Suppressors of T-cell receptor signaling Sts-1 and Sts-2 bind to Cbl and inhibit endocytosis of receptor tyrosine kinases

The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.     

The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation.

Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site-directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane-proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45-deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR-mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand-induced negative regulation of PTPase function. We also demonstrate that the phosphorylation of the C-terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45-deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C-terminal phosphorylated Lck molecules.     

Coronin-1A links cytoskeleton dynamics to TCR alpha beta-induced cell signaling

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of alpha beta T cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-kappaB (I kappa B). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts alpha beta T cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.     

Lck couples Shc to TCR signaling

Recent genetic evidence demonstrated that Shc is a critical molecule for T cell activation and differentiation. However, how Shc is coupled to the T cell antigen receptor (TCR) has not been clearly characterized. Here we report that the tyrosine kinase Lck functions as a connecting molecule for TCR and Shc. Lck plays a critical role in TCR signal transduction by phosphorylating the immuno-receptor tyrosine based activation motif (ITAM). Our data shows that the PTB domain of Shc binds the SH2/3 domains of Lck in a phosphotyrosine-independent manner. Inhibition of the Lck/Shc interaction led to the loss of IL-2 promoter activation, confirming that the role of Shc in IL-2 production requires its interaction with Lck. Together, the data show that Shc is connected to the activated TCR via direct interaction with Lck.     

Galpha(i2) enhances insulin signaling via suppression of protein-tyrosine phosphatase 1B

Suppression of the expression of the heterotrimeric G-protein Galpha(i2) in vivo has been shown to provoke insulin resistance, whereas enhanced insulin signaling is observed when Galpha(i2) is overexpressed in vivo. The basis for Galpha(i2) regulation of insulin signaling was explored in transgenic mice with targeted expression of the GTPase-deficient, constitutively active Q205L Galpha(i2) in fat and skeletal muscle. Phosphorylation of insulin receptor and IRS-1 in response to insulin challenge in vivo was markedly amplified in fat and skeletal muscle expressing Q205L Galpha(i2). The expression and activity of the protein-tyrosine phosphatase 1B (PTP1B), but not protein-tyrosine phosphatases SHP-1, SHP-2, and LAR, were constitutively decreased in tissues expressing the Q205L Galpha(i2), providing a direct linkage between insulin signaling and Galpha(i2). The loss of PTP1B expression may explain, in part, the loss of PTP1B activity in the iQ205L transgenic mice. Activation of Galpha(i2) in mouse adipocytes with lysophosphatidic acid was shown to decrease PTP1B activity, whereas pertussis toxin inactivates Galpha(i2), blocks lysophosphatidic acid-stimulated inhibition of PTP1B activity, and blocks tonic suppression of PTP1B activity by Galpha(i2). Elevation of intracellular cAMP in fat cells is shown to increase PTP1B activity, whereas either depression of cAMP levels or direct activation of Galpha(i2) suppresses PTP1B. These data provide the first molecular basis for the interplay between Galpha(i2) and insulin signaling, i.e. activation of Galpha(i2) can suppress both the expression and activity of PTP1B in insulin-sensitive tissues.     

Selective regulation of TCR signaling pathways by the CD45 protein tyrosine phosphatase during thymocyte development

In CD45-deficient animals, there is a severe defect in thymocyte-positive selection, resulting in an absence of mature T cells and the accumulation of thymocytes at the DP stage of development. However, the signaling defect(s) responsible for the block in development of mature single-positive T cells is not well characterized. Previous studies have found that early signal transduction events in CD45-deficient cell lines and thymocytes are markedly diminished following stimulation with anti-CD3. Nevertheless, there are also situations in which T cell activation and TCR signaling events can be induced in the absence of CD45. For example, CD45-independent TCR signaling can be recovered upon simultaneous Ab cross-linking of CD3 and CD4 compared with cross-linking of CD3 alone. These data suggest that CD45 may differentially regulate TCR signaling events depending on the nature of the signal and/or on the differentiation state of the cell. In the current study, we have assessed the role of CD45 in regulating primary thymocyte activation following physiologic stimulation with peptide. Unlike CD3-mediated stimulation, peptide stimulation of CD45-deficient thymocytes induces diminished, but readily detectable TCR-mediated signaling events, such as phosphorylation of TCR-associated zeta, ZAP70, linker for activation of T cells, and Akt, and increased intracellular calcium concentration. In contrast, phosphorylation of ERK, which is essential for positive selection, is more severely affected in the absence of CD45. These data suggest that CD45 has a selective role in regulating different aspects of T cell activation.     

