Direct Involvement of the Small GTP-binding Protein Rho in lbc Oncogene Function

doi:10.1074/jbc.270.16.9031

Direct Involvement of the Small GTP-binding Protein Rho in lbc Oncogene Function (*)

From the (1) Department of Pharmacology, Schurman Hall, Cornell University, Ithaca, New York 14853, the
(2) CRC Oncogene and Signal Transduction Group, Medical Research Council Laboratories for Molecular Cell Biology, University College, Gower Street, London WC1E, United Kingdom, and the
(3) Department of Hematology/Oncology, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115

↵^§ Current address: Dept. of Physiology, Tufts University, Boston, MA 02111.

Abstract

The lbc oncogene is tumorigenic in nude mice, transforms NIH 3T3 fibroblasts, and encodes a Dbl homology domain found in several transforming gene products including the dbl oncogene product. While both lbc- and dbl-transformed NIH 3T3 foci exhibited a comparable gross appearance, lbc-transformed cell morphology was clearly distinct from that of dbl-transformed cells. Given these differences, we investigated the biochemical activity and target specificity of the Lbc oncoprotein both in vivo and in vitro. Here we show that Lbc associates specifically with the GTP-binding protein Rho in vivo, but not with the Ras, Rac, or Cdc42Hs GTP-binding proteins, and that recombinant, affinity-purified Lbc specifically catalyzes the guanine-nucleotide exchange activity of Rho in vitro. Consistent with an in vivo role for Lbc in Rho regulation, we further demonstrate that micro-injected onco- lbc potently induces actin stress fiber formation in quiescent Swiss 3T3 fibroblasts indistinguishable from that induced by Rho. Finally, lbc-induced NIH 3T3 focus formation is inhibited by co-transfection with a rho dominant-negative mutant. These results strongly indicate that the lbc oncogene encodes a specific guanine nucleotide exchange factor for Rho and causes cellular transformation through activation of the Rho signaling pathway.

Footnotes

↵* This work was supported by Cancer Research Campaign (United Kingdom) funds (to A. H.), a Natural Sciences and Engineering Research Council of Canada fellowship (to M. F. O.), National Institutes of Health Grants GM47458 (to R. A. C.) and R29CA62029 (to D. T.), and an American Cancer Society Faculty Award (to D. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GEF

guanine nucleotide exchange factor

DH

Dbl homology domain

GST

glutathione S-transferase

Ras-GRF

the Ras guanine nucleotide releasing factor (i.e. a Ras-GEF) that is specific for brain

GTPS

guanosine 5′-3- O-(thio)triphosphate.