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J. Biol. Chem., Vol. 279, Issue 24, 25226-25233, June 11, 2004

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Requirement For C-terminal Sequences in Regulation of Ect2 Guanine Nucleotide Exchange Specificity and Transformation^*

Patricia A. Solski, Rhonda S. Wilder, Kent L. Rossman¶, John Sondek¶, Adrienne D. Cox, Sharon L. Campbell¶, and Channing J. Der||

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

Ect2 was identified originally as a transforming protein and a member of the Dbl family of Rho guanine nucleotide exchange factors (GEFs). Like all Dbl family proteins, Ect2 contains a tandem Dbl homology (DH) and pleckstrin homology (PH) domain structure. Previous studies demonstrated that N-terminal deletion of sequences upstream of the DH domain created a constitutively activated, transforming variant of Ect2 (designated N-Ect2 DH/PH/C), indicating that the N terminus served as a negative regulator of DH domain function in vivo. The role of sequences C-terminal to the DH domain has not been established. Therefore, we assessed the consequences of mutation of C-terminal sequences on Ect2-transforming activity. Surprisingly, in contrast to observations with other Dbl family proteins, we found that mutation of the invariant tryptophan residue in the PH domain did not impair N-Ect2 DH/PH/C transforming activity. Furthermore, although the sequences C-terminal to the PH domain lack any known functional domains or motifs, deletion of these sequences (N-Ect2 DH/PH) resulted in a dramatic reduction in transforming activity. Whereas N-Ect2 caused formation of lamellipodia, N-Ect2 DH/PH enhanced actin stress fiber formation, suggesting that C-terminal sequences influenced Ect2 Rho GTPase specificity. Consistent with this possibility, we determined that N-Ect2 DH/PH activated RhoA, but not Rac1 or Cdc42, whereas N-Ect2 DH/PH/C activated all three Rho GTPases in vivo. Taken together, these observations suggest that regions of Ect2 C-terminal to the DH domain alter the profile of Rho GTPases activated in vivo and consequently may contribute to the enhanced transforming activity of N-Ect2 DH/PH/C.

Received for publication, December 17, 2003 , and in revised form, March 17, 2004.

^* This work was supported by National Institutes of Health Grants CA63071 and CA92240 (to C. J. D.), CA67771 (to A. D. C.), GM62299 (to J. S.), and CA89614 (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.

^|| To whom correspondence should be addressed: University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599. Tel.: 919-966-5634; Fax: 919-966-0162; E-mail: cjder{at}med.unc.edu.

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