title: Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity.
abstract: 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.
authors.affiliation: Department of Life Science, College of Natural Science, Daejin University, Kyeonggido 487-711, Korea. jchang@road.daijin.ac.kr
authors.forename: Jong-Soo
authors.initials: JS
authors.lastname: Chang
authors.forename: Heon
authors.initials: H
authors.lastname: Seok
authors.forename: Taeg-Kyu
authors.initials: TK
authors.lastname: Kwon
authors.forename: Do Sik
authors.initials: DS
authors.lastname: Min
authors.forename: Bong-Hyun
authors.initials: BH
authors.lastname: Ahn
authors.forename: Young Han
authors.initials: YH
authors.lastname: Lee
authors.forename: Ju-Won
authors.initials: JW
authors.lastname: Suh
authors.forename: Jong-Woo
authors.initials: JW
authors.lastname: Kim
authors.forename: Shintaro
authors.initials: S
authors.lastname: Iwashita
authors.forename: Akira
authors.initials: A
authors.lastname: Omori
authors.forename: Sachiyo
authors.initials: S
authors.lastname: Ichinose
authors.forename: Osamu
authors.initials: O
authors.lastname: Numata
authors.forename: Jeong-Kon
authors.initials: JK
authors.lastname: Seo
authors.forename: Yong-Seok
authors.initials: YS
authors.lastname: Oh
authors.forename: Pann-Ghill
authors.initials: PG
authors.lastname: Suh
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism; D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry*; D015153:Blotting, Western; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020648:Peptide Elongation Factor 1 / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D051966:Phospholipase C gamma; D010743:Phospholipids / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D017354:Point Mutation; D011485:Protein Binding; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D001798:Blood Proteins; D007527:Isoenzymes; D020648:Peptide Elongation Factor 1; D010455:Peptides; D010743:Phospholipids; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D005982:Glutathione Transferase; D019870:1-Phosphatidylinositol 4-Kinase; D010738:Type C Phospholipases; D051966:Phospholipase C gamma