Direct Activation of Phospholipase C-ϵ by Rho

Michele R Wing,Jason T Snyder,John Sondek,T.Kendall Harden

doi:10.1074/jbc.m306904200

Michele R Wing, Jason T Snyder + Show 2 more

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https://doi.org/10.1074/jbc.m306904200

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Abstract

Unique among the phospholipase C isozymes, the recently identified phospholipase C-epsilon (PLC-epsilon) 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-epsilon, 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-epsilon-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-epsilon with the known regulators Ras, Galpha12/13, or Gbeta1gamma2. 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-epsilon to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Galpha12/13, or Gbeta1gamma2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique approximately 65 amino acid insert within the catalytic core of PLC-epsilon not present in PLC-beta, gamma, delta, or zeta. A PLC-epsilon construct lacking this region was no longer activated by Rho or Galpha12/13 but retained regulation by Gbetagamma and H-Ras. GTP-dependent interaction of Rho with PLC-epsilon was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-epsilon construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-epsilon by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-epsilon imparts responsiveness to Rho, which may signal downstream of Galpha12/13 in the regulation of PLC-epsilon, because activation by both Rho and Galpha12/13 is lost in the absence of this sequence.

Highlights

Unique among the phospholipase C isozymes, the recently identified phospholipase C-⑀ (PLC-⑀) 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
Activation of Phospholipase C (PLC)-⑀ by the Small GTPase Rho—Previous studies illustrated that H-Ras and Rap isozymes interact with the Ras-associating domains of PLC-⑀ to stimulate phospholipase activity [16, 18]
Transfection of PLC-⑀ alone resulted in a small increase in [3H]inositol phosphates that was substantially augmented by cotransfection with G␤1␥2 or GTPase-deficient mutants of G␣12, G␣13, or H-Ras (Fig 1A)

Summary

Introduction

Unique among the phospholipase C isozymes, the recently identified phospholipase C-⑀ (PLC-⑀) 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. A PLC-⑀ construct lacking this region was no longer activated by Rho or G␣12/13 but retained regulation by G␤␥ and H-Ras. GTP-dependent interaction of Rho with PLC-⑀ was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-⑀ construct lacking the unique insert within the catalytic core. GTP-dependent interaction of Rho with PLC-⑀ was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-⑀ construct lacking the unique insert within the catalytic core These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-⑀ by a mechanism independent of the CDC25 or RA domains. In vitro binding experiments illustrate a GTP-dependent and a Y box insert-independent interaction of RhoA with PLC-⑀

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