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The Rho GTPase signaling pathway is required for actin cytoskeletal organization and serum response factor-dependent gene transcription. Lbc is a Rho-specific guanine nucleotide exchange factor that contains a modulatory C-terminal region. To elucidate Lbc regulatory mechanism(s), a yeast two-hybrid screen for proteins that interact with the Lbc C-terminal region was carried out, resulting in multiple isolation of cDNAs encoding the same 734-amino acid Lbc interacting protein. The Lbc interacting protein has homology with the ␣-catenin cell adhesion component and is identical to the ␣-catenin-like ␣-catulin protein of unknown function. The human ␣-catulin gene (CTNNAL1) maps to 9q31-32. Here we identify the predicted endogenous ␣-catulin product, document ␣-catulin and Lbc co-expression in multiple human cell lines, and show ␣-catulin and Lbc subcellular co-fractionation and intracellular localization. The required regions for Lbc and ␣-catulin interaction were mapped, and complex formation between Lbc and ␣-catulin in mammalian cells was detected. Functionally, ␣-catulin co-expression leads to increased Lbc-induced serum response factor activation in vivo as measured by a transcriptional reporter assay. Furthermore, ␣-catulin co-expression enhances Lbcinduced GTP-Rho formation in vivo. These results support the concept that the recently identified ␣-catulin protein may modulate Rho pathway signaling in vivo by providing a scaffold for the Lbc Rho guanine nucleotide exchange factor.

The human lbc oncogene product is a guanine nucleotide exchange factor that specifically activates the Rho small GTP binding protein, thus resulting in biologically active, GTP-bound Rho, which in turn mediates actin cytoskeletal reorganization, gene transcription, and entry into the mitotic S phase. In order to elucidate the mechanism of onco-Lbc transformation, here we report that while proto-and onco-lbc cDNAs encode identical N-terminal dbl oncogene homology (DH) and pleckstrin homology (PH) domains, proto-Lbc encodes a novel C terminus absent in the oncoprotein that includes a predicted ␣-helical region homologous to cyto-matrix proteins, followed by a proline-rich region. The lbc proto-oncogene maps to chromosome 15, and onco-lbc represents a fusion of the lbc proto-oncogene N terminus with a short, unrelated C-terminal sequence from chromosome 7. Both onco-and proto-Lbc can promote formation of GTP-bound Rho in vivo. Proto-Lbc transforming activity is much reduced compared to that of onco-Lbc, and a significant increase in transforming activity requires truncation of both the ␣-helical and proline-rich regions in the proto-Lbc C terminus. Deletion of the chromosome 7-derived C terminus of onco-Lbc does not destroy transforming activity, demonstrating that it is loss of the proto-Lbc C terminus, rather than gain of an unrelated C-terminus by onco-Lbc, that confers transforming activity. Mutations of onco-Lbc DH and PH domains demonstrate that both domains are necessary for full transforming activity. The proto-Lbc product localizes to the particulate (membrane) fraction, while the majority of the onco-Lbc product is cytosolic, and mutations of the PH domain do not affect this localization. The proto-Lbc C-terminus alone localizes predominantly to the particulate fraction, indicating that the C terminus may play a major role in the correct subcellular localization of proto-Lbc, thus providing a mechanism for regulating Lbc oncogenic potential.The family of DH (dbl oncogene homology) domain-encoding oncogenes (8, 40) represents a unique category of transforming genes involved in cellular growth control. The DH domain is associated with guanine nucleotide exchange activation for the Rho/Rac family of small GTP binding proteins (8), resulting in the conversion of the inactive, GDP-bound form of the GTPase to the active, GTP-bound form capable of transducing signals (5,14). In all cases, the DH domain is followed by a pleckstrin homology (PH) domain (5, 34) which can have multiple functions. Thus, these catalytic GDP-GTP exchange factors (GEFs) play a key role in regulating the Rho/Rac GTPase cycle. The Rho/Rac family of small GTPases mediates cytoskeletal reorganization (15), gene transcription (20), and cell cycle progression (36) through unique signal transduction pathways.The 424-amino-acid Lbc oncoprotein is transforming both in vivo and in vitro and contains an N-terminal EF hand motif followed by DH and PH domains (49). We have shown that onco-Lbc activates the Rho small GTP binding protein by catalytica...

Members of the Dbl-homology (DH) family of proteins promote guanine nucleotide exchange on Rho GTPases. Lbc, which speci®cally acts on Rho but not Rac or Cdc42, was isolated as a transforming oncogene and is composed of a DH and a Pleckstrin-homology (PH) domain. We show here that the Lbc DH domain alone is capable of stimulating new DNA synthesis in quiescent ®broblasts and Rho-dependent actin stress ®ber assembly. However, the PH domain is required for subcellular localization of Lbc along actin stress ®bers and for ecient transformation of NIH3T3 cells. The results show that, in contrast to other Dbl-homology proteins, the PH domain of Lbc is dispensable for activation of Rho in vivo. The PH domain-dependent subcellular localization of Lbc may, however, be important for growth factor activation of endogenous Lbc and for oncogenic transformation.