Synergistic regulation of immunoreceptor signaling by SLP-76-related adaptor Clnk and serine/threonine protein kinase HPK-1.

Recently, the identification of Clnk, a third member of the SLP-76 family of adaptors expressed exclusively in cytokine-stimulated hemopoietic cells, has been reported by us and by others. Like SLP-76 and Blnk, Clnk was shown to act as a positive regulator of immunoreceptor signaling. Interestingly, however, it did not detectably associate with known binding partners of SLP-76, including Vav, Nck, and GADS. In contrast, it became complexed in activated T cells and myeloid cells with an as yet unknown tyrosine-phosphorylated polypeptide of approximately 92 kDa (p92). In order to understand better the function of Clnk, we sought to identify the Clnk-associated p92. Using a yeast two-hybrid screen and cotransfection experiments with Cos-1 cells, evidence was adduced that p92 is HPK-1, a serine/threonine-specific protein kinase expressed in hemopoietic cells. Further studies showed that Clnk and HPK-1 were also associated in hemopoietic cells and that their interaction was augmented by immunoreceptor stimulation. A much weaker association was detected between HPK-1 and SLP-76. Transient transfections in Jurkat T cells revealed that Clnk and HPK-1 cooperated to increase immunoreceptor-mediated activation of the interleukin 2 (IL-2) promoter. Moreover, the ability of Clnk to stimulate IL-2 promoter activity could be blocked by expression of a kinase-defective version of HPK-1. Lastly we found that in spite of the differential ability of Clnk and SLP-76 to bind cellular proteins, Clnk was apt at rescuing immunoreceptor signaling in a Jurkat T-cell variant lacking SLP-76. Taken together, these results show that Clnk physically and functionally interacts with HPK-1 in hemopoietic cells. Moreover, they suggest that Clnk is capable of functionally substituting for SLP-76 in immunoreceptor signaling, albeit by using a distinct set of intracellular effectors.

Regulation of SLAM-mediated signal transduction by SAP, the X-linked lymphoproliferative gene product.

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is a short intracellular molecule that is mutated in humans with X-linked lymphoproliferative (XLP) disease. Although the exact role and mechanism of action of SAP are not known, it has the capacity to interact with the cytoplasmic region of SLAM and other related immune cell receptors. As SAP is composed almost exclusively of a Src homology 2 (SH2) domain, it has been proposed that it functions as a natural blocker of SH2 domain--mediated interactions. We report here that the SLAM receptor is capable of triggering a protein tyrosine phosphorylation signal in T cells via a mechanism that is strictly dependent on SAP expression. This signal involves the SH2 domain--containing inositol phosphatase (SHIP); the adaptor molecules Dok2, Dok1 and Shc; and Ras GTPase--activating protein RasGAP. SAP is essential for this pathway because it facilitates the selective recruitment and activation of the Src-related protein tyrosine kinase FynT. We also show that signaling via the SLAM-SAP pathway in an established T cell line can alter the profile of cytokine production during T cell activation. These findings identify a mechanism by which a putative adaptor molecule is required for receptor-mediated signaling events in the immune system. They also provide insights into the pathophysiology of a severe human lymphoproliferative disease.

PTP-PEST, a scaffold protein tyrosine phosphatase, negatively regulates lymphocyte activation by targeting a unique set of substrates.

There is increasing interest in elucidating the mechanisms involved in the negative regulation of lymphocyte activation. Herein, we show that the cytosolic protein tyrosine phosphatase PTP-PEST is expressed abundantly in a wide variety of haemopoietic cell types, including B cells and T cells. In a model B-cell line, PTP-PEST was found to be constitutively associated with several signalling molecules, including Shc, paxillin, Csk and Cas. The interaction between Shc and PTP-PEST was augmented further by antigen receptor stimulation. Overexpression studies, antisense experiments and structure-function analyses provided evidence that PTP-PEST is an efficient negative regulator of lymphocyte activation. This function correlated with the ability of PTP-PEST to induce dephosphorylation of Shc, Pyk2, Fak and Cas, and inactivate the Ras pathway. Taken together, these data suggest that PTP-PEST is a novel and unique component of the inhibitory signalling machinery in lymphocytes.

