Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity.
The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, 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-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.
Mesh Terms:
1-Phosphatidylinositol 4-Kinase, 3T3 Cells, Amino Acid Sequence, Animals, Binding Sites, Blood Proteins, Blotting, Western, Glutathione Transferase, Humans, Isoenzymes, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Peptide Elongation Factor 1, Peptides, Phospholipase C gamma, Phospholipids, Phosphoproteins, Point Mutation, Protein Binding, Protein Biosynthesis, Protein Structure, Secondary, Protein Structure, Tertiary, Rats, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, Signal Transduction, Type C Phospholipases
1-Phosphatidylinositol 4-Kinase, 3T3 Cells, Amino Acid Sequence, Animals, Binding Sites, Blood Proteins, Blotting, Western, Glutathione Transferase, Humans, Isoenzymes, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Peptide Elongation Factor 1, Peptides, Phospholipase C gamma, Phospholipids, Phosphoproteins, Point Mutation, Protein Binding, Protein Biosynthesis, Protein Structure, Secondary, Protein Structure, Tertiary, Rats, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, Signal Transduction, Type C Phospholipases
J. Biol. Chem.
Date: May. 31, 2002
PubMed ID: 11886851
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