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 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.
Departments of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA. elshewy@musc.edu
Hesham M
HM
El-Shewy
Souzan A
SA
Abdel-Samie
Abdelmohsen M
AM
Al Qalam
Mi-Hye
MH
Lee
Kazuyuki
K
Kitatani
Viviana
V
Anelli
Ayad A
AA
Jaffa
Lina M
LM
Obeid
Louis M
LM
Luttrell
D049109:Cell Proliferation; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology / Q000502:physiology*; D008246:Lysophospholipids / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D050527:Mesangial Cells / Q000187:drug effects / Q000502:physiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D064546:Protein Kinase C beta; D021381:Protein Transport; D034622:RNA Interference; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D017527:Receptor, IGF Type 2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000099157:Sphingosine Kinase
D004075:Diglycerides; D020536:Enzyme Activators; D049452:Green Fluorescent Proteins; D007211:Indoles; D007335:Insulin-Like Growth Factor II; D008246:Lysophospholipids; D008301:Maleimides; D017853:Phosphotransferases (Alcohol Group Acceptor); D011493:Protein Kinase C; D064546:Protein Kinase C beta; D017526:Receptor, IGF Type 1; D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D010738:Type C Phospholipases; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate; C088060:bisindolylmaleimide