Phospholipase C and protein kinase C-β 2 mediate insulin-like growth factor II-dependent sphingosine kinase 1 activation

Mol Endocrinol. 2011 Dec;25(12):2144-56. doi: 10.1210/me.2011-0101. Epub 2011 Oct 20.

Abstract

We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.

Publication types

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

MeSH terms

  • Cell Proliferation
  • Diglycerides / metabolism
  • Enzyme Activation*
  • Enzyme Activators / pharmacology
  • Gene Knockdown Techniques
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Indoles / pharmacology
  • Insulin-Like Growth Factor II / pharmacology
  • Insulin-Like Growth Factor II / physiology*
  • Lysophospholipids / metabolism
  • Maleimides / pharmacology
  • Mesangial Cells / drug effects
  • Mesangial Cells / physiology
  • Phosphotransferases (Alcohol Group Acceptor) / antagonists & inhibitors
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism*
  • Protein Kinase C / antagonists & inhibitors*
  • Protein Kinase C / metabolism
  • Protein Kinase C beta
  • Protein Transport
  • RNA Interference
  • Receptor, IGF Type 1 / metabolism
  • Receptor, IGF Type 2 / genetics
  • Receptor, IGF Type 2 / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • Sphingosine / analogs & derivatives
  • Sphingosine / metabolism
  • Sphingosine / pharmacology
  • Tetradecanoylphorbol Acetate / pharmacology
  • Type C Phospholipases / antagonists & inhibitors*
  • Type C Phospholipases / metabolism

Substances

  • Diglycerides
  • Enzyme Activators
  • Indoles
  • Lysophospholipids
  • Maleimides
  • Receptor, IGF Type 2
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • sphingosine 1-phosphate
  • Insulin-Like Growth Factor II
  • Phosphotransferases (Alcohol Group Acceptor)
  • sphingosine kinase
  • Receptor, IGF Type 1
  • Protein Kinase C
  • Protein Kinase C beta
  • Type C Phospholipases
  • bisindolylmaleimide
  • Sphingosine
  • Tetradecanoylphorbol Acetate