Expression of dominant-negative src-homology domain 2-containing protein tyrosine phosphatase-1 results in increased Syk tyrosine kinase activity and B cell activation

The Src-homology domain 2 (SH2)-containing cytoplasmic tyrosine phosphatase, SHP-1 (SH2-containing protein tyrosine phosphatase-1), interacts with several B cell surface and intracellular signal transduction molecules through its SH2 domains. Mice with the motheaten and viable motheaten mutations are deficient in SHP-1 and lack most mature B cells. To define the role of SHP-1 in mature B cells, we expressed phosphatase-inactive SHP-1 (C453S) in a mature B cell lymphoma line. SHP-1 (C453S) retains the ability to bind to both substrates and appropriate tyrosine-phosphorylated proteins and therefore can compete with the endogenous wild-type enzyme. We found that B cells expressing SHP-1 (C453S) demonstrated enhanced and prolonged tyrosine phosphorylation of proteins with molecular masses of 110, 70, and 55-60 kDa after stimulation with anti-mouse IgG. The tyrosine kinase Syk was hyperphosphorylated and hyperactive in B cells expressing SHP-1 (C453S). SHP-1 and Syk were coimmunoprecipitated from wild-type K46 cells, K46 SHP-1 (C453S) cells, and splenic B cells, and SHP-1 dephosphorylated Syk. Cells expressing SHP-1 (C453S) showed increased Ca2+ mobilization, extracellular signal-regulated kinase activation, and homotypic adhesion after B cell Ag receptor engagement. Thus, SHP-1 regulates multiple early and late events in B lymphocyte activation.     

Receptor activator of NF-kappa B ligand stimulates recruitment of SHP-1 to the complex containing TNFR-associated factor 6 that regulates osteoclastogenesis

Receptor activator of NF-kappaB ligand (RANKL) is essential for differentiation and function of osteoclasts. The negative signaling pathways downstream of RANKL are not well characterized. By retroviral transduction of RAW264.7 cells with a dominant negative Src homology 2 domain-containing phosphatase-1 (SHP-1)(C453S), we studied the role of tyrosine phosphatase SHP-1 in RANKL-induced osteoclastogenesis. Over-expression of SHP-1(C453S) significantly enhanced the number of tartrate-resistant acid phosphatase-positive multinuclear osteoclast-like cells in response to RANKL in a dose-dependent manner. RANKL induced the recruitment of SHP-1 to a complex containing TNFR-associated factor (TRAF)6. GST pull down experiments indicated that the association of SHP-1 with TRAF6 is mediated by SHP-1 lacking the two Src homology 2 domains. RANKL-stimulated IkappaB-alpha phosphorylation, IkappaB-alpha degradation and DNA binding ability of NF-kappaB were increased after over-expression of SHP-1(C453S). However, RANKL-induced phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, was unchanged. In addition, SHP-1 regulated RANKL-stimulated tyrosine phosphorylation of p85 subunit of phosphatidylinositol 3 kinase and the phosphorylation of Akt. Increased numbers of osteoclasts contribute to severe osteopenia in Me(v)/Me(v) mice due to mutation of SHP-1. Like RAW264.7 cells expressing SHP-1(C453S), the bone marrow macrophages of Me(v)/Me(v) mice generated much more osteoclast-like cells than that of littermate controls in response to RANKL. Furthermore compared with controls, RANKL induces enhanced association of TRAF6 and RANK in both RAW264.7 cells expressing SHP-1(C453S) and bone marrow macrophages from Me(v)/Me(v) mice. Therefore, SHP-1 plays a role in signals downstream of RANKL by recruitment to the complex containing TRAF6 and these observations may help to understand the mechanism of osteoporosis in Me(v)/Me(v) mice.     

Involvement of the SHP-1 tyrosine phosphatase in regulation of T cell selection.

