Degree Type

Dissertation

Date of Award

2004

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

F. A. Norris

Abstract

Inositol polyphosphate 4-phosphatases (IP4Ps) are enzymes that catalyze the hydrolysis of the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate to form phosphatidylinositol 3-phosphate. Database searches for proteins similar to IP4Ps led to the identification of two novel proteins: P-Rex1 and P-Rex2. These proteins, in addition to sharing similarity with IP4Ps, had a Dbl homology (DH) domain followed by a pleckstrin homology (PH) domain, a characteristic feature of the Dbl family of guanine nucleotide exchange factors (GEFs). P-Rex1 was recently isolated from neutrophils, where it plays a key role in stimulating the neutrophil oxidative burst by activating Rac, a component of the NADPH oxidase complex. Characterization of P-Rex2 revealed that it shares 55% amino acid identity with P-Rex1, and a common but unusual domain architecture with N-terminal DH/PH domains, two DEP domains, two PDZ domains and a C-terminal IP4P-like domain. Furthermore, we identified P-Rex2B an alternate splice variant of P-Rex2 that lacks the IP4P-like domain. Despite its 28% amino acid identity with IP4Ps, phosphatase activity could not be demonstrated for P-Rex2. P-Rex2 was shown to be a GEF specific for Rac, and its GEF activity is conditional on the presence of phosphatidylinositol 3,4,5-trisphosphate or the betagamma subunits of heterotrimeric G proteins, both in vitro and in vivo. Both the lipid and protein effectors bound the PH domain of P-Rex2. Analysis of the interaction of P-Rex2 with Rac revealed the novel and surprising finding that the PH domain of P-Rex2 confers substrate (Rac) recognition and specificity. This function has typically been executed by the DH domain and has never before been attributed to the PH domain of Dbl family GEFs. While DEP domains are domains of unknown function, PDZ domains are domains involved in protein-protein interaction. Binding studies with full-length P-Rex2 revealed an intra-molecular interaction between the PDZ domains and the DEP and PH domains. This interaction obscures the Rac binding site on the PH domain of P-Rex2, and provides an explanation for the inability of P-Rex2 to catalyze exchange on Rac in the absence of effectors. Moreover, it establishes the DEP domains as novel ligands for the PDZ domains.

DOI

https://doi.org/10.31274/rtd-180813-12942

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu

Copyright Owner

Raji Elizabeth Joseph

Language

en

Proquest ID

AAI3145654

File Format

application/pdf

File Size

144 pages

Included in

Biochemistry Commons

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