Endogenous adenosine contributes to renal sympathetic neurotransmission via postjunctional A1 receptor-mediated coincident signaling

Adenosine A(1) receptor antagonists have diuretic/natriuretic activity and may be useful for treating sodium-retaining diseases, many of which are associated with increased renal sympathetic tone. Therefore, it is important to determine whether A(1) receptor antagonists alter renal sympathetic neurotransmission. In isolated, perfused rat kidneys, renal vasoconstriction induced by renal sympathetic nerve simulation was attenuated by 1) 1,3-dipropyl-8-p-sulfophenylxanthine (xanthine analog that is a nonselective adenosine receptor antagonist, but is cell membrane impermeable and thus does not block intracellular phosphodiesterases), 2) xanthine amine congener (xanthine analog that is a selective A(1) receptor antagonist), 3) 1,3-dipropyl-8-cyclopentylxanthine (xanthine analog that is a highly selective A(1) receptor antagonist), and 4) FK453 (nonxanthine analog that is a highly selective A(1) receptor antagonist). In contrast, FR113452 (enantiomer of FK453 that does not block A(1) receptors), MRS-1754 (selective A(2B) receptor antagonist), and VUF-5574 (selective A(3) receptor antagonist) did not alter responses to renal sympathetic nerve stimulation, and ZM-241385 (selective A(2A) receptor antagonist) enhanced responses. Antagonism of A(1) receptors did not alter renal spillover of norepinephrine. 2-Chloro-N(6)-cyclopentyladenosine (highly selective A(1) receptor agonist) increased renal vasoconstriction induced by exogenous norepinephrine, an effect that was blocked by 1,3-dipropyl-8-cyclopentylxanthine, U73122 (phospholipase C inhibitor), GF109203X (protein kinase C inhibitor), PP1 (c-src inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), and OSU-03012 (3-phosphoinositide-dependent protein kinase-1 inhibitor). These results indicate that adenosine formed during renal sympathetic nerve stimulation enhances the postjunctional effects of released norepinephrine via coincident signaling and contributes to renal sympathetic neurotransmission. Likely, the coincident signaling pathway is: phospholipase C → protein kinase C → c-src → phosphatidylinositol 3-kinase → 3-phosphoinositide-dependent protein kinase-1.     

Elevated IL-1beta contributes to antibody suppression produced by stress.

Acute stressor exposure can facilitate innate immunity and suppress acquired immunity. The present study further characterized the potentiating effect of stress on innate immunity, interleukin-1beta (IL-1beta), and demonstrated that stress-induced potentiation of innate immunity may contribute to the stress-induced suppression of acquired immunity. The long-term effect of stress on IL-1beta was measured by using an ex vivo approach. Sprague-Dawley rats were challenged with lipopolysaccharide (LPS) in vivo, and the IL-1beta response was measured in vitro. Splenocytes, mesenteric lymphocytes, and peritoneal cavity cells had a dose- and time-dependent ex vivo IL-1beta response to LPS. Rats that were exposed to inescapable shock (IS, 100 1.6 mA, 5-s tail shocks, 60-s intertrial interval) and challenged with a submaximal dose of LPS 4 days later had elevated IL-1beta measured ex vivo. To test whether the acute stress-induced elevation in IL-1beta contributes to the long-term suppression in acquired immunity, IL-1beta receptors were blocked for 24 h after stress. Serum anti-keyhole limpet hemocyanin (KLH) immunoglobulin (Ig) was measured. In addition, the acute elevation (2 h post-IS) of splenic IL-1beta in the absence of antigen was verified. Interleukin-1 receptor antagonist prevented IS-induced suppression in anti-KLH Ig. These data support the hypothesis that stress-induced increases in innate immunity (i.e., IL-1beta) may contribute to stress-induced suppression in acquired immunity (i.e., anti-KLH Ig).     