The selection events shaping T cell development in the thymus represent the outcome of TCR-driven intracellular signaling cascades evoked by Ag receptor interaction with cognate ligand. In view of data indicating TCR-evoked thymocyte proliferation to be negatively modulated by the SHP-1 tyrosine phosphatase, a potential role for SHP-1 in regulating selection processes was investigated by analysis of T cell development in H-Y TCR transgenic mice rendered SHP-1 deficient by introduction of the viable motheaten mutation or a dominant negative SHP-1-encoding transgene. Characterization of thymocyte and peripheral T cell populations in H-Y TCR-viable motheaten mice revealed TCR-evoked proliferation as well as the positive and negative selection of H-Y-specific thymocytes to be enhanced in these mice, thus implicating SHP-1 in the negative regulation of each of these processes. T cell selection processes were also augmented in H-Y TCR mice carrying a transgene driving lymphoid-restricted expression of a catalytically inert, dominant-negative form of SHP-1. SHP-1-negative effects on thymocyte TCR signaling were not influenced by co-cross-linking of the CD28 costimulatory and/or CTLA-4 inhibitory receptors and appear, accordingly, to be realized independently of these comodulators. These observations indicate that SHP-1 raises the signaling threshold required for both positive and negative selection and reveal the inhibitory effects of SHP-1 on TCR signaling to be cell autonomous. The demonstrated capacity for SHP-1 to inhibit TCR-evoked proliferation and selection indicate SHP-1 modulatory effects on the magnitude of TCR-generated signal to be a key factor in determining the cellular consequences of TCR-ligand interaction.     

Negative regulation of myeloid cell proliferation and function by the SH2 domain-containing tyrosine phosphatase-1

The SH2 domain containing tyrosine phosphatase SHP-1 has been implicated in the regulation of a multiplicity of signaling pathways involved in hemopoietic cell growth, differentiation, and activation. A pivotal contribution of SHP-1 in the modulation of myeloid cell signaling cascades has been revealed by the demonstration that SHP-1 gene mutation is responsible for the overexpansion and inappropriate activation of myelomonocytic populations in motheaten mice. To investigate the role of SHP-1 in regulation of myeloid leukocytes, an HA epitope-tagged dominant negative (interfering) SHP-1 (SHP-1C453S) was expressed in the myelo-monocytic cell line U937 using the pcDNA3 vector. Overexpression of this protein in SHP-1C453S transfectants was demonstrated by Western blot analysis and by detection of decreased specific activity. Growth, proliferation, and IL-3-induced proliferative responses were substantially increased in the SHP-1C453S-overexpressing cells relative to those in control cells. The results of cell cycle analysis also revealed that the proportion of cells overexpressing SHP-1C453S in S phase was greater than that of control cells. The SHP-1C453S-expressing cells also displayed diminished rates of apoptosis as detected by flow cytometric analysis of propidium iodide-stained cells and terminal deoxynucleotidyltransferase-mediated fluorescein-dUTP nick end-labeling assay. While motility and phagocytosis were not affected by SHP-1C453S overexpression, adhesion and the oxidative burst in response to PMA were enhanced in the SHP-1C453S compared with those in the vector alone transfectants. Taken together, these results suggest that SHP-1 exerts an important negative regulatory influence on cell proliferation and activation while promoting spontaneous cell death in myeloid cells.     

Cd81 Interacts with the T Cell Receptor to Suppress Signaling

CD81 (TAPA-1) is a ubiquitously expressed tetraspanin protein identified as a component of the B lymphocyte receptor (BCR) and as a receptor for the Hepatitis C Virus. In an effort to identify trans-membrane proteins that interact with the T-cell antigen receptor (TCR), we performed a membrane yeast two hybrid screen and identified CD81 as an interactor of the CD3delta subunit of the TCR. We found that in the absence of CD81, in thymocytes from knockout mice, TCR engagement resulted in stronger signals. These results were recapitulated in T cell lines that express low levels of CD81 through shRNA mediated silencing. Increased signaling did not result from alterations in the levels of TCR on the surface of T lymphocytes. Although CD81 is not essential for normal T lymphocyte development, it plays an important role in regulating TCR and possibly pre-TCR signal transduction by controlling the strength of signaling. CD81 dependent alterations in thymocyte signaling are evident in increased CD5 expression on CD81 deficient double positive (DP) thymocytes. We conclude that CD81 interacts with the T cell receptor to suppress signaling.     

