Andrew M. Scharenberg

Recruitment of Tyrosine Phosphatase HCP by the Killer Cell Inhibitory Receptor

Cytolysis of target cells by natural killer (NK) cells and by some cytotoxic T cells occurs unless prevented by inhibitory receptors that recognize MHC class I on target cells. Human NK cells express a p58 inhibitory receptor specific for HLA-C. We report association of the tyrosine phosphatase HCP with the p58 receptor in NK cells. HCP association was dependent on tyrosine phosphorylation of p58. Phosphotyrosyl peptides corresponding to the p58 tail bound and activated HCP in vitro. Furthermore, introduction of an inactive mutant HCP into an NK cell line prevented the p58-mediated inhibition of target cell lysis. These data imply that the inhibitory function of p58 is dependent on its tyrosine phosphorylation and on recruitment and activation of HCP.

Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B‐cell receptor activation

Brutons tyrosine kinase (Btk) is essential for B‐lineage development and represents an emerging family of non‐receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B‐cell antigen receptor (BCR) cross‐linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk‐deficient human B‐cell lines. Btk and phospholipase Cγ (PLCγ) co‐expression resulted in tyrosine phosphorylation of PLCγ and required the same Btk domains as those for Btk‐dependent calcium influx. Receptor‐dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)‐sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg‐sensitive, IP3‐gated calcium store(s) that regulates store‐operated calcium entry. Overexpression of dominant‐negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co‐cross‐linking of the BCR and the inhibitory receptor, FcγRIIb1, completely abrogated Btk‐dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.

Activation of BTK by a Phosphorylation Mechanism Initiated by SRC Family Kinases

Brutons tyrosine kinase (BTK) is pivotal in B cell activation and development through its participation in the signaling pathways of multiple hematopoietic receptors. The mechanisms controlling BTK activation were studied here by examination of the biochemical consequences of an interaction between BTK and SRC family kinases. This interaction of BTK with SRC kinases transphosphorylated BTK on tyrosine at residue 551, which led to BTK activation. BTK then autophosphorylated at a second site. The same two sites were phosphorylated upon B cell antigen receptor cross-linking. The activated BTK was predominantly membrane-associated, which suggests that BTK integrates distinct receptor signals resulting in SRC kinase activation and BTK membrane targeting.

The FcεRIβ Subunit Functions as an Amplifier of FcεRIγ-Mediated Cell Activation Signals

The high affinity IgE receptor (Fc(epsilon)RI) plays a central role in the initiation of allergic responses. Fc(epsilon)RI is multimeric and is expressed as either (alpha)(gamma2) trimers or (alpha)(beta)(gamma2) tetramers. Recently, polymorphisms of the beta chain gene have been associated with the development of various allergic phenotypes. Until now, the role of beta in Fc(epsilon)RI-dependent signaling was largely unknown. For that reason, we compared the signaling characteristics of various wild-type and mutant (alpha)(gamma2) and (alpha)(beta)(gamma2) Fc(epsilon)RI complexes. These studies demonstrate that the gamma dimer functions as an autonomous activation module, while beta functions as an amplifier that provides a gain of 5- to 7-fold, as measured by Syk activation and calcium mobilization.

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.

Regulation of Btk Function by a Major Autophosphorylation Site Within the SH3 Domain

Brutons tyrosine kinase (Btk) plays a crucial role in B cell development. Overexpression of Btk with a Src family kinase increases tyrosine phosphorylation and catalytic activity of Btk. This occurs by transphosphorylation at Y551 in the Btk catalytic domain and the enhancement of Btk autophosphorylation at a second site. A gain-of-function mutant called Btk* containing E41 to K change within the pleckstrin homology domain induces fibroblast transformation. Btk* enhances the transphosphorylation of Y551 by endogenous Src family tyrosine kinases and autophosphorylation at the second site. We mapped the major Btk autophosphorylation site to Y223 within the SH3 domain. Mutation of Y223 to F blocks Btk autophosphorylation and dramatically potentiates the transforming activity of Btk* in fibroblasts. The location of Y223 in a potential ligand-binding pocket suggests that autophosphorylation regulates SH3-mediated signaling by Btk.

The Emerging Field of Receptor-Mediated Inhibitory Signaling: SHP or SHIP?

which results in interaction of CTLA-4 with its countereceptors CD80 or CD86 (Figure 1C, right panel). Since In the past two decades, tremendous advances have mice which lack CTLA-4 have hyperactivated T cells been made in understanding the molecular mechanisms and are prone to lymphoproliferative diseases, it is used by various types of cell surface receptors to transthought that CTLA-4 mediates an inhibitory signal which duce signals. Nearly all of these advances have come provides an important negative feedback control for profrom the study of model systems where a receptor “actiliferation and cytokine production induced by T-cell revates” cells togenerate a well defined response because ceptor activation signals (Marengere et al., 1996). such systems were most amenable to study. As knowlWhile each of these systems is unique in terms of the edge about activating model systems has increased, it manner in which the activating and inhibitory signals has become clear that there aremany situations in which are engaged, two common features exist among them. the activating signal sent from one receptor is moduFirst, each involves activating signals mediated by holated as the direct result of a “negative” or “inhibitory” mologous cytoplasmic tail motifs known as immunoresignal sent by another cell surface receptor. While the ceptor tyrosine based activation motifs (ITAMs, restudy of this type of signaling is generally in its infancy, viewed in (Weiss and Littman, 1994)). These motifs several recent papers have begun to shed light on the become tyrosine phosphorylated by src family kinases molecular mechanisms which underly receptor-mediwhen the activating receptors are engaged by clustering ated inhibitory signals in immunologic systems. Given stimuli, resulting in the recruitment toengaged receptors the tendency of nature toutilize signaling functions modof both src and syk/zap70 family non-receptor tyrosine ularly in a variety of signaling pathways, the paradigms kinases. Downstream propagation of the activation sigoutlined by these systems may have implications for the nal is then mediated by activation of these tyrosine kistudy of inhibitory or deactivating signals in nonimmunonases and the resulting phosphorylation of specific sublogic situations as well. In addition, the study of these strates. These three systems also share a second signals may add new dimensions to our understanding feature: the inhibitory signals are mediated by separate of other widely utilized signaling pathways. receptors, such as FcgRIIb1, KIR, and CTLA-4, which are Descriptions of three representative systems utilized engaged in concert with the activating receptor when in these recent studies are useful for understanding the an appropriate stimulus is present. When the inhibitory nature of each inhibitory signal and areoutlined in Figure receptors are appropriately engaged, they become 1. Briefly, the surface immunoglobulin receptor (sIg) phosphorylated on specific cytoplasmic tail tyrosines complex and FcgRIIb1 (a low affinity receptor for IgG) by src family kinases (Bindstadt et al., 1996; Burshtyn are both normally present on B-cell surfaces (Figure 1A, et al., 1996; Marengere et al., 1996; Muta et al., 1994), left panel). When sIg receptors are clustered on contact which, as will be described in more detail below, results with antigen (Figure 1A, middle panel), they typically in the recruitment of signaling molecules which are inproduce a cell activation signal which induces B-cell hibitory in function. proliferation. However, if the same B cells are stimulated The initial breakthrough in understanding the inhibiso that the sIg receptors are coclustered with FcgRIIb1 tory signals sent by these receptors was the demonstrareceptors (for example by contact of the B cell with an tion that a thirteen amino acid sequence present in the immune complex of cognate antigen and IgG, Figure FcgRIIb1 tail was necessary and sufficient to convey 1A, right panel), B cells fail to proliferate and in some inhibitory activity—in essence defining an immunorecases may apoptose (Ashman et al., 1996). In the natural ceptor tyrosine based inhibitory motif (ITIM) (Amigorena killer (NK) cell system, a number of cell surface receptors et al., 1992; Muta et al., 1994). A phosphorylated version are able to initiate NK cell cytolysis, one of which is of the FcgRIIb1 ITIM was then shown to bind to SH2FcgRIII (Figure 1B, left panel). When an NK cell encouncontaining tyrosine phosphatase-1 (SHP-1, previously ters a target cell, it recognizes and kills the target cell called HCP, SHPTP1, PTP1C, see Figure 2), as well as if the target cell lacks class I MHC molecules. One of activate SHP-1 in vitro (D’Ambrosio et al., 1995; rethe ways in which NKcells recognize target cells is when viewed in Tonks and Neel, 1996). This was rapidly folIgG bound to the target cell surface binds to FcgRIII on lowed by the recognition that KIR had similar motifs, NK cells (Figure 1B, middle panel). If the target cells that phosphopeptides derived from KIR ITIMs were also express appropriate class I MHC molecules which can able to bind to and activate SHP-1 in vitro, and that a be recognized by appropriate killer cell inhibitory recepdominant negative form of SHP-1 could block the KIR tors (KIR) on the NK cell, they are protected from cytolyinhibitory signal (Burshtyn et al., 1996). In addition, resis (Figure 1C, right panel). In the T-cell system, the cruitment of SH2-containing tyrosine phosphatase-2 T-cell antigen receptor (TCR) and CD4 and/or CD8 co(SHP-2, previously known as syp, SHPTP2, see Figure

Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering.

High affinity IgE receptor (Fc epsilon RI) signaling after contact with antigen occurs in response to receptor clustering. This paper describes methodology, based on vaccinia virus driven protein expression, for probing signaling pathways and its application to Fc epsilon RI interactions with the lyn and syk tyrosine kinases. Reconstitution of the complete tetrameric Fc epsilon RI receptor, lyn and syk in a non‐hematopoietic ‘null’ cell line is sufficient to reconstruct clustering‐controlled receptor tyrosine phosphorylation and activation of syk, without apparent requirement for hematopoietic specific phosphatases. The src family kinase lyn phosphorylates Fc epsilon RI in response to receptor clustering, resulting in syk binding to the phosphorylated Fc epsilon RI. Lyn also participates in the tyrosine phosphorylation and activation of syk in a manner which is dependent on phosphorylated Fc epsilon RI. Using overexpression of active and dominant negative syk proteins in a mast cell line which naturally expresses Fc epsilon RI, we corroborate syks role downstream of receptor phosphorylation, and demonstrate that syk SH2 domains protect receptor ITAMs from ongoing dephosphorylation. Based on these results, we propose that receptor clustering controls lyn‐mediated Fc epsilon RI tyrosine phosphorylation by shifting a balance between phosphorylation and dephosphorylation towards accumulation of tyrosine phosphorylated Fc epsilon RI. Fc epsilon RI tyrosine phosphorylation functions to bring syk into a microenvironment where it becomes tyrosine phosphorylated and activated, thereby allowing clustering to indirectly control syk activity.

Quantitative distribution of protein kinase C α, β, γ, and ϵ mRNAS in the hippocampus of control and nictitating membrane conditioned rabbits

Abstract We used oligonucleotide in situ hybridization and film autoradiography to quantitate the distributions of protein kinase C (PKC) α, β, γ, and ϵ mRNAs in subregions of rabbit hippocampus. Levels of each of the hippocampal PKC isozyme mRNAs and patterns of their regional distributions were remarkably invariant between individuals. Within stratum pyramidale, the highest levels of PKC α mRNA were in the CA2 region, while PKC β mRNA was maximally expressed in CA1, and PKC ϵ mRNA in CA3; PKC γ mRNA was abundantly expressed throughout Ammons horn. Previous experiments employing quantitative autoradiography for [ 3 H]PDBU (Olds et al., Science , 245 (1989) 866–869) revealed an increase in membrane-bound PKC in the CA1 region of rabbit hippocampus up to 3 days following classical conditioning of the nictitating membrane response. We report here that there were no differences in levels of PKC α, β, γ, or ϵ mRNA between conditioned and control rabbits in any hippocampal region one day after training. These data are consistent with the hypothesis that PKC is post-translationally activated and translocated to the membrane during memory storage.

Molecular mechanism of paired immunoglobulin­ like receptor B (PIR-B)-mediated inhibitory signal

B cell express paired immunoglobulin-like receptor B (PIR-B) contains four potential ITIMs (immunoreceptor tyrosine-based inhibitory motifs) in the cytoplasmic domain. Coligation of PIR-B with B cell antigen receptor (BCR) blocks antigen-induced B cell activation. This inhibition is mediated in part by recruitment of SH2-containing tyrosine phosphatases SHP-1 and SHP-2 to the phosphorylated ITIMs in the cytoplasmic domain of PIR-B. PIR-B ligation inhibits the BCR-induced tyrosine phosphorylation of Igα/Igβ, Syk, Btk, and phospholipase C (PLQ-γ2. Overexpression of a catalytically inactive form of SHP-1 prevents the PIR-B-mediated inhibition of tyrosine phosphorylation of Syk, Btk, and PLC-γ2. Dephosphorylation of Syk and Btk mediated by SHP-1 leads to a decrease of their kinase activity, which in turn inhibits tyrosine phosphorylation of PLC-γ2. In addition, Lyn is required for tyrosine phosphorylation of PIR-B.