Deregulation of DUSP activity in EGFR-mutant lung cancer cell lines contributes to sustained ERK1/2 signaling

Lung cancers demonstrate loss of cellular signaling control pathways. EGFR-mutant non-small cell lung cancer cell lines constitutively express active ERK1/2 and require ERK activity for survival. DUSP4 is a negative regulator of ERK activity and is up-regulated in EGFR-mutant lung cancer cell lines relative to K-ras mutant cells. Both DUSP4 and family member, DUSP1, can bind ERK in vitro. However, only DUSP1 has detectable binding to ERK in vivo in cell lines of either genotype. Depletion of DUSP4 in EGFR-mutant cells unexpectedly results in loss of pERK whereas loss of DUSP4 in K-ras mutant cells predictably yields increased pERK. These data support a role for DUSP4, and perhaps DUSP1, as a positive activator of ERK in EGFR-mutant lung cancer cell lines independent of the ability to bind to ERK.     

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-1(PGM)) has a potent phosphatase activity that contributes to the suppression of TCR signaling. The function of Sts-2(PGM) 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-2(PGM) 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-2(PGM) triple mutant in which these three amino acids are altered to their counterparts in Sts-1(PGM) 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.     

Nore1B regulates TCR signaling via Ras and Carma1

Nore1A was originally identified as a potential Ras effector, and Nore1B is an alternatively spliced isoform. Both share a Ras/Rap association domain (RA domain) but only Nore1A contains sequence motifs that predict SH3 domain binding and diacylglycerol/phorbol ester binding in the amino-terminal region. Here we report that Carma1 binds to Nore1A and Nore1B through the RA domain and that Carma1 interacts with active Ras in the presence of Nore1B. RNA interference against Nore1B attenuates NF-kappaB activation induced by T cell receptor (TCR) ligation, but not NF-kappaB activation induced by TNFalpha or lipoteichoic acid. In addition, Nore1B is also required for KiRas GV12-mediated ERK1 activation and Elk1 reporter activity in T cells. We also provide evidence that knockdown of Nore1B also impairs polarized redistribution of Ras at the B cell-T cell immune interface. Together, these findings suggest that endogenous Nore1B recruits active Ras to the APC-T cell interface and mediates the interaction between Ras and Carma1.     

Notch1 signaling contributes to the oncogenic effect of HBx on human hepatic cells

Hepatocellular carcinoma (HCC) is a malignant tumor and hepatitis B virus X protein (HBx) plays a crucial role in its pathogenesis. The Notch1 signaling pathway is involved in various malignant tumors including liver cancers and down-regulation of Notch-1 may exert anti-tumor effects. Here, we demonstrate that inhibition of Notch1 by plasmid-based shRNA suppresses growth of human hepatic cells transfected with HBx through G0/G1 cell cycle arrest and apoptosis inhibition, possibly linked to the promoted expression of cyclin-dependent kinase inhibitor, P16, and decreased expression of apoptosis inhibitor, Bcl-2. The anti-proliferative and pro-apoptotic effects of Notch1 shRNA in HBx-transformed L02 cell may be partly mediated by down-regulation of nuclear factor-kappaB (NF-κB) binding activities, demonstrating possible cross-talk between Notch-1 and NF-κB signaling pathways. The oncogene HBx may therefore induce malignant transformation of human hepatic cells via Notch1 pathway, indicating that Notch1 plays a crucial role in HBx-related liver cancer and could be an effective therapeutic target for HCC.     