Fine tuning of TCR signaling by CD5

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5(-/-) mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5(-/-)) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5(-/-) phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.     

Src homology region 2 (SH2) domain-containing phosphatase-1 dephosphorylates B cell linker protein/SH2 domain leukocyte protein of 65 kDa and selectively regulates c-Jun NH2-terminal kinase activation in B cells

Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk. Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.     

TCR signaling thresholds regulating T cell development and activation are dependent upon SHP-1

An examination of thymocytes and peripheral T cells from SHP-1-deficient motheaten mice possessing a transgenic MHC class I-restricted TCR has implicated SHP-1 in regulating TCR signaling thresholds at three checkpoints in T cell development and activation. First, in the population of CD4-CD8- double negative thymocytes, SHP-1 appears capable of regulating signals from TCR complexes that control the maturation and proliferation of double negative thymocytes. Second, the loss of SHP-1 increased the number of CD4+CD8+ double positive thymocytes capable of maturing as TCRhigh single positive thymocytes. Third, the loss of SHP-1 altered the basal level of activation of naive lymph node T cells. Accordingly, SHP-1-deficient lymph node T cells bearing the transgenic TCR demonstrated a hyperresponsiveness to stimulation with cognate peptide. However, the loss of SHP-1 did not alter the cytolytic ability of mature effector cytotoxic T lymphocytes. Together these results suggest that SHP-1 contributes to establishing thresholds for TCR signaling in thymocytes and naive peripheral T cells.     

Developmental and functional impairment of T cells in mice lacking CD3 zeta chains.

CD3 zeta is a component of the T cell antigen receptor (TCR) complex and is important for signal transduction. We have established mice selectively lacking CD3 zeta but able to express CD3 eta, a polypeptide produced from the same locus through alternative splicing, using the method of gene targeting in embryonic stem cells. In homozygous mutant mice, the numbers of thymocytes and peripheral T cells were greatly reduced and the expression levels of TCR on these cells were 5-fold lower than those on wild-type cells. By contrast, TCR gamma delta+ intestinal intraepithelial lymphocytes were not obviously affected by the mutation. T cells from homozygous mutants exhibited an impaired proliferative response. These results imply that CD3 zeta has a critical role in the development and signal transduction of T cells in vivo.     

Protein kinase C-theta is required for efficient positive selection

Protein kinase C-theta (PKCtheta) is critical for TCR-initiated signaling in mature T cells, but initial reports found no requirement for PKCtheta in thymocyte development. Thymocytes and peripheral T cells utilize many of the same signaling components and, given the significant role of PKCtheta in peripheral T cells, it was surprising that it was not involved at all in TCR signaling in thymocytes. We decided to re-evaluate the role of PKCtheta in thymocyte development using the well-characterized class II-restricted n3.L2 TCR-transgenic TCR model. Analysis of n3.L2 PKCtheta(-/-) mice revealed a defect in thymocyte-positive selection, resulting in a 50% reduction in the generation of n3.L2 CD4 single-positive thymocytes and n3.L2 CD4 mature T cells. Competition between n3.L2 WT and n3.L2 PKCtheta(-/-) thymocytes in bone marrow chimeras revealed a more dramatic defect, with a >80% reduction in generation of n3.L2 CD4 single-positive thymocytes derived from PKCtheta(-/-) mice. Inefficient positive selection of n3.L2 PKCtheta(-/-) CD4 single-positive cells resulted from "weaker" signaling through the TCR and correlated with diminished ERK activation. The defect in positive selection was not complete in the PKCtheta(-/-) mice, most likely accounted for by compensation by other PKC isoforms not evident in peripheral cells. Similar decreased positive selection of both CD4 and CD8 single-positive thymocytes was also seen in nontransgenic PKCtheta(-/-) mice. These findings now place PKCtheta as a key signaling molecule in the positive selection of thymocytes as well as in the activation of mature T cells.     

