Dual activation of phospholipase C-epsilon by Rho and Ras GTPases

J Biol Chem. 2008 Oct 31;283(44):29690-8. doi: 10.1074/jbc.M805038200. Epub 2008 Sep 2.

Abstract

Phospholipase C-epsilon (PLC-epsilon) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-epsilon to stimulate its phospholipase activity. Purified PLC-epsilon was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P(2)-containing phospholipid vesicles. Whereas mutation of two critical lysine residues within the second Ras-association domain of PLC-epsilon prevented K-Ras-dependent activation of the purified enzyme, guanine nucleotide-dependent activation by RhoA was retained. Deletion of a loop unique to PLC-epsilon eliminated its activation by RhoA but not H-Ras. In contrast, removal of the autoinhibitory X/Y-linker region of the catalytic core of PLC-epsilon markedly activates the enzyme (Hicks, S. N., Jezyk, M. R., Gershburg, S., Seifert, J. P., Harden, T. K., and Sondek, J. (2008) Mol. Cell, 31, 383-394), but PLC-epsilon lacking this regulatory region retained activation by both Rho and Ras GTPases. Additive activation of PLC-epsilon by RhoA and K- or H-Ras was observed in intact cell studies, and this additivity was recapitulated in experiments in which activation of purified PLC-epsilon was quantified with PtdIns(4,5)P(2)-containing phospholipid vesicles reconstituted with purified, isoprenylated GTPases. A maximally effective concentration of activated RhoA also increased the sensitivity of purified PLC-epsilon to activation by K-Ras. These results indicate that PLC-epsilon can be directly and concomitantly activated by both RhoA and individual Ras GTPases resulting in diverse upstream control of signaling cascades downstream of PLC-epsilon.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Enzyme Activation
  • Gene Expression Regulation, Enzymologic
  • Genes, ras
  • Lysine / chemistry
  • Models, Biological
  • Mutation
  • Open Reading Frames
  • Phosphoinositide Phospholipase C / metabolism*
  • Rats
  • Signal Transduction
  • ras Proteins / metabolism*
  • rho GTP-Binding Proteins / metabolism*

Substances

  • Phosphoinositide Phospholipase C
  • phospholipase C epsilon
  • ras Proteins
  • rho GTP-Binding Proteins
  • Lysine