Convergence of Schiff base costimulatory signaling and TCR signaling at the level of mitogen-activated protein kinase ERK2.
Schiff base formation on specialized T cell surface amines provides a costimulatory signal to T cells through a mechanism that activates Na+ and K+ transport, substantially enhancing TCR-dependent IL-2 production. Schiff base-forming molecules that mimic the natural carbonyl donor potently enhance immune responses and provide the first mechanism-based, orally active immunopotentiatory agents. In the present study, costimulation by the Schiff base-forming molecule tucaresol was investigated at the level of mitogen-activated protein kinase (MAPK) in T cell lines. Both TCR-directed stimulation by anti-CD3 and Schiff base stimulation by tucaresol produced a distinct mobility shift in MAPK, characterized by direct immunoblotting of cell lysate proteins subjected to SDS-PAGE, that corresponded with increased phosphorylation. Combined TCR-CD3 and tucaresol stimulation substantially enhanced and prolonged the MAPK response, providing a biochemical basis for the costimulatory nature of the pathway utilized by Schiff base signaling. The MAPK affected was identified by immunoprecipitation as ERK2. Both the direct effects and the TCR signal-enhancing effects of tucaresol on MAPK activation were also demonstrated in a functional MAPK assay measuring substrate phosphorylation. Borohydride reduction of tucaresol's Schiff base-forming carbonyl group abolished both enhancement of MAPK phosphorylation and IL-2 production, as did a selective inhibitor of the MAPKK, MEK1. Tucaresol had no effect on TCR-mediated rises in intracellular free Ca2+ or inositol 1,4,5-triphosphate generation, while tucaresol signaling occurred normally in the lck-deficient J.CaM1.6 T cell line, consistent with convergence of tucaresol- and TCR-induced signals downstream of early TCR-mediated events.
Chen
H
H
Immunology Unit, Glaxo Wellcome Medicines Research Centre, Stevenage, United Kingdom.
Hall
S
S
Heffernan
B
B
Thompson
N T
NT
Rogers
M V
MV
Rhodes
J
J
D001547:Benzaldehydes / Q000737:chemistry / Q000494:pharmacology*; D001565:Benzoates / Q000737:chemistry / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D001894:Borohydrides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000473:pathology; D008213:Lymphocyte Activation / Q000502:physiology*; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D016853:Muromonab-CD3 / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000187:drug effects / Q000276:immunology*; D012545:Schiff Bases; D015398:Signal Transduction / Q000502:physiology*; D012964:Sodium / Q000378:metabolism; D014407:Tumor Cells, Cultured
D001547:Benzaldehydes; D001565:Benzoates; D001894:Borohydrides; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007376:Interleukin-2; D016853:Muromonab-CD3; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D012545:Schiff Bases; C025364:sodium borohydride; D012964:Sodium; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; C059629:tucaresol; D011188:Potassium; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium