Calcium-Induced p56Lck Phosphorylation in Human T Lymphocytes via Calmodulin Dependent Kinase

We report that stimulation of both primary human and Jurkat T lymphocytes with the calcium ionophore ionomycin, or A23187, results in the phosphorylation of p56Lck as determined by shifts in mobility of p56Lck on immunoblots. The shifts in the mobility of p56Lck induced by ionomycin could be blocked by preincubation of the cells with EGTA, demonstrating the requirement for extracellular calcium in this response. Although increases in intracellular calcium have been shown to modulate CD45 activity, phosphorylation of p56Lck was not mediated via CD45. Ionomycin stimulation of J45.01 cells, a CD45-negative Jurkat cell derivative, also resulted in p56Lck mobility shifts. Instead, this response appears to be mediated via a calmodulin-dependent kinase. This response could be blocked by calmidazolium, an inhibitor of calmodulin, and KN-93, an inhibitor of calmodulin-dependent kinases (CaM-Kinase). KN-92, an inactive analog of KN-93, failed to block this response. These studies demonstrate a new role for calcium and CaM-Kinase in human T-lymphocytes and describe a novel mechanism by which p56Lck can be modulated.