Proximal protein tyrosine kinases in immunoreceptor signaling.

Immunoreceptor engagement results in the sequential activation of several classes of protein tyrosine kinases, including the Src and Syk/Zap-70 families. Recent progress has been made in our understanding of the regulation and function of these molecules. First, it was revealed that membrane compartmentation of protein tyrosine kinases may be essential for their proper biological function. Second, Src family kinases were found to act not only as positive regulators, but also as inhibitors of cell activation. Third, it was appreciated that Csk, a potent inhibitor of Src kinases, is regulated by an assortment of protein-protein interactions. Fourth, differences in the regulation of Syk and Zap-70 were observed, suggesting significant distinctions in the purpose of these two kinases in immunoreceptor signaling. And fifth, it was suggested that proximal kinases implicated in immunoreceptor-mediated signal transduction may be regulated by protein degradation via binding to c-Cbl, a ubiquitin ligase.

SH2 domain-mediated interaction of inhibitory protein tyrosine kinase Csk with protein tyrosine phosphatase-HSCF.

The protein tyrosine kinase (PTK) Csk is a potent negative regulator of several signal transduction processes, as a consequence of its exquisite ability to inactivate Src-related PTKs. This function requires not only the kinase domain of Csk, but also its Src homology 3 (SH3) and SH2 regions. We showed previously that the Csk SH3 domain mediates highly specific associations with two members of the PEP family of nonreceptor protein tyrosine phosphatases (PTPs), PEP and PTP-PEST. In comparison, the Csk SH2 domain interacts with several tyrosine phosphorylated molecules, presumed to allow targetting of Csk to sites of Src family kinase activation. Herein, we attempted to understand better the regulation of Csk by identifying ligands for its SH2 domain. Using a modified yeast two-hybrid screen, we uncovered the fact that Csk associates with PTP-HSCF, the third member of the PEP family of PTPs. This association was documented not only in yeast cells but also in a heterologous mammalian cell system and in cytokine-dependent hemopoietic cells. Surprisingly, the Csk-PTP-HSCF interaction was found to be mediated by the Csk SH2 domain and two putative sites of tyrosine phosphorylation in the noncatalytic portion of PTP-HSCF. Transfection experiments indicated that Csk and PTP-HSCF synergized to inhibit signal transduction by Src family kinases and that this cooperativity was dependent on the domains mediating their association. Finally, we obtained evidence that PTP-HSCF inactivated Src-related PTKs by selectively dephosphorylating the positive regulatory tyrosine in their kinase domain. Taken together, these results demonstrate that part of the function of the Csk SH2 domain is to mediate an inducible association with a PTP, thereby engineering a more efficient inhibitory mechanism for Src-related PTKs. Coupled with previously published observations, these data also establish that Csk forms complexes with all three known members of the PEP family.

Regulation of SOCS-1 expression by translational repression.

Accumulating evidence demonstrates that cytokine receptor signaling is negatively regulated by a family of Src homology 2 domain-containing adaptor molecules termed SOCS (suppressor of cytokine signaling). Previous studies have indicated that the expression of SOCS-related molecules is tightly controlled at the level of transcription. Furthermore, it has been reported that SOCS polypeptides are relatively unstable in cells, unless they are associated with elongins B and C. Herein, we document the existence of a third mechanism of regulation of SOCS function. Our data showed that expression of SOCS-1, a member of the SOCS family, is strongly repressed at the level of translation initiation. Structure-function analyses indicated that this effect is mediated by the 5' untranslated region of socs-1 and that it relates to the presence of two upstream AUGs in this region. Further studies revealed that socs-1 translation is cap-dependent and that it is modulated by eIF4E-binding proteins. In combination, these results uncover a novel level of regulation of SOCS-related molecules. Moreover, coupled with previous findings, they suggest that SOCS expression is tightly regulated through multiple mechanisms, in order to avoid inappropriate interference with cytokine-mediated effects.

Dok-3, a novel adapter molecule involved in the negative regulation of immunoreceptor signaling.

