Insulin-induced phospholipase D1 and phospholipase D2 activity in human embryonic kidney-293 cells mediated by the phospholipase C gamma and protein kinase C alpha signalling cascade

Biochem J. 2000 Nov 1;351 Pt 3(Pt 3):613-9.

Abstract

Phospholipase D (PLD)1 is quiescent in vitro and in vivo until stimulated by classical protein kinase C (PKC) isoforms, ADP-ribosylation factor or Rho family members. By contrast, PLD2 has high basal activity, and the mechanisms involved in agonist-induced activation of PLD2 are poorly understood. Using transiently transfected human embryonic kidney (HEK)-293 cells as a model system, we report in the present study that PLD2 overexpressed in HEK-293 cells exhibits regulatory properties similar to PLD1 when stimulated in response to insulin and phorbol ester. Co-expression of PLD1 or PLD2 with PKC alpha results in constitutive activation of both PLD isoforms, which cannot be further stimulated by insulin. Co-expression of PLD1 with phospholipase C (PLC)gamma has the same effect, while co-expression of PLD2 with PLC gamma allows PLD2 activity to be stimulated in an insulin-dependent manner. The PKC-specific inhibitors bisindolylmaleimide and Gö 6976 abolish insulin-induced PLD2 activation in HEK-293 cells co-expressing the insulin receptor, PLC gamma and PLD2, confirming that not only PLD1, but PLD2 as well, is regulated in a PKC-dependent manner. Finally, we provide evidence that PKC alpha is constitutively associated with PLD2. In summary, we demonstrate that insulin treatment results in activation of both PLD1 and PLD2 in appropriate cell types when the appropriate upstream intermediate signalling components, i.e. PKC alpha and PLC gamma, are expressed at sufficient levels.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line
  • Humans
  • Insulin / pharmacology*
  • Isoenzymes / metabolism*
  • Kidney / cytology
  • Kidney / enzymology*
  • Phospholipase C gamma
  • Phospholipase D / biosynthesis*
  • Phospholipase D / metabolism
  • Protein Binding
  • Protein Kinase C / metabolism*
  • Protein Kinase C-alpha
  • Signal Transduction*
  • Type C Phospholipases / metabolism*

Substances

  • Insulin
  • Isoenzymes
  • PRKCA protein, human
  • Protein Kinase C
  • Protein Kinase C-alpha
  • Type C Phospholipases
  • phospholipase D2
  • Phospholipase C gamma
  • Phospholipase D
  • phospholipase D1