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Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity.
J Biol Chem. 2002 May 31; 277(22):19697-702.JB

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+Show Affiliations

Department of Life Science, College of Natural Science, Daejin University, Kyeonggido 487-711, Korea. jchang@road.daijin.ac.krNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11886851

Citation

Chang, Jong-Soo, et al. "Interaction of Elongation Factor-1alpha and Pleckstrin Homology Domain of Phospholipase C-gamma 1 With Activating Its Activity." The Journal of Biological Chemistry, vol. 277, no. 22, 2002, pp. 19697-702.
Chang JS, Seok H, Kwon TK, et al. Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity. J Biol Chem. 2002;277(22):19697-702.
Chang, J. S., Seok, H., Kwon, T. K., Min, D. S., Ahn, B. H., Lee, Y. H., Suh, J. W., Kim, J. W., Iwashita, S., Omori, A., Ichinose, S., Numata, O., Seo, J. K., Oh, Y. S., & Suh, P. G. (2002). Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity. The Journal of Biological Chemistry, 277(22), 19697-702.
Chang JS, et al. Interaction of Elongation Factor-1alpha and Pleckstrin Homology Domain of Phospholipase C-gamma 1 With Activating Its Activity. J Biol Chem. 2002 May 31;277(22):19697-702. PubMed PMID: 11886851.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity. AU - Chang,Jong-Soo, AU - Seok,Heon, AU - Kwon,Taeg-Kyu, AU - Min,Do Sik, AU - Ahn,Bong-Hyun, AU - Lee,Young Han, AU - Suh,Ju-Won, AU - Kim,Jong-Woo, AU - Iwashita,Shintaro, AU - Omori,Akira, AU - Ichinose,Sachiyo, AU - Numata,Osamu, AU - Seo,Jeong-Kon, AU - Oh,Yong-Seok, AU - Suh,Pann-Ghill, Y1 - 2002/03/08/ PY - 2002/3/12/pubmed PY - 2002/7/3/medline PY - 2002/3/12/entrez SP - 19697 EP - 702 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 277 IS - 22 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/11886851/Interaction_of_elongation_factor_1alpha_and_pleckstrin_homology_domain_of_phospholipase_C_gamma_1_with_activating_its_activity_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=11886851 DB - PRIME DP - Unbound Medicine ER -