Adapters are typically viewed as molecules coordinating the recruitment of positive effectors of cell signaling. Herein, we report the identification of Dok-3, a novel adapter molecule belonging to the Dok family. Our studies show that Dok-3 is highly expressed in several hemopoietic cell types, including B cells and macrophages. It undergoes rapid tyrosine phosphorylation in response to immunoreceptor-mediated cellular activation, seemingly as a result of the action of Src family kinases. This phosphorylation induces the binding of Dok-3 to at least two inhibitory molecules, the 5' inositol phosphatase SHIP and the protein tyrosine kinase Csk. We also demonstrate that augmented expression of wild-type Dok-3 in a B-cell line results in an inhibition of immunoreceptor-mediated nuclear factor of activated T-cells (NFAT) activation and cytokine release, while introduction of a Dok-3 mutant with impaired ability to associate with SHIP and Csk enhances B-cell responsiveness. Taken together, these results indicate that Dok-3 is an adapter involved in the recruitment of inhibitory molecules and that it may play a significant role in the negative regulation of immunoreceptor signaling in hemopoietic cells such as B cells and macrophages.

The noncatalytic domain of protein-tyrosine phosphatase-PEST targets paxillin for dephosphorylation in vivo.

The noncatalytic domain of protein-tyrosine phosphatase (PTP)-PEST contains a binding site for the focal adhesion-associated protein paxillin. This binding site has been narrowed to a 52-residue sequence that is composed of two nonoverlapping, weak paxillin binding sites. The PTP-PEST binding site on paxillin has been mapped to the two carboxyl-terminal LIM (lin11, isl-1, and mec-3) domains. Transient expression of PTP-PEST reduced tyrosine phosphorylation of p130(cas), as anticipated. A PTP-PEST mutant defective for binding p130(cas) does not cause a reduction in its tyrosine phosphorylation in vivo. Expression of PTP-PEST also caused a reduction of phosphotyrosine on paxillin. Expression of mutants of PTP-PEST with deletions in the paxillin-binding site did not associate with paxillin in vivo and failed to cause a reduction in the phosphotyrosine content of paxillin. These results demonstrate that paxillin can serve as a PTP-PEST substrate in vivo and support the model that a noncatalytic domain interaction recruits paxillin to PTP-PEST to facilitate its dephosphorylation.

Clnk, a novel SLP-76-related adaptor molecule expressed in cytokine-stimulated hemopoietic cells.

We have identified a novel Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76)-related molecule which we have termed Clnk (for cytokine-dependent hemopoietic cell linker). Unlike its relatives SLP-76 and B cell linker protein (Blnk), Clnk is not expressed uniformly within a given hemopoietic cell lineage. Even though it can be detected in several cell types, including T cells, natural killer cells, and mast cells, its expression seems to be strictly dependent on sustained exposure to cytokines such as interleukin (IL)-2 and IL-3. Strong support for the notion that Clnk is involved in immunoreceptor signaling was provided by the observation that it inducibly associated with at least one tyrosine-phosphorylated polypeptide (p92) in response to immunoreceptor stimulation. Moreover, transient expression of Clnk caused an increase in immunoreceptor-mediated signaling events in a T cell line. Taken together, these results show that Clnk is a novel member of the SLP-76 family selectively expressed in cytokine-stimulated hemopoietic cells. Furthermore, they suggest that Clnk may be involved in a cross-talk mechanism between cytokine receptor and immunoreceptor signaling.

TCR alpha beta-independent CD28 signaling and costimulation require non-CD4-associated Lck.

Whether the sequelae of signals generated through CD28 either directly or in circumstances of costimulation require proximal events mediated by p56lck remains contentious. We demonstrate that CD4-, but not CD4+ clonal variants respond to CD28-specific mAb with both early and late indicators of activation. Forced expression of A418/A420-mutated CD4 or wild-type CD4 in the CD4- variant recapitulated the CD28-mediated responses of the CD4- and CD4+ variants, respectively. The implicated involvement of non-CD4-associated Lck is formally demonstrated by overexpressing S20/S23 Lck or wild-type Lck in CD4+ variants. The former, but not latter, rescues direct CD28 signaling, and supports costimulation. The results demonstrate that constitutive levels of non-CD4-associated Lck functionally limit CD28-mediated signaling.

