Differential interaction of Cb1 with Grb2 and CrkL in CD2-mediated NK cellactivation

Natural killer (NK) cells participate in both innate and adoptive immunity by their prompt secretion of cytokines and by their ability to lyse virally infected cells or tumor cells. CD2 is surface glycoprotein receptors and c rucial for NK cell activation. However, molecular events involved in CD2-me diated NK cell activation have not been fully elucidated. Cbl-Grb2 and Cbl- CrkL interactions have been implicated in T cell and B cell receptor, and c ytokine receptor signaling, Here we analyzed tyrosine phosphorylation and i nteractions of Cbl with adapter proteins, Grb2 and CrkL, in NK3.3 cells, CD 2 crosslinking results in the marked tyrosine phosphorylation of Cbl in an antibody concentration- and time-dependent manner. Immunodepletion studies reveal that Grb2-associated tyrosine phosphorylated p120 kDa protein is Cbl . In vitro binding studies using GST-fusion proteins demonstrate that Cbl c onstitutively associates with the SH3 domains of Grb2, with a preference fo r the amino-terminal domain. In addition, we demonstrate that CrkL associat es with a large portion of tyrosine phosphorylated Cbl after CD2 stimulatio n of NK3.3 cells. In contrast to constitutive Cbl association with Grb2, ty rosine phosphorylated Cbl interacts with CrkL via its SH2 domain only after CD2 stimulation, Although the precise roles of interactions of Cbl with Gr b2 and CrkL in NK cell activation remains to be elucidated, their tyrosine phosphorylation, in addition to the multiple protein interactions described here, strongly suggest that interactions of Cbl with Grb2 and CrkL may pla y pivotal roles in CD2-mediated NK cell activation. (C) 2001 Elsevier Scien ce Ltd. All rights reserved.