Deficiency of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) causes enrichment of CD4+CD25+ regulatory T cells

A subpopulation of T cells, named regulatory T cells (T(reg) cells), has been shown to play a key role in tolerance and the prevention of autoimmunity. It is not known how changes in TCR signal strength during thymic T cell development affect the generation of a T(reg) population. In this study, we took two different strategies to modulate the TCR signal strength: an intrinsic approach, where signaling was enhanced by the loss of a negative regulator, and an extrinsic approach, where signaling strength was altered through variations in the concentrations of the selecting peptide. The tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a known negative regulator of TCR-mediated signaling. motheaten mice, lacking expression of SHP-1, showed a 2- to 3-fold increase in the percentage of CD4(+)CD25(+) T(reg) cells within the CD4(+) T cells. Similarly, the percentage of T(reg) cells was heightened in fetal thymic organ cultures (FTOCs) derived from motheaten mice compared with wild-type FTOCs, thus establishing the thymic origin of these T(reg) cells. Using FTOCs derived from DO11.10 TCR transgenic mice, we demonstrated that exposure to increasing concentrations of the cognate OVA peptide favored the appearance of T(reg) cells. Our data suggest that the development of CD4(+)CD25(+) T(reg) cells is intrinsically different from non-T(reg) cells and that T(reg) cells are selectively enriched under conditions of enhanced negative selection. Our data also reveal a key role for the SHP-1-mediated regulation of TCR signal strength in influencing the ratio of T(reg) vs non-T(reg) cells.     

Inhibition of Rho at different stages of thymocyte development gives different perspectives on Rho function.

Development of thymocytes can be staged according to the levels of expression of the cell-surface markers CD4, CD8, CD44, CD25 and CD2. Thymocyte development is regulated by a complex signalling network [1], one component of which is the GTPase Rho. The bacterial enzyme C3 transferase from Clostridium botulinum selectively ADP-ribosylates Rho in its effector-binding domain and thereby abolishes its biological function [2,3]. To explore the function of Rho in thymocyte development, we previously used the proximal promoter of the gene encoding the Src-family kinase p56lck to make transgenic mice that selectively express C3 transferase in the thymus [4,6]. In these mice, which lack Rho function from the earliest thymocyte stages, thymocyte numbers are reduced by approximately 50- to 100-fold. Here, we describe transgenic mice that express C3 transferase under the control of the locus control region (LCR) of the CD2 gene; this regulatory element drives expression at a later stage of thymocyte development than the lck proximal promoter [7]. In these mice, thymocyte numbers were also reduced by 50- to 100-fold, but unlike the lck-C3 mice, in which the reduction predominantly results from defects in cell survival of CD25(+) thymocyte progenitors, the CD2-C3 transgenic mice had a pre-T-cell differentiation block at the CD25(+) stage after rearrangement of the T-cell receptor (TCR) beta chains. Analysis of CD2-C3 mice demonstrated that Rho acts as an intracellular switch for TCR beta selection, the critical thymic-differentiation checkpoint. These results show that Rho-mediated survival signals for CD25(+) pre-T cells are generated by the extracellular signals that act on earlier thymocyte precursors and also that temporal cell-type-specific elimination of Rho can reveal different functions of this GTPase in vivo.     

Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant-negative p21ras protein (H-rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck-H-rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR-derived signals in mature CD4+8- or CD8+4- thymocytes. In contrast, some TCR-derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant-negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H-Y-specific TCR and lck-H-rasN17 demonstrated that antigen-specific negative selection occurs normally in the presence of p21H-rasN17. Superantigen-induced negative selection in vivo also proceeded unhindered in H-rasN17 thymocytes. In contrast, positive selection of thymocytes in the H-Y mice was severely compromised by the presence of p21H-rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.     