Interactions of CD45-associated protein with the antigen receptor signaling machinery in T-lymphocytes.

CD45 is a transmembrane protein tyrosine phosphatase playing an essential role during T-cell activation. This function relates to the ability of CD45 to regulate p56(lck), a cytoplasmic protein tyrosine kinase necessary for T-cell antigen receptor (TCR) signaling. Previous studies have demonstrated that CD45 is constitutively associated in T-lymphocytes with a transmembrane molecule termed CD45-AP (or lymphocyte phosphatase-associated phosphoprotein). Even though the exact role of this polypeptide is unclear, recent analyses of mice lacking CD45-AP have indicated that its expression is also required for optimal T-cell activation. Herein, we wished to understand better the function of CD45-AP. The results of our studies showed that in T-cells, CD45-AP is part of a multimolecular complex that includes not only CD45, but also TCR, the CD4 and CD8 coreceptors, and p56(lck). The association of CD45-AP with TCR, CD4, and CD8 seemed to occur via the shared ability of these molecules to bind CD45. However, binding of CD45-AP to p56(lck) could take place in the absence of other lymphoid-specific components, suggesting that it can be direct. Structure-function analyses demonstrated that such an interaction was mediated by an acidic segment in the cytoplasmic region of CD45-AP and by the kinase domain of p56(lck). Interestingly, the ability of CD45-AP to interact with Lck in the absence of other lymphoid-specific molecules was proportional to the degree of catalytic activation of p56(lck). Together, these findings suggest that CD45-AP is an adaptor molecule involved in orchestrating interactions among components of the antigen receptor signaling machinery. Moreover, they raise the possibility that one of the functions of CD45-AP is to recognize activated Lck molecules and bring them into the vicinity of CD45.

Cooperative inhibition of T-cell antigen receptor signaling by a complex between a kinase and a phosphatase.

Antigen receptor-triggered T-cell activation is mediated by the sequential action of the Src and Syk/Zap-70 families of protein tyrosine kinases (PTKs). Previously, we reported that another PTK termed p50(csk) was a potent negative regulator of T-cell receptor (TCR) signaling because of its ability to inactivate Src-related kinases. This inhibitory effect required the catalytic activity of Csk, as well as its Src homology (SH)3 and SH2 domains. Subsequent studies uncovered that, via its SH3 domain, p50(csk) was associated with PEP, a proline-enriched protein tyrosine phosphatase (PTP) of unknown function expressed in hemopoietic cells. Herein, we have attempted to identify the role of the Csk-PEP complex in T lymphocytes. The results of our experiments showed that, like Csk, PEP was a strong repressor of TCR signaling. This property was dependent on the phosphatase activity of PEP, as well as on the sequence mediating its binding to p50(csk). Through reconstitution experiments in Cos-1 cells, evidence was obtained that Csk and PEP act synergistically to inhibit protein tyrosine phosphorylation by Src-related kinases, and that this effect requires their association. Finally, experiments with a substrate-trapping mutant of PEP suggested that PEP functions by dephosphorylating and inactivating the PTKs responsible for T-cell activation. In addition to giving novel insights into the mechanisms involved in the negative regulation of T-cell activation, these findings indicate that the association of an inhibitory PTK with a PTP constitutes a more efficient means of inhibiting signal transduction by Src family kinases in vivo.

The carboxyl-terminal region of biliary glycoprotein controls its tyrosine phosphorylation and association with protein-tyrosine phosphatases SHP-1 and SHP-2 in epithelial cells.

