Substrate Specificity and Recognition Is Conferred by the Pleckstrin Homology Domain of the Dbl Family Guanine Nucleotide Exchange Factor P-Rex2

Raji E Joseph,F.A Norris

doi:10.1074/jbc.m412495200

Raji E Joseph, F.A Norris

Open Access

https://doi.org/10.1074/jbc.m412495200

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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.

Highlights

The Rho family of G proteins are members of the Ras superfamily [1, 2]
The Rac1W56F Mutation Does Not Affect P-Rex2 Binding— Comparison of the crystal structure complexes of Tiam1 and Rac1 with other Rho GTPase-guanine nucleotide exchange factors (GEFs) complexes has identified the Trp56 residue of Rac1 as a key determinant of specificity for Rac-specific GEFs [27]. This residue of Rac1 was shown to interact with a conserved Ile in the Dbl homology (DH) domain of Rac-specific GEFs
Because mutation of the Trp56 residue of Rac1 to Phe is sufficient to abolish binding by the DHPH domain of the Rac1specific GEF Tiam1, we decided to test whether this mutation had any effect on the binding ability of P-Rex2 [27]

Summary

The abbreviations used are

Diffuse B-cell lymphoma; GEF, guanine nucleotide exchange factor; DH, Dbl homology; PH, pleckstrin homology; GST, glutathione S-transferase; Dbs, big sister of Dbl; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate. The crystal structures of the Rac1-Tiam, Cdc42-Dbs, Dbs-RhoA, and Cdc42-Intersectin complexes show that the CR1 and CR3 regions of the DH domain of the GEF make extensive contact with the ␤2␤3 strands of the GTPases [25,26,27,28] These regions were predicted to determine the specificity of these interactions. Based on these crystal structure complexes, Trp of Rac was identified as one of the critical residues for recognition by Rac-specific GEFs [27] Mutation of this residue to Phe, the corresponding residue in Cdc, was sufficient to abolish binding by Tiam. Helix 3 of Rac is sufficient to confer binding to a chimeric GTPase, the helix by itself is not sufficient to allow P-Rex to catalyze nucleotide exchange This suggests that substrate recognition and catalysis by the P-Rex GEF have distinct requirements

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION