Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation

Ernesto J Fuentes; Antoine E Karnoub; Michelle A Booden; Channing J Der; Sharon L Campbell

doi:10.1074/jbc.M211792200

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Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation

Journal article

Open access

Peer reviewed

Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation

Ernesto J Fuentes, Antoine E Karnoub, Michelle A Booden, Channing J Der and Sharon L Campbell

The Journal of biological chemistry, Vol.278(23), pp.21188-21196

06/06/2003

DOI: 10.1074/jbc.M211792200

PMID: 12637530

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Abstract

Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo. In this study, we evaluated the role of phosphoinositide binding to the PH domain in regulating the DH domain function of Dbs in vitro and in vivo. We determined that mutation of basic amino acids located within the beta1-beta2 and beta3-beta4 loops of the PH domain resulted in impaired phospholipid binding in vitro, yet full guanine nucleotide exchange activity in vitro was retained for RhoA and Cdc42. Surprisingly, these mutants were compromised in their ability to activate Rho GTPases in vivo and to cause transformation of NIH 3T3 cells. However, Dbs subcellular localization was impaired by these PH domain mutations, supporting a role for phospholipid interactions in facilitating membrane association. Despite the importance of phospholipid binding for Dbs function in vivo, we found that Dbs signaling and transforming activity was not stimulated by phosphatidylinositol 3-kinase activation. We suggest that the PH domain of Dbs facilitates two distinct roles in the regulation of DH domain function, one critical for GTPase association and activation in vitro and one critical for phosphoinositide binding and GTPase interaction in vivo, that together promote Dbs association with membranes.

Protein Structure, Tertiary

Amino Acid Sequence

Catalytic Domain

Guanine Nucleotide Exchange Factors - genetics

Molecular Sequence Data

cdc42 GTP-Binding Protein - metabolism

rhoA GTP-Binding Protein - metabolism

Phosphoproteins - genetics

Cell Division - physiology

Stress Fibers - metabolism

Animals

GTP Phosphohydrolases - metabolism

Mutagenesis

Blood Proteins - genetics

Guanine Nucleotide Exchange Factors - metabolism

Phosphatidylinositols - metabolism

Signal Transduction - physiology

Cell Membrane - metabolism

Mice

In Vitro Techniques

Binding Sites

3T3 Cells

Details

Title: Subtitle: Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation
Creators: Ernesto J Fuentes - Department of Biochemistry and Biophysics, University of North Carolina, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599, USA
Antoine E Karnoub
Michelle A Booden
Channing J Der
Sharon L Campbell
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.278(23), pp.21188-21196
DOI: 10.1074/jbc.M211792200
PMID: 12637530
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Language: English
Date published: 06/06/2003
Academic Unit: Biochemistry and Molecular Biology
Record Identifier: 9984025395902771

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