Wadzinski B E
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600, USA.
J Biol Chem. 1999 Jan 8;274(2):687-92. doi: 10.1074/jbc.274.2.687.
A growing body of evidence indicates that regulation of protein-serine/threonine phosphatase 2A (PP2A) involves its association with other cellular and viral proteins in multiprotein complexes. PP2A-containing protein complexes may exist that contribute to PP2A's important regulatory role in many cellular processes. To identify such protein complexes, PP2A was partially purified from rat brain soluble extracts following treatment with a reversible cross-linker to stabilize large molecular size forms of PP2A. Compared with native (uncross-linked) PP2A, cross-linked PP2A revealed an enrichment of p70 S6 kinase and two p21-activated kinases (PAK1 and PAK3) in the PP2A complex, indicating these kinases may associate with PP2A. The existence of protein kinase-PP2A complexes in rat brain soluble extracts was further substantiated by the following results: 1) independent immunoprecipitation of the kinases revealed that PP2A co-precipitated with p70 S6 kinase and the two PAK isoforms; 2) glutathione S-transferase fusion proteins of p70 S6 kinase and PAK3 each isolated PP2A; and 3) PAK3 and p70 S6 kinase bound to microcystin-Sepharose (an affinity resin for PP2A-PP1). Cumulatively, these findings provide evidence for association of PP2A with p70 S6 kinase, PAK1, and PAK3 in the context of the cellular environment. Moreover, together with the recent reports describing associations of PP2A with Ca2+/calmodulin-dependent protein kinase IV (Westphal, R. S., Anderson, K. A., Means, A. R., and Wadzinski, B. E. (1998) Science 280, 1258-1261) and casein kinase IIalpha (Heriche, J. K., Lebrin, F., Rabilloud, T., Leroy, D., Chambaz, E. M., and Goldberg, Y. (1997) Science 276, 952-955), the present data provide compelling evidence for the existence of protein kinase-PP2A signaling modules as a new paradigm for the control of various intracellular signaling cascades.
越来越多的证据表明，蛋白质丝氨酸/苏氨酸磷酸酶2A（PP2A）的调节涉及它在多蛋白复合物中与其他细胞和病毒蛋白的结合。可能存在含PP2A的蛋白复合物，它们在许多细胞过程中对PP2A的重要调节作用有贡献。为了鉴定这样的蛋白复合物，在用可逆交联剂处理大鼠脑可溶性提取物后对PP2A进行了部分纯化，以稳定大分子形式的PP2A。与天然（未交联）PP2A相比，交联的PP2A显示PP2A复合物中富含p70 S6激酶和两种p21激活激酶（PAK1和PAK3），表明这些激酶可能与PP2A结合。大鼠脑可溶性提取物中存在蛋白激酶-PP2A复合物通过以下结果得到进一步证实：1）对激酶进行独立免疫沉淀显示PP2A与p70 S6激酶和两种PAK同工型共沉淀；2）p70 S6激酶和PAK3的谷胱甘肽S-转移酶融合蛋白各自分离出PP2A；3）PAK3和p70 S6激酶与微囊藻毒素-琼脂糖凝胶（一种PP2A-PP1亲和树脂）结合。总的来说，这些发现为细胞环境中PP2A与p70 S6激酶、PAK1和PAK3的结合提供了证据。此外，连同最近描述PP2A与Ca2+/钙调蛋白依赖性蛋白激酶IV（Westphal，R.S.，Anderson，K.A.，Means，A.R.，和Wadzinski，B.E.（1998）Science 280，1258 - 1261）以及酪蛋白激酶IIα（Heriche，J.K.，Lebrin，F.，Rabilloud，T.，Leroy，D.，Chambaz，E.M.，和Goldberg，Y.（1997）Science 276，952 - 955）结合的报道，目前的数据为存在蛋白激酶-PP2A信号模块作为控制各种细胞内信号级联反应的新范例提供了令人信服的证据。
