SERINE-16 OF STATHMIN AS A CYTOSOLIC TARGET FOR CA2+ CALMODULIN-DEPENDENT KINASE-II AFTER CD2 TRIGGERING OF HUMAN T-LYMPHOCYTES/

We investigated specific signaling events initiated after T cell trigg ering through the costimulatory surface receptors CD2 and CD28 as comp ared with activation via the Ag receptor (TCR/CD3), We therefore follo wed the phosphorylation of stathmin, a ubiquitous cytoplasmic phosphop rotein proposed as a general relay integrating diverse intracellular s ignaling pathways through the combinatorial phosphorylation of serines 16, 25, 38, and 63, the likely physiologic substrates for Ca2+/calmod ulin (CaM)-dependent kinases, mitogen-activated protein (MAP) kinase, cyclin-dependent kinases (cdks), and protein kinase A, respectively. W e addressed the specific protein kinase systems involved in the CD2 pa thway of T cell activation through the analysis of stathmin phosphoryl ation patterns in exponentially growing Jurkat T cells, as revealed by phosphopeptide mapping. Stimulation via CD2 activated multiple signal transduction pathways, resulting in phosphorylation of distinct sites of stathmin, the combination of which only partially overlaps the CD3 - and CD28-induced patterns. The partial redundancy of the three T cel l activation pathways was evidenced by the phosphorylation of Ser(25) and Ser(38), substrates of MAP kinases and of the cdk family kinase(s) , respectively. Conversely, the phosphorylation of Ser(16) of stathmin was observed in response to both CD2 and CD28 triggering, but not CD3 triggering, with a kinetics compatible with the lasting activation of CaM kinase II in response to CD2 triggering. In vitro, Ser(16) of rec ombinant human stathmin was phosphorylated also by purified CaM kinase IT, and in vivo, CaM kinase II activity was indeed stimulated in CD2- triggered Jurkat cells. Altogether, our results favor an association o f Cah I kinase II activity with costimulatory signals of T lymphocyte activation and phosphorylation of stathmin on Ser(16).