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J. Biol. Chem., Vol. 279, Issue 46, 47992-47997, November 12, 2004

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RhoA Activates Purified Phospholipase C- by a Guanine Nucleotide-dependent Mechanism^*

Jason P. Seifert, Michele R. Wing, Jason T. Snyder, Svetlana Gershburg, John Sondek¶||, and T. Kendall Harden**

From the Department of Pharmacology, the Department of Biochemistry and Biophysics, and ^¶Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Phospholipase C- (PLC-) is a recently identified PLC isoform activated by subunits of heterotrimeric G proteins (G₁₂, G₁₃, and G) as well as by the low molecular weight GTPases, Rho and Ras. To define the enzymatic activity and substrate specificity of PLC- as well as its potential direct activation by Rho family GTPases, a major fragment of PLC- encompassing the catalytic core (EF-hand repeats through the tandem Ras-associating domains; 118 kDa) was purified to near homogeneity and assayed after reconstitution under various conditions. Similar to the enzymatic profiles of previously purified PLC- isozymes, the purified fragment of PLC- maximally hydrolyzed phosphatidylinositol 4-phosphate at a rate of 10 µmol/mg of protein/min, exhibited phospholipase activity dependent on the concentration of free calcium, and favored phosphatidylinositol 4,5-bisphosphate as substrate relative to other phosphoinositides. Furthermore, in mixed detergent phospholipid micelles, RhoA stimulated the phospholipase activity of the PLC- fragment in both a concentration-dependent and guanosine 5'-O-(3-thiotriphosphate)-dependent manner. This activation was abolished by the deletion of a unique 65 amino acid-insert within the catalytic core of PLC-. Although Rac1 activated purified PLC-2ina guanine nucleotide-dependent manner, Rac1 failed to promote guanine nucleotide-dependent activation of purified PLC-. These results indicate that PLC- is a direct downstream effector for RhoA and that RhoA-dependent activation of PLC- depends on a unique insert within the catalytic core of the phospholipase.

Received for publication, June 24, 2004 , and in revised form, August 17, 2004.

^* This work was supported in part by National Institutes of Health Grants GM29316, GM57391, and GM65533. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Supported by the Pew Charitable Trusts.

^** To whom correspondence should be addressed: University of North Carolina, School of Medicine, Dept. of Pharmacology, CB# 7365, Chapel Hill, NC 27599-7365. E-mail: tkh{at}med.unc.edu.

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