Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization.
The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P1) phosphorylation was studied. Activation of S1P1 receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P1 phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P1 phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P1 phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and β isoforms in S1P1 immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or β and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P1 phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P1.
Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México D.F. 04510, Mexico.
Marco Antonio
MA
Morquecho-León
Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México D.F. 04510, Mexico.
Silvana
S
Bazúa-Valenti
Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México D.F. 04510, Mexico.
M Teresa
MT
Romero-Ávila
Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México D.F. 04510, Mexico. Electronic address: agarcia@ifc.unam.mx.
J Adolfo
JA
García-Sáinz
D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D049349:Receptors, Lysosphingolipid / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D007527:Isoenzymes; D050505:Mutant Proteins; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D049349:Receptors, Lysosphingolipid; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium
G protein-coupled receptor kinase; GFP; GRK; PKC; Protein kinase C; Receptor phosphorylation; S1P; S1P(1); Sphingosine-1-phosphate; Sphingosine-1-phosphate receptor 1; green fluorescent protein; protein kinase C; shRNA; short hairpin RNA; sphingosine-1-phosphate; sphingosine-1-phosphate receptor 1