Requirement for C-terminal sequences in regulation of Ect2 guanine nucleotide exchange specificity and transformation

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Abstract

Ect2 was identified originally as a transforming protein and a member of the Dbl family of Rho guanine nucleotide exchange factors (GEFs). Like all Db1 family proteins, Ect2 contains a tandem Db1 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 ΔN-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 Db1 family proteins, we found that mutation of the invariant tryptophan residue in the PH domain did not impair ΔN-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 (ΔN-Ect2 DH/PH) resulted in a dramatic reduction in transforming activity. Whereas ΔN-Ect2 caused formation of 1a-mellipodia, ΔN-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 ΔN-Ect2 DH/PH activated RhoA, but not Rac1 or Cdc42, whereas ΔN-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 ΔN-Ect2 DH/PH/C.

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Solski, P. A., Wilder, R. S., Rossman, K. L., Sondek, J., Cox, A. D., Campbell, S. L., & Der, C. J. (2004). Requirement for C-terminal sequences in regulation of Ect2 guanine nucleotide exchange specificity and transformation. Journal of Biological Chemistry, 279(24), 25226–25233. https://doi.org/10.1074/jbc.M313792200

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