Nick Carpino

Essential, Nonredundant Role for the Phosphoinositide 3-Kinase p110δ in Signaling by the B-Cell Receptor Complex

ABSTRACT Many receptor and nonreceptor tyrosine kinases activate phosphoinositide 3-kinases (PI3Ks). To assess the role of the δ isoform of the p110 catalytic subunit of PI3Ks, we derived enzyme-deficient mice. The mice are viable but have decreased numbers of mature B cells, a block in pro-B-cell differentiation, and a B1 B-cell deficiency. Both immunoglobulin M receptor-induced Ca2+ flux and proliferation in response to B-cell mitogens are attenuated. Immunoglobulin levels are decreased substantially. The ability to respond to T-cell-independent antigens is markedly reduced, and the ability to respond to T-cell-dependent antigens is completely eliminated. Germinal center formation in the spleen in response to antigen stimulation is disrupted. These results define a nonredundant signaling pathway(s) utilizing the δ isoform of p110 PI3K for the development and function of B cells.

SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation.

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non‐essential role in embryo development. Rescued SOCS3‐deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3‐deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho‐1) suppresses giant cell differentiation. Conversely, SOCS3‐deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3‐deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation.

The Ornithine Decarboxylase Gene Is Essential for Cell Survival during Early Murine Development

ABSTRACT Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring a disrupted ODC gene.ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation, loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly, analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development, and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation.

Absence of an Essential Role for Thymic Stromal Lymphopoietin Receptor in Murine B-Cell Development

ABSTRACT The murine cytokine thymic stromal lymphopoietin (TSLP) supports the development of B220+ IgM+ immature B cells and induces thymocyte proliferation in vitro. Human TSLP, by contrast, activates CD11c+ dendritic cells, but not B or T cells. Recent studies have demonstrated that the receptor for TSLP consists of a heterodimer of the interleukin 7 (IL-7) α chain and a novel protein that resembles the hematopoietic cytokine receptor common γ chain. We examined signal transduction by the γ-like chains using chimeric receptor proteins. The cytoplasmic domain of the human, but not of the murine, γ-like chain, activates Jak2 and Stat5 and supports the proliferation of hematopoietic cell lines. In order to assess the role of the murine γ-like chain in vivo, we generated γ-like chain-deficient mice. Receptor-deficient mice are unresponsive to TSLP but exhibit no obvious phenotypic defects. In particular, hematopoietic cell development appeared normal. B-cell development, including the IgM+ compartment, was unaffected by loss of the TSLP pathway, as were T lymphopoiesis and lymphocyte proliferation in vitro. Cytokine receptors that utilize the common γ chain signal through the lymphocyte-specific kinase Jak3. Mice deficient in Jak3 exhibit a SCID phenotype but harbor a residual B220+ splenic lymphocyte population. We demonstrate here that this residual lymphocyte population is lost in mice lacking both the γ-like chain and Jak3.

A small amphipathic α‐helical region is required for transcriptional activities and proteasome‐dependent turnover of the tyrosine‐phosphorylated Stat5

Cytokines induce the tyrosine phosphorylation and associated activation of signal transducers and activators of transcription (Stat). The mechanisms by which this response is terminated are largely unknown. Among a variety of inhibitors examined, the proteasome inhibitors MG132 and lactacystin affected Stat4, Stat5 and Stat6 turnover by significantly stabilizing the tyrosine‐phosphorylated form. However, these proteasome inhibitors did not affect downregulation of the tyrosine‐phosphorylated Stat1, Stat2 and Stat3. With Stat5 isoforms, we have observed that tyrosine‐phosphorylated carboxyl‐truncated forms of Stat5 proteins were considerably more stable than phosphorylated wild‐type forms of the protein. Also, the C‐terminal region of Stat5 could confer proteasome‐dependent downregulation to Stat1. With a series of C‐terminal deletion mutants, we have defined a relatively small, potentially amphipathic α‐helical region that is required for the rapid turnover of the phosphorylated Stat5 proteins. The region is also required for transcriptional activation, suggesting that the functions are linked. The results are consistent with a model in which the transcriptional activation domain of activated Stat5 is required for its transcriptional activity and downregulation through a proteasome‐dependent pathway.

Regulation of ZAP-70 Activation and TCR Signaling by Two Related Proteins, Sts-1 and Sts-2

T cells play a central role in the recognition and elimination of foreign pathogens. Signals through the T cell receptor (TCR) control the extent and duration of the T cell response. To ensure that T cells are not inappropriately activated, signaling pathways downstream of the TCR are subject to multiple levels of positive and negative regulation. Herein, we describe two related proteins, Sts-1 and Sts-2, that negatively regulate TCR signaling. T cells from mice lacking Sts-1 and Sts-2 are hyperresponsive to TCR stimulation. The phenotype is accompanied by increased Zap-70 phosphorylation and activation, including its ubiquitinylated forms. Additionally, hyperactivation of signaling proteins downstream of the TCR, a marked increase in cytokine production by Sts1/2(-/-) T cells, and increased susceptibility to autoimmunity in a mouse model of multiple sclerosis is observed. Therefore, Sts-1 and Sts-2 are critical regulators of the signaling pathways that regulate T cell activation.

