PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phorbol 12-myristate 13-acetate (PMA).
Sphingosine-1-phosphate (S1P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S1P generation is regulated via sphingosine kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S1P extracellularly. The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK1 (hSK1) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK1. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C(6)-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S1P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S1P, allowing for autocrine/paracrine signaling.
Johnson
Korey R
KR
Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Becker
Kevin P
KP
Facchinetti
Maria Marta
MM
Hannun
Yusuf A
YA
Obeid
Lina M
LM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008246:Lysophospholipids; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D000099157:Sphingosine Kinase
D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D011493:Protein Kinase C; D011993:Recombinant Fusion Proteins; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate