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J. Biol. Chem., Vol. 278, Issue 23, 21188-21196, June 6, 2003

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Critical Role of the Pleckstrin Homology Domain in Dbs Signaling and Growth Regulation^*

Ernesto J. Fuentes  , Antoine E. Karnoub ¶ ||, Michelle A. Booden ¶ **, Channing J. Der ¶  and Sharon L. Campbell  

From the Departments of Biochemistry and Biophysics and ^¶Pharmacology, University of North Carolina, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599

Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo. In this study, we evaluated the role of phosphoinositide binding to the PH domain in regulating the DH domain function of Dbs in vitro and in vivo. We determined that mutation of basic amino acids located within the ₁-₂ and ₃-₄ loops of the PH domain resulted in impaired phospholipid binding in vitro, yet full guanine nucleotide exchange activity in vitro was retained for RhoA and Cdc42. Surprisingly, these mutants were compromised in their ability to activate Rho GTPases in vivo and to cause transformation of NIH 3T3 cells. However, Dbs subcellular localization was impaired by these PH domain mutations, supporting a role for phospholipid interactions in facilitating membrane association. Despite the importance of phospholipid binding for Dbs function in vivo, we found that Dbs signaling and transforming activity was not stimulated by phosphatidylinositol 3-kinase activation. We suggest that the PH domain of Dbs facilitates two distinct roles in the regulation of DH domain function, one critical for GTPase association and activation in vitro and one critical for phosphoinositide binding and GTPase interaction in vivo, that together promote Dbs association with membranes.

Received for publication, November 19, 2002 , and in revised form, March 3, 2003.

^* This work was supported by National Institutes of Health Grants CA63071 (to C. J. D.) and CA84480 (to S. L. C). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Lineberger Comprehensive Cancer Center Training Grant from the National Institutes of Health; recipient of a National Science Foundation Minority Fellowship.

^|| Recipient of a Susan G. Komen Breast Cancer Foundation Postdoctoral Fellowship.

^** Supported by the Leukemia and Lymphoma Society.

To whom correspondence may be addressed: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Tel.: 919-966-5634; Fax: 919-966-0162; E-mail: cjder{at}med.unc.edu. To whom correspondence may be addressed: Dept. of Biochemistry and Biophysics, 530 Mary Ellen Jones Bldg., CB 7260, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-7210; Fax: 919-966-2852; E-mail: campbesl{at}med.unc.edu.

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