FAST TRACK RACK1 regulates Ki-Ras-mediated signaling and morphological transformation of NIH 3T3 cells Bodil Bjørndal 1 *   , Line M. Myklebust 1  , Ken Roger Rosendal 1 , Frøydis D. Myromslien 1 , James B. Lorens 2 , Garry Nolan 3 , Ove Bruland 4 and Johan R. Lillehaug 1 1 Department of Molecular Biology, University of Bergen, Bergen, Norway 2 Department of Biomedicine, University of Bergen, Bergen, Norway 3 Department of Immunology and Microbiology, Stanford University School of Medicine, Stanford, CA 4 Department of Medical Genetics, Haukeland University Hospital, Bergen, Norway Activating Ras mutations are involved in a significant fraction of human tumors. A suppressor screen using a retroviral mouse fibroblast cDNA library was performed to identify novel factors in Ras-mediated transformation. We identified a novel potent inhibi- tor of Ras-mediated morphological transformation encoded by a truncated version of the receptor for activated C-kinase (RACK1). The truncated protein, designated RACK1DWD1, lacked the N-terminal 49 amino acids encoding the first of the 7 WD40 repeats in RACK1. RACK1DWD1 expression restored con- tact inhibition, stress fiber formation and reduced ERK phospho- rylation in Ki-Ras transformed NIH 3T3 cells. We demonstrate that truncated RACK1 is involved in complexes consisting of wild- type RACK1 and protein kinase C isoforms a, bI and d, compro- mising the transduction of an activated Ras signal to the Raf- MEK-ERK pathway. The cellular localization of RACK1DWD1 differed from wtRACK1, indicating that signaling complexes con- taining the truncated version of RACK1 are incorrectly localized. Notably, 12-O-tetradecanoyl-13-phorbol acetate (TPA) mediated intracellular translocation of RACK1-interacting PKC a and d was abrogated in RACK1DWD1-expressing cells. Our data sup- port a model where RACK1 acts as a key factor in Ki-Ras-medi- ated morphological transformation. ' 2006 Wiley-Liss, Inc. Key words: ERK; PKC; RACK1; Ras; transformation Members of the ras gene family (Ki-ras, Ha-ras and N-ras) are structurally related and encode proteins (p21) known to play an important role in the regulation of normal signal transduction and cell growth. A range of ras mutations are present with high fre- quency in different human tumors. The mechanism by which mutated Ras contributes to cancer development and morphological transformation of cells is still not completely understood. Ras gen- erates downstream effects through Raf, Rac and Rho affecting cell growth, lamellipodia formation and stress fiber formation, respec- tively. 1–3 Activated Ras recruits Raf-1 from the cytosol to the cell membrane, where Raf-1 activation takes place through dephos- phorylation of inhibitory sites by protein phosphatase 2A (PP2A) as well as the phosphorylation of activating sites by a range of ki- nases. Activated Raf-1 phosphorylates and activates MEK (ERK/ MAPK kinase), which in turn phosphorylates and activates extrac- ellular-signal-regulated kinase (ERK/MAPK). Activated ERK has many substrates in the cytosol, and in the nucleus it controls gene expression by phosphorylating transcription factors such as Elk-1 and other Ets-family proteins. 4–6 The activation of the ERK/ MAPK pathway is central in Ras signaling, and expression of active ERK is sufficient to transform 3T3 cells. 7 Importantly, mutations in B-Raf are frequently observed in human cancers (melanomas). In addition, the c-Jun N-terminal kinase (JNK)/ MAPK pathway, 8,9 the PI3-K/Akt 10–13 and Src 14,15 pathways have been linked to cellular transformation, while the p38 MAPK path- way provides negative feedback of Ras transformation by block- ing activation of JNK. 16 Oncogenic Ras signaling can be blocked by inhibiting Ras pal- mitoylation and farnesylation. 17 Dominant-negative mutations of Raf-1 18 and MEK 19 effectively block Ras oncogenicity. Similarly, the membrane-anchored glycoprotein RECK can function down- stream of Ras to block Ras-mediated morphological transforma- tion. 20,21 Spry2 of the Sprouty family of human homologs to dro- sophila Sprouty was shown to block Ras signaling by inhibiting Raf activation. 22 In an attempt to identify and characterize novel factors inhibiting the transforming signal of mutated Ki-Ras, we used a retroviral cDNA expression screen in NIH 3T3 cells. Here, we show that a truncated version of receptor for activated C-ki- nase (RACK1) is able to block the Ki-Ras transforming signal. The importance of correct spatial and temporal organization of the individual components in signal complexes is increasingly rec- ognized. Based on homology to the G b -subunit of heterotrimeric G-proteins, the 7 WD40 repeats of RACK1 are postulated to form a propeller structure, where the blades potentially bind different proteins. 23,24 This will provide a platform for functional specificity and bring together components of one or several signaling path- ways. It has recently been shown that dimerized RACK1 binds activated Ras and affects Ras-dependent signaling. 25 Importantly, RACK1 binds a subset of protein kinase C (PKC) isoforms in their activated form and is thus essential in increasing the PKC sub- strate specificity. 26–28 Here, we demonstrate that the truncated form of RACK1 lack- ing the first WD40 repeat is an effective inhibitor of Ki-Ras-medi- ated transformation of 3T3 cells and restores cellular contact inhi- bition and actin fiber formation. Our studies show that the MAP kinase pathway is important in Ki-Ras-mediated morphological transformation, and that the activation of ERK by mutated Ki-Ras is significantly decreased in cells coexpressing N-terminally trun- cated RACK1. In addition, translocation of PKC, ERK phospho- rylation and filopodia-formation in response to 12-O-tetradeca- noyl-13-phorbol acetate (TPA) treatment are inhibited by trun- cated RACK1. We further demonstrate that RACK1DWD1 interacts with wtRACK1 and PKC isoforms and inhibits the cellu- lar translocation of PKC in response to TPA. Our present findings   The first 2 authors contributed equally to this work. Grant sponsors: Norwegian Cancer Society; Norwegian Research Council; The Locus on Cancer Research, Faculty of Medicine, University of Bergen. *Correspondence to: Department of Molecular Biology, University of Bergen, 5020 Bergen, Norway. Fax: 147-55-58-96-83. E-mail: Received 3 July 2006; Accepted after revision 31 August 2006 DOI 10.1002/ijc.22373 Published online 5 December 2006 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: ERK/MAPK, extracellular signal-regulated kinase/mito- gen-activated protein kinase; JNK, c-Jun N-terminal kinase; MEK, mito- gen-activated protein/extracellular signal-regulated kinase; pERK, phos- phorylated ERK; PI3-K, phosphatidylinositol-3-OH kinase; PKC, protein kinase C; PLD, phospholipase D; RACK1, receptor for activated C-kinase; RT-PCR, reverse transcriptase polymerase chain reaction; TPA, 12-O-tet- radecanyl phorbol-13-acetate (phorbol ester); SDS-PAGE, SDS-polyacry- lamid gel electrophoresis. Int. J. Cancer: 120, 961–969 (2006) ' 2006 Wiley-Liss, Inc. Publication of the International Union Against Cancer