Biliary glycoprotein (Bgp, C-CAM, or CD66a) is an immunoglobulin-like cell adhesion molecule and functions as a tumor suppressor protein. We have previously shown that the Bgp1 isoform responsible for inhibition of colonic, liver, prostate, and breast tumor cell growth contains within its cytoplasmic domain two tyrosine residues positioned in immunoreceptor tyrosine-based inhibition motif (ITIM) consensus sequences. Moreover, we determined that these residues, upon phosphorylation, associate with the protein-tyrosine phosphatase SHP-1. In this report, we have further evaluated the structural bases of the association of Bgp1 with Tyr phosphatases. First, we demonstrate that Bgp1 also associates with the SHP-2 Tyr phosphatase, but not with an unrelated Tyr phosphatase, PTP-PEST. Association of Bgp1 and SHP-2 involves the Tyr residues within the Bgp1 ITIM sequences, Val at position +3 relative to the second Tyr (Tyr-515), and the SHP-2 N-terminal SH2 domain. In addition, our results indicate that residues +4, +5, and +6 relative to Tyr-515 in the Bgp1 cytoplasmic domain play a significant role in these interactions, as their deletion reduced Bgp1 Tyr phosphorylation and association with SHP-1 and SHP-2 by as much as 80%. Together, these results indicate that both SHP-1 and SHP-2 interact with the Bgp1 cytoplasmic domain via ITIM-like sequences. Furthermore, they reveal that the C-terminal amino acids of Bgp1 are critical for these interactions.

Interleukin 2-mediated uncoupling of T cell receptor alpha/beta from CD3 signaling.

T cell activation and clonal expansion is the result of the coordinated functions of the receptors for antigen and interleukin (IL)-2. The protein tyrosine kinase p56(lck) is critical for the generation of signals emanating from the T cell antigen receptor (TCR) and has also been demonstrated to play a role in IL-2 receptor signaling. We demonstrate that an IL-2-dependent, antigen-specific CD4(+) T cell clone is not responsive to anti-TCR induced growth when propagated in IL-2, but remains responsive to both antigen and CD3epsilon-specific monoclonal antibody. Survival of this IL-2-dependent clone in the absence of IL-2 was supported by overexpression of exogenous Bcl-xL. Culture of this clonal variant in the absence of IL-2 rendered it susceptible to anti-TCR-induced signaling, and correlated with the presence of kinase-active Lck associated with the plasma membrane. The same phenotype is observed in primary, resting CD4(+) T cells. Furthermore, the presence of kinase active Lck associated with the plasma membrane correlates with the presence of ZAP 70-pp21zeta complexes in both primary T cells and T cell clones in circumstances of responsive anti-TCR signaling. The results presented demonstrate that IL-2 signal transduction results in the functional uncoupling of the TCR complex through altering the subcellular distribution of kinase-active Lck.

Zinc is essential for binding of p56(lck) to CD4 and CD8alpha.

Binding of the protein tyrosine kinase p56(lck) to T-cell co-receptors CD4 and CD8alpha is necessary for T-lymphocyte development and activation. Association of p56(lck) with CD4 requires two conserved cysteine residues in the cytosolic domain of CD4 and two in the amino terminus of p56(lck), consistent with the notion that these four residues coordinate a single metal atom (1-5). Here we demonstrate that Zn2+ is essential for complex formation. In an in vitro binding reaction, Zn2+ mediates p56(lck) association with a glutathione S-transferase (GST) fusion protein containing the cytosolic domains of CD4 or CD8alpha; no other metals tested support binding. Treatment of preformed GST-CD4.p56(lck) dimers with the Zn2+ chelators 1,10-O-phenanthroline or 8-hydroxyquinoline-5-sulfonic acid results in dissociation of GST-CD4 from p56(lck), consistent with the finding of Huse et al. (5) that Zn2+ is contained within similar complexes. Furthermore, we show that, within live cells, CD4.p56(lck) and CD8alpha.p56(lck) interactions occur in a zinc-dependent fashion. Specifically, pretreatment of the human Jurkat T-cell line with membrane permeable zinc chelators disrupts CD4.p56(lck) complexes, and treatment of COS cells co-expressing CD8alpha and p56(lck) with such chelators likewise leads to dissociation of CD8alpha.p56(lck) complexes. CD4. p56(lck) and CD8alpha.p56(lck) represent the first examples of intracellular proteins that require zinc as a bridge for heterodimerization.

