title: Substrate specificity and recognition is conferred by the pleckstrin homology domain of the Dbl family guanine nucleotide exchange factor P-Rex2.
abstract: Dbl family guanine nucleotide exchange factors (GEFs) are characterized by the presence of a catalytic Dbl homology domain followed invariably by a lipid-binding pleckstrin homology (PH) domain. To date, substrate recognition and specificity of this family of GEFs has been reported to be mediated exclusively via the Dbl homology domain. Here we report the novel and unexpected finding that, in the Dbl family Rac-specific GEF P-Rex2, it is the PH domain that confers substrate specificity and recognition. Moreover, the beta3beta4 loop of the PH domain of P-Rex2 is the determinant for Rac1 recognition, as substitution of the beta3beta4 loop of the PH domain of Dbs (a RhoA- and Cdc42-specific GEF) with that of P-Rex2 confers Rac1-specific binding capability to the PH domain of Dbs. The contact interface between the PH domain of P-Rex2 and Rac1 involves the switch loop and helix 3 of Rac1. Moreover, substitution of helix 3 of Cdc42 with that of Rac1 now enables the PH domain of P-Rex2 to bind this Cdc42 chimera. Despite having the ability to recognize this chimeric Cdc42, P-Rex2 is unable to catalyze nucleotide exchange on Cdc42, suggesting that recognition of substrate and catalysis are two distinct events. Thus substrate recognition can now be added to the growing list of functions that are being attributed to the PH domain of Dbl family GEFs.
authors.affiliation: Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
authors.forename: Raji E
authors.initials: RE
authors.lastname: Joseph
authors.forename: F A
authors.initials: FA
authors.lastname: Norris
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry*; D002384:Catalysis; D018076:DNA, Complementary / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry*; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013997:Time Factors; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism
chemical_list: D001798:Blood Proteins; D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C057811:MCF2 protein, human; C490712:PREX2 protein, human; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; C039077:platelet protein P47; D005982:Glutathione Transferase; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein