Phospholipase C and Protein Kinase C-β 2 Mediate Insulin-Like Growth Factor II-Dependent Sphingosine Kinase 1 Activation

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 PKC alpha, beta 1, beta 2, delta, and zeta, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKC beta 2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKC beta 2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKC beta 2 (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 PKC beta 2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKC beta 2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKC beta 2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKC beta 2 by the IGF-II/M6P receptor are required for the activation of SK1. (Molecular Endocrinology 25:2144-2156, 2011)