High expression of inhibitory receptor SHPS-1 and its association with protein-tyrosine phosphatase SHP-1 in macrophages.

SHPS-1 (or SIRP) is a member of the immunoglobulin (Ig) superfamily abundantly expressed in neurons and other cell types. Within its cytoplasmic domain, it possesses at least two immunoreceptor tyrosine-based inhibitory motifs, which are targets for tyrosine phosphorylation and mediate the recruitment of SHP-2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase. Since other immunoreceptor tyrosine-based inhibitory motifs-containing receptors have critical roles in the negative regulation of hemopoietic cell functions, we wanted to examine the expression of SHPS-1 in cells of hematological lineages. By analyzing a panel of hemopoietic cell lines, evidence was provided that SHPS-1 is abundantly expressed in macrophages and, to a lesser extent, in myeloid cells. No expression was detected in T-cell or B-cell lines. Expression of SHPS-1 could also be documented in normal ex vivo peritoneal macrophages. Further studies showed that SHPS-1 was an efficient tyrosine phosphorylation substrate in macrophages. However, unlike in non-hemopoietic cells, tyrosine-phosphorylated SHPS-1 in macrophages associated primarily with SHP-1 and not SHP-2. Finally, our analyses allowed us to identify several isoforms of SHPS-1 in mouse cells. In part, this heterogeneity was due to differential glycosylation of SHPS-1. Additionally, it was caused by the production of at least two distinct shps-1 transcripts, coding for SHPS-1 polypeptides having different numbers of Ig-like domains in the extracellular region. Taken together, these findings indicate that SHPS-1 is likely to play a significant role in macrophages, at least partially as a consequence of its capacity to recruit SHP-1.

De novo-developed T cells have compromised response to existing alloantigens: using Ld-specific transgenic 2C T cells as tracers in a mouse heart transplantation model.

In this study, the phenotype, TCR signaling events, and function of T cells developed de novo during adulthood in the presence of extrathymic alloantigen were investigated. C57BL/6 mice(H-2b) were first transplanted heterotopically with BALB/c hearts (H-2d) and treated with rapamycin for 2 wk to create a tolerant status. Three weeks postoperation, the mice were whole body irradiated and transplanted with bone marrow cells from 2C mice, which are transgenic for TCR, and most of their T cells are Ld-specific CD8 cells. The 2C T cells developed de novo in the C57BL/6 mice were not able to reject the heart allograft. No clonal deletion, TCR down-regulation, or CD8 down-regulation was found in the tolerized 2C T cells. There was no characteristic phenotype of these cells in terms of CD25, ICAM-1, CD44, and MEL-14 expression. Early TCR signaling events such as intracellular calcium concentration flux, tyrosine phosphorylation, Lck and Fyn kinase activities, and Lck and Fyn protein levels in the tolerized 2C T cells were comparable to their normal counterparts, but the tolerized T cells were defective in IL-2 production and proliferation upon H-2d alloantigen stimulation in vitro. Exogenous IL-2 could not reverse the compromised proliferation. The results of this study indicate that during adulthood, the de novo-developed T cells become tolerant to extrathymic Ag without clonal deletion. These newly minted T cells are functionally defective although they are indistinguishable from normal T cells in phenotypes and in some early signaling events.

Regulation of mouse PECAM-1 tyrosine phosphorylation by the Src and Csk families of protein-tyrosine kinases.

