Ron D, Chen C H, Caldwell J, Jamieson L, Orr E, Mochly-Rosen D
Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305-5332.
Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):839-43. doi: 10.1073/pnas.91.3.839.
Protein kinase C (PKC) translocates from the soluble to the cell particulate fraction on activation. Intracellular receptors that bind activated PKC in the particulate fraction have been implicated by a number of studies. Previous work identified 30- to 36-kDa proteins in the particulate fraction of heart and brain that bound activated PKC in a specific and saturable manner. These proteins were termed receptors for activated C-kinase, or RACKs. In the following study, we describe the cloning of a cDNA encoding a 36-kDa protein (RACK1) that fulfills the criteria for RACKs. (i) RACK1 bound PKC in the presence of PKC activators, but not in their absence. (ii) PKC binding to the recombinant RACK1 was not inhibited by a pseudosubstrate peptide or by a substrate peptide derived from the pseudosubstrate sequence, indicating that the binding did not reflect simply PKC association with its substrate. (iii) Binding of PKC to RACK1 was saturable and specific; two other protein kinases did not bind to RACK1. (iv) RACK1 contains two short sequences homologous to a PKC binding sequence previously identified in annexin I and in the brain PKC inhibitor KCIP. Peptides derived from these sequences inhibited PKC binding to RACK1. Finally, RACK1 is a homolog of the beta subunit of G proteins, which were recently implicated in membrane anchorage of the beta-adrenergic receptor kinase [Pitcher, J., Inglese, L., Higgins, J. B., Arriza, J. A., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G. & Lefkowitz, R. J. (1992) Science 257, 1264-1267]. Our in vitro data suggest a role for RACK1 in PKC-mediated signaling.
蛋白激酶C(PKC)在激活时从可溶性部分转位至细胞颗粒部分。多项研究表明,细胞内存在能与颗粒部分中活化的PKC结合的受体。此前的研究在心脏和大脑的颗粒部分鉴定出了30至36 kDa的蛋白质,这些蛋白质能以特异性和可饱和的方式结合活化的PKC。这些蛋白质被称为活化C激酶受体(RACKs)。在接下来的研究中,我们描述了一种编码36 kDa蛋白质(RACK1)的cDNA的克隆,该蛋白质符合RACKs的标准。(i)在PKC激活剂存在的情况下,RACK1能结合PKC,而在其不存在时则不能。(ii)PKC与重组RACK1的结合不受假底物肽或源自假底物序列的底物肽的抑制,这表明这种结合并非仅仅反映PKC与其底物的关联。(iii)PKC与RACK1的结合是可饱和且特异性的;另外两种蛋白激酶不与RACK1结合。(iv)RACK1包含两个与先前在膜联蛋白I和脑PKC抑制剂KCIP中鉴定出的PKC结合序列同源的短序列。源自这些序列的肽能抑制PKC与RACK1的结合。最后,RACK1是G蛋白β亚基的同源物,最近有研究表明G蛋白β亚基与β - 肾上腺素能受体激酶的膜锚定有关[Pitcher, J., Inglese, L., Higgins, J. B., Arriza, J. A., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G. & Lefkowitz, R. J. (1992) Science 257, 1264 - 1267]。我们的体外数据表明RACK1在PKC介导的信号传导中发挥作用。