The pleckstrin homology domain regulates phosphoinositide-specific phospholipase C delta-1 activity in cells

P. A. Bromann*, R. A. Litwinski, Jon W Lomasney

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

The pleckstrin homology (FH) domain has been postulated to serve as an anchor for enzymes which operate at a lipid-water interface. We have previously characterized the activities of human PLC dl and three PH domain mutants in vitro : K32G, E54K, and H356L. To study enzyme activity in cells, five stable Chinese hamster ovary (CHO) ceil lines were made. Four clones which expressed a relatively high level of expression were chosen for further analysis: PLC dl.16, K32G.7, E54K.10, and H356L.8. PLC dl and mutant protein expression levels were similar for the four stable cell lines (about 8 mg/mg total protein). Rat thrombin-stimulated (1.0 U/ml) [3JH-PI hydrolysis was measured, and fold-stimulation was as follows: pCDNA3=1.4; PLC dl.16=1.8; K32G.7=1.5; E54K.1 -42.4; and H356I-=1.7. Similar values were obtained with human thrombin. Further, ionomycin (2 mM) stimulated activity was higher than thrombin and correlates with PLC dl expression levels. We also investigated the PH domain as a membrane targeting domain. The PH domains of PLC dl, K32G, and E54K were cloned into the pEGFP Nl expression vor lor in-frame with the gene for green fluorescent protein (GFP). The pattorn of fusion protein expression observed via fluorescence microscopy. Wild-type PLC dl PH-GFP fusion protein localised to iho plasma membrane and cytosol. K;i2G PH-GFP was found only in the rylosol. E54K PH-GFP fusion protein was predominantly localized to thr plasma membrane and to a greater extent than PLC dl PH-GFP. These studies show that the PH domain of PLC dl can regulate enzyme activity as well as membrane association in cells.

Original languageEnglish (US)
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Dec 1 1998

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

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