PECAM-1 is an adhesion molecule expressed on hemopoietic and endothelial cells. Recently, it was observed that PECAM-1 becomes tyrosine-phosphorylated in response to a variety of physiological stimuli. Furthermore, tyrosine-phosphorylated PECAM-1 was shown to associate with SHP-2, a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase expressed ubiquitously. In light of the significance of tyrosine protein phosphorylation as a regulatory mechanism, we wished to understand better the nature and impact of the protein-tyrosine kinases (PTKs) mediating PECAM-1 tyrosine phosphorylation. Through reconstitution experiments in COS-1 cells, we determined that mouse PECAM-1 could be tyrosine-phosphorylated by Src-related PTKs and Csk-related PTKs, but not by other kinases such as Syk, Itk, and Pyk2. Using site-directed mutagenesis and peptide phosphorylation studies, we found that these PTKs were efficient at phosphorylating Tyr-686, but not Tyr-663, of PECAM-1. Src-related enzymes also phosphorylated mouse PECAM-1 at one or more yet to be identified sites. In other studies, we demonstrated that phosphorylation of PECAM-1 by Src or Csk family kinases was sufficient to trigger its association with SHP-2. Moreover, it was able to promote binding of PECAM-1 to SHP-1, a SHP-2-related protein-tyrosine phosphatase expressed in hemopoietic cells. Taken together, these findings indicated that the Src and Csk families of kinases are strong candidates for mediating tyrosine phosphorylation of PECAM-1 and triggering its association with SH2 domain-containing phosphatases under physiological circumstances.

A unique insert in the linker domain of Syk is necessary for its function in immunoreceptor signalling.

Accumulating data indicate that the 'linker' region of Syk, which lies between its tandem Src homology 2 (SH2) domains and kinase region, provides a critical function for the biological activity of Syk. This importance has been ascribed to the presence of tyrosine phosphorylation sites capable of mediating the recruitment of cellular effectors. We and others previously identified an alternatively spliced variant of Syk, termed SykB, which lacks a 23 amino acid sequence in the linker domain. As this 'linker insert' is also not present in the closely related enzyme Zap-70, it seems plausible that Syk possesses this unique sequence for functional reasons. To understand its role better, we have compared the abilities of Syk and SykB to participate in immunoreceptor-triggered signal transduction. The results of our experiments revealed that, unlike Syk, SykB was inefficient at coupling stimulation of FcepsilonRI on basophils or the antigen receptor on T cells to the early and late events of cellular activation. Further studies showed that the functional defect in SykB was not caused by the absence of crucial tyrosine phosphorylation sites, or by a reduced intrinsic kinase activity. Rather, it correlated with the reduced ability of SykB to bind phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) in vitro and in vivo. In combination, these results demonstrated that the unique insert in the linker domain of Syk is crucial for its capacity to participate in immunoreceptor signalling. Furthermore, they provided evidence that the linker region can regulate the ability of Syk to bind ITAMs, thus identifying a novel function for this domain.

The Syk protein tyrosine kinase is essential for Fcgamma receptor signaling in macrophages and neutrophils.

The cytoplasmic protein tyrosine kinase Syk has two amino-terminal SH2 domains that engage phosphorylated immunoreceptor tyrosine-based activation motifs in the signaling subunits of immunoreceptors. Syk, in conjunction with Src family kinases, has been implicated in immunoreceptor signaling in both lymphoid and myeloid cells. We have investigated the role of Syk in Fcgamma receptor (FcgammaR)-dependent and -independent responses in bone marrow-derived macrophages and neutrophils by using mouse radiation chimeras reconstituted with fetal liver cells from Syk-/- embryos. Chimeric mice developed an abdominal hemorrhage starting 2 to 3 months after transplantation that was ultimately lethal. Syk-deficient neutrophils derived from the bone marrow were incapable of generating reactive oxygen intermediates in response to FcgammaR engagement but responded normally to tetradecanoyl phorbol acetate stimulation. Syk-deficient macrophages were defective in phagocytosis induced by FcgammaR but showed normal phagocytosis in response to complement. The tyrosine phosphorylation of multiple cellular polypeptides, including the FcgammaR gamma chain, as well as Erk2 activation, was compromised in Syk-/- macrophages after FcgammaR stimulation. In contrast, the induction of nitric oxide synthase in macrophages stimulated with lipopolysaccharide and gamma interferon was not dependent on Syk. Surprisingly, Syk-deficient macrophages were impaired in the ability to survive or proliferate on plastic petri dishes. Taken together, these results suggest that Syk has specific physiological roles in signaling from FcgammaRs in neutrophils and macrophages and raise the possibility that in vivo, Syk is involved in signaling events other than those mediated by immunoreceptors.