Identification, cDNA Cloning, and Targeted Deletion of p70, a Novel, Ubiquitously Expressed SH3 Domain-Containing Protein

ABSTRACT In a screen for proteins that interact with Jak2, we identified a previously uncharacterized 70-kDa protein and cloned the corresponding cDNA. The predicated sequence indicates that p70 contains an SH3 domain and a C-terminal domain with similarities to the catalytic motif of phosphoglycerate mutase. p70 transcripts were found in all tissues examined. Similarly, when an antibody raised against a C-terminal peptide to analyze p70 protein expression was used, all murine tissues examined were found to express p70. To investigate the in vivo role of p70, we generated a p70-deficient mouse strain. Mice lacking p70 are viable, develop normally, and do not display any obvious abnormalities. No differences were detected in various hematological parameters, including bone marrow colony-forming ability, in response to cytokines that utilize Jak2. In addition, no impairment in B- and T-cell development and proliferative ability was detected.

A novel histidine tyrosine phosphatase, TULA-2, associates with Syk and negatively regulates GPVI signaling in platelets

T-cell ubiquitin ligand-2 (TULA-2) is a recently discovered histidine tyrosine phosphatase thought to be ubiquitously expressed. In this work, we have investigated whether TULA-2 has a key role in platelet glycoprotein VI (GPVI) signaling. This study indicates that TULA-2 is expressed in human and murine platelets and is able to associate with Syk and dephosphorylate it. Ablation of TULA-2 resulted in hyperphosphorylation of Syk and its downstream effector phospholipase C-γ2 as well as enhanced GPVI-mediated platelet functional responses. In addition, shorter bleeding times and a prothrombotic phenotype were observed in mice lacking TULA-2. We therefore propose that TULA-2 is the primary tyrosine phosphatase mediating the dephosphorylation of Syk and thus functions as a negative regulator of GPVI signaling in platelets.

TULA proteins regulate activity of the protein tyrosine kinase Syk

TULA belongs to a two‐member family: TULA (STS‐2) is a lymphoid protein, whereas STS‐1/TULA‐2 is expressed ubiquitously. TULA proteins were implicated in the regulation of signaling mediated by protein tyrosine kinases (PTKs). The initial experiments did not fully reveal the molecular mechanism of these effects, but suggested that both TULA proteins act in a similar fashion. It was shown recently that STS‐1/TULA‐2 dephosphorylates PTKs. In this study, we analyzed the effects of TULA proteins on Syk, a PTK playing an important role in lymphoid signaling. First, we have shown that TULA‐2 decreases tyrosine phosphorylation of Syk in vivo and in vitro and that the intact phosphatase domain of TULA‐2 is essential for this effect. We have also shown that TULA‐2 exhibits a certain degree of substrate specificity. Our results also indicate that inactivated TULA‐2 increases tyrosine phosphorylation of Syk in cells co‐transfected to overexpress these proteins, thus acting as a dominant‐negative form that suppresses dephosphorylation of Syk caused by endogenous TULA‐2. Furthermore, we have demonstrated that phosphatase activity of TULA is negligible as compared to that of TULA‐2 and that this finding correlates with an increase in Syk tyrosine phosphorylation in cells overexpressing TULA. This result is consistent with the dominant‐negative effect of inactivated TULA‐2, arguing that TULA acts in this system as a negative regulator of TULA‐2‐dependent dephosphorylation. To summarize, our findings indicate that TULA proteins may exert opposite effects on PTK‐mediated signaling and suggest that a regulatory mechanism based on this feature may exist. J. Cell. Biochem. 104: 953–964, 2008.

Sts-2 Is a Phosphatase That Negatively Regulates Zeta-associated Protein (ZAP)-70 and T Cell Receptor Signaling Pathways

T cell activity is controlled in large part by the T cell receptor (TCR). The TCR detects the presence of foreign pathogens and activates the T cell-mediated immune reaction. Numerous intracellular signaling pathways downstream of the TCR are involved in the process of T cell activation. Negative regulation of these pathways helps prevent excessive and deleterious T cell responses. Two homologous proteins, Sts-1 and Sts-2, have been shown to function as critical negative regulators of TCR signaling. The phosphoglycerate mutase-like domain of Sts-1 (Sts-1PGM) has a potent phosphatase activity that contributes to the suppression of TCR signaling. The function of Sts-2PGM as a phosphatase has been less clear, principally because its intrinsic enzyme activity has been difficult to detect. Here, we demonstrate that Sts-2 regulates the level of tyrosine phosphorylation on targets within T cells, among them the critical T cell tyrosine kinase Zap-70. Utilizing new phosphorylated substrates, we demonstrate that Sts-2PGM has clear, albeit weak, phosphatase activity. We further pinpoint Sts-2 residues Glu-481, Ser-552, and Ser-582 as specificity determinants, in that an Sts-2PGM triple mutant in which these three amino acids are altered to their counterparts in Sts-1PGM has substantially increased activity. Our results suggest that the phosphatase activities of both suppressor of TCR signaling homologues cooperate in a similar but independent fashion to help set the threshold for TCR-induced T cell activation.