Calcium-induced p56(Lck) phosphorylation in human T lymphocytes via calmodulin dependent kinase

We report that stimulation of both primary human and Jurkat T lymphocytes w ith the calcium ionophore ionomycin, or A23187, results in the phosphorylat ion of p56(Lck) as determined by shifts in mobility of p56(Lck) On immunobl ots. The shifts in the mobility of p56(Lck) induced by ionomycin could be b locked by preincubation of the cells with EGTA, demonstrating the requireme nt for extracellular calcium in this response. Although increases in intrac ellular calcium have been shown to modulate CD45 activity, phosphorylation of p56(Lck) was not mediated via CD45, Ionomycin stimulation of J45.01 cell s, a CD45-negative Jurkat cell derivative, also resulted in p56(Lck) mobili ty shifts. Instead, this response appears to be mediated via a calmodulin-d ependent kinase, This response could be blocked by calmidazolium, an inhibi tor 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 resp onse. These studies demonstrate a new role for calcium and CaM-Kinase in hu man T-lymphocytes and describe a novel mechanism by which p56(Lck) can be m odulated. (C) 1999 Academic Press.