Figure 5.

(A) Genetic deficiency of p56lck prevents cellular tyrosine phosphorylation upon treatment with 10 μM C6-ceramide, whereas retransfection of p56lck restores the tyrosine phosphorylation of cellular proteins by C6-ceramide. JCaM1.6 or JCaM1.6/Lck⁺ cells were stimulated with C6-ceramide and lysed, and the whole-cell lysates were analyzed for tyrosine phosphorylation as above. (B) Tyrosine phosphorylation of the n-K⁺ channel by C6-ceramide depends on the functional expression of p56lck in JCaM1.6/Lck⁺ cells and is absent in p56lck-deficient JCaM1.6 cells. JCaM1.6/Lck⁺ or p56lck-deficient JCaM1.6 cells were stimulated with 10 μM C6-ceramide for the indicated time, and the n-K⁺ channel protein was immunoprecipitated and tested for tyrosine phosphorylation by Western blotting. The strips show similar amounts of K_v1.3 protein in all lanes (small blots). (C) The tyrosine kinase p56lck is stimulated by C6-ceramide in JCaM1.6/Lck⁺ cells, whereas no signal could be detected in the p56lck genetically deficient JCaM1.6 cells. Kinase activity was determined by autophosphorylation as above.