Kathryn M. Brumbaugh

Sequential Involvement of Lck and SHP-1 with MHC-Recognizing Receptors on NK Cells Inhibits FcR-Initiated Tyrosine Kinase Activation

Recognition of major histocompatibility (MHC) class I complexes on target cells by killer cell inhibitory receptors (KIR) blocks natural killer (NK) and T cell cytotoxic function. The inhibitory effect of KIR ligation requires the phosphotyrosine-dependent association of KIR with the cytoplasmic SH2-containing protein tyrosine phosphatase SHP-1. Using a somatic genetic model, we first define a requirement for the Src family protein tyrosine kinase (PTK) Lck in mediating KIR tyrosine phosphorylation. We then investigate how KIR ligation interrupts PTK-dependent NK cell activation signals. Specifically, we show that KIR ligation inhibits the Fc receptor (FcR)-induced tyrosine phosphorylation of the FcR-associated zeta signaling chain, the PTK ZAP-70, and phospholipase C gamma. Overexpression of catalytically inactive SHP-1 (acting as a dominant negative) restores the tyrosine phosphorylation of these signaling events and reverses KIR-mediated inhibition of NK cell cytotoxic function. These results suggest sequential roles for Lck and SHP-1 in the inhibition of PTK following MHC recognition by NK cells.

ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses.

Genotoxic stress triggers the activation of checkpoints that delay cell-cycle progression to allow for DNA repair. Studies in fission yeast implicate members of the Rad family of checkpoint proteins, which includes Rad17, Rad1, Rad9 and Hus1, as key early-response elements during the activation of both the DNA damage and replication checkpoints. Here we demonstrate a direct regulatory linkage between the human Rad17 homologue (hRad17) and the checkpoint kinases, ATM and ATR. Treatment of human cells with genotoxic agents induced ATM/ATR-dependent phosphorylation of hRad17 at Ser 635 and Ser 645. Overexpression of a hRad17 mutant (hRad17AA) bearing Ala substitutions at both phosphorylation sites abrogated the DNA-damage-induced G2 checkpoint, and sensitized human fibroblasts to genotoxic stress. In contrast to wild-type hRad17, the hRad17AA mutant showed no ionizing-radiation-inducible association with hRad1, a component of the hRad1–hRad9–hHus1 checkpoint complex. These findings demonstrate that ATR/ATM-dependent phosphorylation of hRad17 is a critical early event during checkpoint signalling in DNA-damaged cells.

Signal transduction by human NK cell MHC-recognizing receptors

Summary: Cells may be protected from natural killer (NK)‐cell‐mediated killing by the expression of specific MHC class I complexes. This protective effect is due to the expression on NK cells of MHC class I‐recognizing receptors which, upon ligation, transduce potent inhibitory signals into the NK cells. The molecular signalling mechanisms employed by the human NK‐cell MHC‐recognizing killer cell inhibitory receptors (KIR) and CD94 are the focus of this review, A sequential model of KIR signalling involving lck‐dependent tyrosine phosphorylation of KIR and subsequent association of KIR with the SH2 ‐containing tyrosine phosphatase, SHP‐1, is presented. We explore how engagement of either KIR m CD94 modulates the protein tyrosine kinase‐dependent biochemical signals responsible for activation of NK‐ceil cytotoxic function. Additionally, we discuss models of inhibitory signalling proposed for each of the lymphocyte lineages, emphasizing that disparate molecular mechanisms may be utilized by ceils to produce similar biological responses.

Signal Transduction During NK Cell Activation: Balancing Opposing Forces

Significant progress has been made in our understanding of the basic signaling mechanisms regulating NK cell activation. Advances have been fueled in part by the molecular characterization of specific activating receptors (e.g., the Fc gamma RIII multi-subunit complex) and inhibitory receptors (e.g., novel MHC-recognizing inhibitory receptors). However, certain aspects of these analyses are complicated by the heterogeneous nature of the receptor-ligand interactions utilized during the development of a cytotoxic response. Future advances will depend in part on the further molecular characterization of the involved receptors and second messengers and on the development of experimental models for genetically manipulating the signaling elements. It will remain important to understand both activating and inhibitory signaling pathways as the emerging theme is that the balance of these two opposing forces determines the functional outcome of an NK cells interaction with its target.