RACK1, a Receptor for Activated C Kinase and a Homolog of the β Subunit of G Proteins, Inhibits Activity of Src Tyrosine Kinases and Growth of NIH 3T3 Cells

Abstract

To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the ␤ subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 M) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested. Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G 0 /G 1 than do wild-type cells. Prolongation of G 0 /G 1 could account for the increased doubling time of RACK1-overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.The cellular gene c-src and its viral homolog v-src (the transforming gene of Rous sarcoma virus) encode 60-kDa, cytoplasmic, membrane-associated protein-tyrosine kinases (reviewed in reference 6). For the viral protein (v-Src) or for transforming mutants of the cellular protein (c-Src or Src), a close correlation exists between elevated specific kinase activity and cell transformation. At least four factors are known to influence Src activity: (i) mutation within the coding region of the src gene, (ii) phosphorylation on Tyr 527 and Tyr 416 of Src, (iii) subcellular localization of Src and its substrates, and (iv) association of Src with other cellular proteins.Compelling evidence indicates that Src-binding proteins can regulate Src activity (reviewed in reference 6). A number of interacting proteins that upregulate Src activity have been identified. The prototype is middle T antigen (mT), the transforming protein of polyomavirus. Src complexed with mT has elevated specific activity due to dephosphorylation of Tyr 527 (5,8,11,16). Activation of Src is required for the induction of mammary tumors in mT transgenic mice (24). Characterization of the Src-mT complex led to discovery of the fundamental mechanism by which the cellular Src protein is converted to a transforming protein and defined the requirement of Src for polyomavirus transformation. Thus, characterization of a single Src-binding protein contributed substantially to our understanding of both RNA and DNA tumor virology.While a number of interacting proteins that upregulate Src activity have been identified, few that downregulate Src activity have been identifi...

Three‐dimensional fibroblast–tumor cell interaction causes downregulation of RACK1 mRNA expression in breast cancer cells in vitro

“…14 Accordingly, simultaneous translocation of Rack1 with ␤IIPKC after activation to its subcellular site of action was documented, indicating that Rack1 functions as a shuttle protein for the PKC enzyme to reach close proximity to its appropriate substrate. 29 Rack1 also interacts with a number of other proteins, e.g., phospholipase C␥, 30 Src, 31 dynamin, 32 integrin ␤ subunit, 33 cAMP-specific phosphodiesterase PDE4D5 isoform, 34 p65 synaptotagmin, 35 type I IFN receptor 36 or p120 GAP . 37 Here, its WD40 repeat structure interacts with SH2 domains (e.g., in Src and phospholipase C␥) or with the pleckstrin homology domain (e.g., in p120 GAP ).…”

RACK1 inhibits the serum‐ and anchorage‐independent growth of v‐Src transformed cells

“…We identified RACK1 as a Src substrate and an inhibitor of c-Src kinase activity and NIH 3T3 cell growth [7][8][9]. However, it is not yet known what influence RACK1 has on v-Src kinase activity or cell transformation.…”

“…NIH(pmvsrc/foc/EP)A1 cells were transfected with 1 lg pcDNA3-HA-RACK1 or pcDNA3 using Lipofectamine (Gibco-BRL) according to the manufacturer's protocol and as previously described [7]. Colonies were selected for neomycin resistance with the addition of G418 (400 lg/ml) and subclones were examined for stable expression of HA-RACK1 by immunoblotting with anti-RACK1 [2,7].…”

“…Lysate proteins or immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting with the specified mouse MAb or rabbit polyclonal antibody, as previously described [2,[7][8][9]. Methods to evaluate the phosphorylating activity of Src by kinase assays in vitro have also been described [2,[7][8][9].…”

“…Construction of pcDNA3-RACK1 and pcDNA3-HA-RACK1 (by insertion of the influenza virus hemagglutinin (HA) tag into the BglII site of pcDNA3-RACK1) has been previously described [7]. NIH(pmvsrc/foc/EP)A1 cells were transfected with 1 lg pcDNA3-HA-RACK1 or pcDNA3 using Lipofectamine (Gibco-BRL) according to the manufacturer's protocol and as previously described [7].…”