T-Cell receptor signaling pathway exerts a negative control on thrombin-mediated increase in [Ca2+]i and p38 MAPK activation in Jurkat T cells: implication of the tyrosine kinase p56Lck.
Activation of the mitogen-activated protein kinase (Erk) and c-Jun terminal kinase is a well-documented mechanism for the seven transmembrane spanning receptors. We have previously shown that thrombin stimulation of the T-leukemic cell line Jurkat induced a transient increase in [Ca2+]i and tyrosine phosphorylation of several cellular proteins. Here, we have analyzed p42-44 MAPK, JNK and p38 MAPK activation using Jurkat T-cell lines deficient in either the tyrosine kinase p56Lck (JCaM1) or the tyrosine phosphatase CD45 (J45.01). Our results demonstrate that p56Lck and CD45 exert a negative control on thrombin-induced p38 MAPK activation and [Ca2+]i release in Jurkat cells. Thrombin receptor expression was identical on the different cell lines as assessed by FACS analysis. Tyrosine phosphorylation of p38 MAPK was drastically increased after thrombin stimulation of JCaM1 or J45.01 cells, as compared with parental cells (JE6.1). P42-44 MAPK and JNK activity also enhanced after thrombin treatment of JE6.1 and JCaM1 cell lines, whereas basal kinase activity was higher in J45.01 cells and was not further stimulated by thrombin. Thrombin and thrombin receptor agonist peptide-induced [Ca2+]i mobilization paralleled p38 MAPK activation in JCaM1 and J45.01 cells. Moreover, reconstitution of J45.01 and JCaM1 cell lines with either CD45 or Lck is accompanied by restoration of a normal thrombin-induced [Ca2+]i response and p38MAPK phosphorylation. These data show that a component of the T-cell receptor signaling pathway exerts a negative control on thrombin-induced responses in Jurkat T cells. Accordingly, we found that thrombin enhanced tyrosine phosphorylation of p56Lck and decreased p56Lck kinase activity in J45.01 cells. Our results are consistent with a negative role for p56Lck on thrombin-induced [Ca2+]i release and p38 MAPK activation in Jurkat T-cell lines.
Maulon
L
L
CJF INSERM 96.05, Activation des Cellules Hématopoiétiques, Faculté de Médecine, Nice Cédex, France.
Guérin
S
S
Ricci
J E
JE
Breittmayer
D F
DF
Auberger
P
P
D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000378:metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases; D008970:Molecular Weight; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D018179:Receptors, Thrombin / Q000378:metabolism; D015398:Signal Transduction; D013499:Surface Properties; D013601:T-Lymphocytes / Q000201:enzymology*; D013917:Thrombin / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
D011948:Receptors, Antigen, T-Cell; D018179:Receptors, Thrombin; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D017493:Leukocyte Common Antigens; D013917:Thrombin; D002118:Calcium