The Functional Role of B7 Molecules on the Induction of Thymocyte Activation and Apoptosis

To evaluate the role of B7 on thymocyte activation and apoptosis, we took advantage of TCR transgenic mice in which the majority of thymocytes express a uniform TCR that is specific for ovalbumin. We also prepared Chinese hamster ovary (CHO) cells expressing B7 and appropriate class II molecules. We found that the apoptosis of double-positive thymocytes by TCR-mediated signaling, which presumably represents negative selection, requires a costimulatory signal provided by B7-1 or B7-2. The requirement of B7-1 costimulation for the apoptosis of thymocytes does not change in either low or high antigenic peptide loading. We also demonstrated that two signals through TCR and CD28 augmented the proliferation of thymocytes, and the requirement of CD28-mediated signal by B7-1 or B7-2 for thymocyte proliferation became less evident when high doses of antigenic peptide were loaded, indicating that the intensity of TCR-mediated signal determines the requirement of B7-mediated second signal for thymocyte proliferation.     

Effects of a leukemia-associated gain-of-function mutation of SHP-2 phosphatase on interleukin-3 signaling

Mutations in SHP-2 phosphatase that cause hyperactivation of its catalytic activity have been identified in human leukemias, particularly juvenile myelomonocytic leukemia, which is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce hematopoietic malignancies are not fully understood. Our previous studies have shown that SHP-2 plays an essential role in IL-3 signal transduction in both catalytic-dependent and -independent manners and that overexpression (5-6-fold) of wild type (WT) SHP-2 attenuates IL-3-mediated hematopoietic cell function through accelerated dephosphorylation of STAT5. These results raised the possibility that SHP-2-associated leukemias are not solely attributed to the increased catalytic activity of GOF mutant SHP-2. GOF mutant SHP-2 must have gained additional capacities. To test this possibility, we investigated effects of a GOF mutation of SHP-2 (SHP-2 E76K) on hematopoietic cell function and IL-3 signal transduction by comparing with those of overexpressed WT SHP-2. Our results showed that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreased hematopoietic potential of the transduced cells in recipient animals. The E76K mutation in the N-terminal Src homology 2 domain increased interactions of mutant SHP-2 with Grb2, Gab2, and p85, leading to hyperactivation of IL-3-induced Erk and phosphatidylinositol 3-kinase (PI3K) pathways. In addition, despite the substantial increase in the catalytic activity, dephosphorylation of STAT5 by SHP-2 E76K was dampened. Furthermore, catalytically inactive SHP-2 E76K with an additional C459S mutation retained the capability to increase the interaction with Gab2 and to enhance the activation of the PI3K pathway. Taken together, these studies suggest that in addition to the elevated catalytic activity, fundamental changes in physical and functional interactions between GOF mutant SHP-2 and signaling partners also play an important role in SHP-2-related leukemigenesis.     

Caspase enzyme activity is not essential for apoptosis during thymocyte development

Caspases, a family of cysteine proteases, are critical mediators of apoptosis. To address the importance of caspases in thymocyte development, we have generated transgenic mice that express the baculovirus protein p35, a viral caspase inhibitor, specifically in the thymus. p35 expression inhibited Fas (CD95)-, CD3-, or peptide-induced caspase activity in vitro and conferred resistance to Fas-induced apoptosis. However, p35 did not block specific peptide-induced negative selection in OT1 and HY TCR transgenic mouse models. Even the potent pharmacological caspase inhibitor zVAD-FMK (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone) could not prevent peptide-induced deletion of OT1 thymocytes, although it improved basal thymocyte survival in vitro. Moreover, the developmental block observed in rag1-/- thymocytes, which lack pre-TCR signaling, was also not rescued by p35 expression. These results indicate that caspase-independent signal transduction pathways can mediate thymocyte death during normal T cell development.     

Positive regulation of c-Jun N-terminal kinase and TNF-alpha production but not histamine release by SHP-1 in RBL-2H3 mast cells

