Journal
EMBO JOURNAL
Volume 20, Issue 4, Pages 743-754Publisher
OXFORD UNIV PRESS
DOI: 10.1093/emboj/20.4.743
Keywords
GTP-binding protein Ras; GTP exchange factor; phospholipase C; Raf-1; Ras-binding domain
Categories
Funding
- NIDDK NIH HHS [DK56294, R01 DK056294] Funding Source: Medline
- NIGMS NIH HHS [R01 GM053536, GM53536] Funding Source: Medline
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Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLC beta, PLC gamma and PLC delta, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLC epsilon, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2, The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras-binding domain of Raf-1; however, the RA1 domain binds H-Ras with a low affinity in a GTP-independent manner. While G alpha (q), G beta gamma or, surprisingly, H-Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H-Ras stimulates PLC epsilon co-expressed in COS-7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site-directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras-effector interaction and a unique profile of activation by Ras effector domain mutants, These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.
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