Interaction of elongation factor-1 alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity
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- Interaction of elongation factor-1 alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity
- Chang, JS; Seok, H; Kwon, TK; Min, DS; Ahn, BH; Lee, YH; Suh, JW; Kim, JW; Iwashita, S; Omori, A; Ichinose, S; Numata, O; Seo, Jeong Kon; Oh, YS; Suh, Pann-Ghill
- PHOSPHATIDYLINOSITOL 4-KINASE; TYROSINE KINASE; SIGNALING MOLECULES; FACTOR 1-ALPHA; COMMON FOLD; C ISOZYMES; PH DOMAINS; MEMBRANE; BINDING; CELLS
- Issue Date
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
- JOURNAL OF BIOLOGICAL CHEMISTRY, v.277, no.22, pp.19697 - 19702
- The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-γ1 has two putative PH domains, an NH2-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-γ1 with translational elongation factor (EF)-1α, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-γ1 followed by peptide sequence analysis, we identified EF-1α as a binding partner of a split PH domain of PLC-γ1. Analysis by site-directed mutagenesis of the PH domain revealed that the β2-sheet of a split PH domain is critical for the interaction with EF-1α. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP2). So the PH domain of PLC-γ1 binds to both EF-1α and PlP2. The binding affinity of EF-1α to the GST·PH domain fusion protein increased in the presence of PIP2, although PIP2 does not bind to EF-1α directly. This suggests that EF-1α may control the binding affinity between the PH domain and PIP2. PLC-γ1 is substantially activated in the presence of EF-1α with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-γ1 (Y509A/F510A) that lost its binding affinity to EF-1α shows basal level activity. Taken together, our data show that EF-1α plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-γ1 activity via a split PH domain.
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