Abraham, Robert T.

Abstract Protein tyrosine kinases play fundamental roles in the transduction of signals that regulate cell growth, differentiation, and functional responses to a diversity of external stimuli. It is therefore likely that understanding protein tyrosine kinase activity in NK cells will be crucial in further defining the intracellular regulation of their unique and specialized functions. We investigated the role of protein tyrosine phosphorylation in receptor-mediated signal transduction using stimuli known to play major roles in regulating NK cell activation. Immunoblot analyses with antiphosphotyrosine antibodies demonstrated that IL-2, a potent stimulus for NK cell proliferation and an agent that enhances NK cytotoxic function, induced the tyrosine phosphorylation of at least eight proteins in clonal CD16+/CD3-human NK cells. In contrast, IL-4, which modulates NK cell function without inducing proliferation, had no apparent effect on protein tyrosine phosphorylation. Because protein kinase C (PKC) activation plays a prominent, yet distinct role in NK cell-mediated cytolytic reactions, we next investigated whether PKC activation affects NK cell protein tyrosine phosphorylation. Surprisingly, PKC-activating agents, including the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and 4 beta-phorbol 12, 13-didecanoate, as well as the synthetic diacylglycerol,1-oleoyl-2-acetylglycerol, also induced the tyrosine phosphorylation of a distinct set of proteins. The 4 beta-phorbol 12,13-didecanoate homolog, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, also failed to induce protein tyrosine phosphorylation. Further, the PKC inhibitor, 1-O-hexadecyl-2-O-methylglycerol blocked tyrosine phosphorylation induced by 1-oleoyl-2-acetylglycerol. In subsequent studies, both CD8+ and CD8- NK clones were found to express the src-family tyrosine kinase, p56lck, which was detected by immunoblot analysis with anti-p56lck antiserum. In both types of clonal NK cell lines, IL-2 and 12-O-tetradecanoyl-phorbol appeared to stimulate the differential phosphorylation of p56lck as evidenced by the appearance of higher molecular mass isoforms on SDS-polyacrylamide gels. Thus, our results identify and characterize a potential role for tyrosine phosphorylation and for the lymphocyte-specific tyrosine kinase p56lck in the signaling events that regulate NK cell activation.

Abstract NK cells mediate both direct cytotoxicity against a variety of tumor cells and indirect (FcR-dependent) cytotoxicity against antibody-coated targets. When cloned human NK cells (CD16+/CD3-) were exposed to NK-sensitive targets for 30 min, the level of inositol phosphates rose two to five times above background. The rise in inositol phosphates induced by NK-sensitive targets was paralleled by an increase in intracellular free calcium concentration ([Ca2+]i). A panel of tumor cells that were resistant to NK cell lysis did not stimulate significant levels of inositol phosphate production and did not induce an elevation of intracellular free calcium. Ligation of the FcR (CD16) with the mAb 3G8 also triggered phosphoinositide turnover. Kinetic experiments demonstrated that stimulation by either susceptible target cells or by FcR ligation led to rapid (less than 1 min) generation of the Ca2+-mobilizing second messenger, inositol trisphosphate, with slower accumulation of inositol bisphosphate and inositol monophosphate. Previous studies have demonstrated that activation of the cAMP-dependent second messenger pathway strongly inhibits NK cell-mediated cytotoxic functions. Treatment of NK effector cells with forskolin to elevate intracellular cAMP levels resulted in a concentration-dependent inhibition of phosphoinositide hydrolysis induced by both NK-sensitive targets and 3G8-mediated FcR ligation. These results suggest that phosphoinositide turnover represents a critical early event in the human NK cell cytolytic process. Moreover, the potent inhibitory effect of cAMP on NK cell cytotoxicity may be explained by the uncoupling of NK receptors from phospholipase C-mediated phosphoinositide hydrolysis.

Abstract Interleukin-2 (IL-2) potently stimulates natural killer (NK) cell proliferation and cytotoxic function. However, the molecular mechanisms by which IL-2 delivers activation signals from the IL-2 receptor to the NK cell interior are incompletely understood. Previous studies demonstrated that IL-2 stimulation induced the tyrosine phosphorylation of multiple proteins in NK cells, together with a prominent reduction in the elec- trophoretic mobility of p56lac. The present studies indicate that IL-2 induces a rapid (≤1 min) increase in the catalytic activity of p56lck, as measured by increases in protein tyrosine kinase activity in vitro. Furthermore, in response to IL-2, p56lck itself undergoes complex alterations in serine and tyrosine phosphorylation. Cyanogen bromide cleavage maps indicate that IL-2 stimulates a pronounced increase in the phosphorvlation of the NH2-terminal region of p56lck containing multiple known sites of serine phosphorylation. In addition, IL-2 induced a marked increase in the phosphorylation of a COOH-terminal peptide containing the regulatory Tyr-505 residue of p56lck. These results suggest that p56lck serves as a substrate for both protein serine and tyrosine kinases activated during stimulation of this cell type with IL-2. Furthermore, these results indicate that the pleiotropic effects of IL-2 on NK cell physiology are initiated and regulated by a complex and multitiered interaction of different protein kinases including p56lck.

Abstract We have shown previously that Wiskott-Aldrich syndrome protein (WASP) activation at the site of T cell-APC interaction is a two-step process, with recruitment dependent on the proline-rich domain and activation dependent on binding of Cdc42-GTP to the GTPase binding domain. Here, we show that WASP recruitment occurs through binding to the C-terminal Src homology 3 domain of Nck. In contrast, WASP activation requires Vav-1. In Vav-1-deficient T cells, WASP recruitment proceeds normally, but localized activation of Cdc42 and WASP is disrupted. The recruitment and activation of WASP are coordinated by tyrosine-phosphorylated Src homology 2 domain-containing leukocyte protein of 76 kDa, which functions as a scaffold, bringing Nck and WASP into proximity with Vav-1 and Cdc42-GTP. Taken together, these findings reconstruct the signaling pathway leading from TCR ligation to localized WASP activation.