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Molecular and Cellular Biology, October 2003, p. 6823-6835, Vol. 23, No. 19
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.19.6823-6835.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Functional Analysis of the Caenorhabditis elegans UNC-73B PH Domain Demonstrates a Role in Activation of the Rac GTPase In Vitro and Axon Guidance In Vivo

Terrance J. Kubiseski,1 Joe Culotti,1,2 and Tony Pawson1,2*

Samuel Lunenfeld Research Institute of Mt. Sinai Hospital, Toronto, Ontario M5G 1X5,1 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada2

Received 16 May 2003/ Returned for modification 17 May 2003/ Accepted 1 July 2003

The Caenorhabditis elegans UNC-73B protein regulates axon guidance through its ability to act as a guanine nucleotide exchange factor (GEF) for the CeRAC/MIG-2 GTPases. Like other GEFs for Rho family GTPases, UNC-73B has a Dbl homology (DH) catalytic domain, followed by a C-terminal pleckstrin homology (PH) domain. We have explored whether the PH domain cooperates with the adjacent DH domain to promote UNC-73B GEF activity and axonal pathfinding. We show that the UNC-73B PH domain binds preferentially to monophosphorylated phosphatidylinositides in vitro. Replacement of residues Lys1420 and Arg1422 with Glu residues within the PH domain impaired this phospholipid binding but did not affect the in vitro catalytic activity of the DH domain. In contrast, a mutant UNC-73B protein with a Trp1502-to-Ala substitution in the PH domain still interacted with phosphorylated phosphatidylinositides but had lost its GEF activity. UNC-73B minigenes containing these mutations were microinjected into C. elegans and transferred to unc-73(e936) mutant worms. Unlike the wild-type protein, neither PH domain mutant was able to rescue the unc-73 axon guidance defect. These results suggest that the UNC-73B PH domain plays distinct roles in targeting and promoting GEF activity towards the Rac GTPase, both of which are important for the directed movements of motorneurons in vivo.

* Corresponding author. Mailing address: Samuel Lunenfeld Research Institute of Mt. Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. Phone: (416) 586-8262. Fax: (416) 586-8869. E-mail: pawson{at}mshri.on.ca.

Molecular and Cellular Biology, October 2003, p. 6823-6835, Vol. 23, No. 19
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.19.6823-6835.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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