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Molecular and Cellular Biology, August 2005, p. 7289-7302, Vol. 25, No. 16
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.16.7289-7302.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Regulation of Neurite Outgrowth in N1E-115 Cells through PDZ-Mediated Recruitment of Diacylglycerol Kinase {zeta}

Yury Yakubchyk,1,3 Hanan Abramovici,1,3 Jean-Christian Maillet,1,3 Elias Daher,1,3 Christopher Obagi,1,3 Robin J. Parks,2,3,4 Matthew K. Topham,5 and Stephen H. Gee1,3*

Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada,1 Molecular Medicine Program, Ottawa Health Research Institute, Ottawa, Ontario K1H 8L6, Canada,2 Centre for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario, Canada,3 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada,4 Huntsman Cancer Institute and Department of Internal Medicine, University of Utah, Salt Lake City, Utah 841125

Received 20 February 2004/ Returned for modification 6 April 2004/ Accepted 19 May 2005

Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase {zeta} (DGK-{zeta}), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-{zeta} and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-{zeta} in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-{zeta} kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-{zeta} mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-{zeta} promotes neurite outgrowth through association with the GTPase Rac1. DGK-{zeta} colocalized with Rac1 in neuronal processes and DGK-{zeta}-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-{zeta} directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-{zeta} mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1V12) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-{zeta} with Rac1V12, suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-{zeta} binding to active Rac1. Collectively, these findings suggest DGK-{zeta}, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells.

* Corresponding author. Mailing address: Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario K1H 8M5, Canada. Phone: (613) 562-5800, ext. 8079. Fax: (613) 562-5645. E-mail: stevegee{at}uottawa.ca.

Molecular and Cellular Biology, August 2005, p. 7289-7302, Vol. 25, No. 16
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.16.7289-7302.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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