Direct Activation of Phospholipase C-ε by Rho

Abstract

Unique among the phospholipase C isozymes, the recently identified phospholipase C-ε (PLC-ε) contains an amino-terminal CDC25 domain capable of catalyzing nucleotide exchange on Ras family GTPases as well as a tandem array of Ras-associating (RA) domains near its carboxyl terminus that are effector binding sites for activated H-Ras and Rap. To determine whether other small GTPases activate PLC-ε, we measured inositol phosphate accumulation in COS-7 cells expressing a broad range of GTPase-deficient mutants of Ras superfamily proteins. RhoA, RhoB, and RhoC all markedly stimulated inositol phosphate accumulation in PLC-ε-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-ε with the known regulators Ras, Gα 12/13, or Gβ1γ2. In contrast, little effect was observed with the other Rho family members Rac1, Rac2, Rac3, and Cdc42. Truncation of the two carboxyl-terminal RA domains caused loss of responsiveness to H-Ras but not to Rho. Truncation of PLC-ε to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Gα12/13, or Gβ1γ 2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique ∼65 amino acid insert within the catalytic core of PLC-ε not present in PLC-β, γ, δ, or ζ. A PLC-ε construct lacking this region was no longer activated by Rho or Gα12/13 but retained regulation by Gβγ and H-Ras. GTP-dependent interaction of Rho with PLC-ε was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-ε construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-ε by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-ε imparts responsiveness to Rho, which may signal downstream of Gα12/13 in the regulation of PLC-ε, because activation by both Rho and Gα12/13 is lost in the absence of this sequence.