Direct activation of phospholipase C-epsilon by Rho.
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.
Wing
Michele R
MR
Department of Pharmacology, the Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Snyder
Jason T
JT
Sondek
John
J
Harden
T Kendall
TK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D054801:Phosphoinositide Phospholipase C; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012984:Software; D010738:Type C Phospholipases / Q000378:metabolism*; D020687:cdc25 Phosphatases / Q000378:metabolism; D020668:rap GTP-Binding Proteins / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D007295:Inositol Phosphates; D020033:Protein Isoforms; D005982:Glutathione Transferase; D020687:cdc25 Phosphatases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; D020668:rap GTP-Binding Proteins; D018631:ras Proteins; D020741:rho GTP-Binding Proteins