The SH2-containing protein tyrosine phosphatase1 (SHP-1) is important for signaling from immune receptors. To investigate the role of SHP-1 in mast cells we overexpressed the wild-type and the phosphatase-inactive forms of SHP-1 in rat basophilic leukemia 2H3 (RBL-2H3) mast cell line. The phosphatase-inactive SHP-1 (C453S or D419A) retains its ability to bind tyrosine phosphorylated substrates and thereby competes with the endogenous wild-type enzyme. Overexpression of wild-type SHP-1 decreased the FcepsilonRI aggregation-induced tyrosine phosphorylation of the beta and gamma subunits of the receptor whereas the dominant negative SHP-1 enhanced phosphorylation. There were also similar changes in the tyrosine phosphorylation of Syk. However, receptor-induced histamine release in the cells expressing either wild-type or dominant negative SHP-1 was similar to that in the parental control cells. In contrast, compared with the parental RBL-2H3 cells, FcepsilonRI-induced c-Jun N-terminal kinase phosphorylation and the level of TNF-alpha mRNA was increased in the cells overexpressing wild-type SHP-1 whereas the dominant negative SHP-1 had the opposite effect. The substrate-trapping mutant SHP1/D419A identified pp25 and pp30 as two major potential substrates of SHP-1 in RBL-2H3 cells. Therefore, SHP-1 may play a role in allergy and inflammation by regulating mast cell cytokine production.     

Human 70-kDa SHP-1L differs from 68-kDa SHP-1 in its C-terminal structure and catalytic activity.

The tyrosine phosphatase SHP-1 functions as a negative regulator in hematopoietic cell development, proliferation, and receptor-mediated cellular activation. In Jurkat T cells, a major 68-kDa band and a minor 70-kDa band were immunoprecipitated by a monoclonal antibody against the SHP-1 protein-tyrosine phosphatase domain, while an antibody against the SHP-1 C-terminal 19 amino acids recognized only the 68-kDa SHP-1. The SDS-gel-purified 70-kDa protein was subjected to tryptic mapping and microsequencing, which was followed by molecular cloning. It revealed that the 70-kDa protein, termed SHP-1L, is a C-terminal alternatively spliced form of SHP-1. SHP-1L is 29 amino acids longer than SHP-1, and its 66 C-terminal amino acids are different from SHP-1. The C terminus of SHP-1L contains a proline-rich motif PVPGPPVLSP, a potential Src homology 3 domain-binding site. In contrast to SHP-1, tyrosine phosphorylation of SHP-1L is not detected upon stimulation in Jurkat T cells. This is apparently due to the lack of a single in vivo tyrosine phosphorylation site, which only exists in the C terminus of SHP-1 (Y564). COS cell-expressed glutathione S-transferase-SHP-1L can dephosphorylate tyrosine-phosphorylated ZAP70. At pH 7.4, SHP-1L was shown to be more active than SHP-1 in the dephosphorylation of ZAP70. At pH 5.4, SHP-1L and SHP-1 exhibited similar catalytic activity. It is likely that these two isoforms play different roles in the regulation of hematopoietic cell signal transduction.     

Opening a window on thymic positive selection: developmental changes in the influence of cosignaling by integrins and CD28 on selection events induced by TCR engagement

How TCR and non-TCR signals are integrated by thymocytes to generate a decision to undergo either positive or negative selection remains incompletely understood. Recent evidence suggests that TCR signal transduction changes its quality during thymocyte maturation, but whether the contributions of various cosignaling or costimulatory pathways to thymocyte selection also are modified during development is unclear. Questions also remain about the possible selective roles of specific costimulatory pathways in induction of differentiation vs death among thymocytes at any given stage of maturity. To address these issues, a quantitative in vitro analysis of initiation of CD4+CD8+ thymocyte differentiation as measured by CD69 up-regulation/coreceptor down-modulation was conducted in parallel with an analysis of induction of death. Using transfected cells varying in their surface display of ICAM-1 or B7.1 along with antibody blocking experiments, we demonstrate here that ICAM-1 provides a selective boost to signaling for differentiation without substantially affecting induction of death among CD4+CD8+ cells, a property that is lost as thymocytes mature further. In contrast, B7 engagement enhances both cell activation and death in parallel. Based on these data, we propose that the high level of ICAM-1 on cortical epithelial cells plays a special role in opening a window between TCR signaling for differentiation vs death, permitting efficient initiation of positive selection on epithelial ligands. In contrast, late CD28-dependent cosignaling on hemopoietic cells in the medulla would help enforce negative selection by augmenting the effects of TCR engagement by low levels of high affinity ligands.