Active ERK contributes to protein translation by preventing JNK-dependent inhibition of protein phosphatase 1

Human alveolar macrophages, central to immune responses in the lung, are unique in that they have an extended life span in contrast to precursor monocytes. We have shown previously that the ERK MAPK (ERK) pathway is constitutively active in human alveolar macrophages and contributes to the prolonged survival of these cells. We hypothesized that ERK maintains survival, in part, by positively regulating protein translation. In support of this hypothesis, we have found novel links among ERK, JNK, protein phosphatase 1 (PP1), and the eukaryotic initiation factor (eIF) 2alpha. eIF2alpha is active when hypophosphorylated and is essential for initiation of protein translation (delivery of initiator tRNA charged with methionine to the ribosome). Using [(35)S]methionine labeling, we found that ERK inhibition significantly decreased protein translation rates in alveolar macrophages. Decreased protein translation resulted from phosphorylation (and inactivation) of eIF2alpha. We found that ERK inhibition increased JNK activity. JNK in turn inactivated (via phosphorylation) PP1, the phosphatase responsible for maintaining the hypophosphorylated state of eIF2alpha. As a composite, our data demonstrate that in human alveolar macrophages, constitutive ERK activity positively regulates protein translation via the following novel pathway: active ERK inhibits JNK, leading to activation of PP1alpha, eIF2alpha dephosphorylation, and translation initiation. This new role for ERK in alveolar macrophage homeostasis may help to explain the survival characteristic of these cells within their unique high oxygen and stress microenvironment.     

Wip1 phosphatase modulates ATM-dependent signaling pathways

Deletion of Ppm1d, the gene encoding the Wip1 phosphatase, renders cells resistant to transformation and mice resistant to tumor development. Here, we report that deficiency of Wip1 resulted in activation of the ataxia-telangiectasia mutated (ATM) kinase. In turn, overexpression of Wip1 was sufficient to reduce activation of the ATM-dependent signaling cascade after DNA damage. Wip1 dephosphorylated ATM Ser1981, a site critical for ATM monomerization and activation, and was critical for resetting ATM phosphorylation as cells repaired damaged DNA. We propose that the Wip1 phosphatase is an integral component of an ATM-dependent signaling pathway.     

Hyperosmotic stress contributes to mouse colonic inflammation through the methylation of protein phosphatase 2A

There are several reports suggesting hyperosmotic contents in the feces of patients suffering from inflammatory bowel disease (IBD). Previous works have documented that hyperosmolarity can cause inflammation attributable to methylation of the catalytic subunit of protein phosphatase 2A (PP2A) and subsequent NF-kappaB activation resulting in cytokine secretion. In this study, we demonstrate that dextran sulfate sodium (DSS) induces colitis due to hyperosmolarity and subsequent PP2A activation. Mice were randomized and fed with increased concentrations of DSS (0 mOsm, 175 mOsm, 300 mOsm, and 627 mOsm) for a duration of 3 wk or with hyperosmotic concentrations of DSS (627 mOsm) or mannitol (450 mOsm) for a duration of 12 wk. Long-term oral administration of hyposmotic DSS or mannitol had no demonstrable effect. Hyperosmotic DSS or mannitol produced a significant increase in colonic inflammation, as well as an increase in the weight of sacral lymph nodes and in serum amyloid A protein levels. Similar results were obtained through the ingestion of comparable osmolarities of mannitol. Hyperosmolarity induces the methylation of PP2A, nuclear p65 NF-kappaB activation. and cytokine secretion. The rectal instillation of okadaic acid, a well-known PP2A inhibitor, reverses the IBD. Short inhibiting RNAs (siRNAs) targeted toward PP2Ac reverse the effect of hyperosmotic DSS. The present study strongly suggests that DSS-induced chronic colitis is a consequence of the methylation of PP2Ac induced by hyperosmolarity.     

The Clp1/Cdc14 phosphatase contributes to the robustness of cytokinesis by association with anillin-related Mid1

Cdc14 phosphatases antagonize cyclin-dependent kinase-directed phosphorylation events and are involved in several facets of cell cycle control. We investigate the role of the fission yeast Cdc14 homologue Clp1/Flp1 in cytokinesis. We find that Clp1/Flp1 is tethered at the contractile ring (CR) through its association with anillin-related Mid1. Fluorescent recovery after photobleaching analyses indicate that Mid1, unlike other tested CR components, is anchored at the cell midzone, and this physical property is likely to account for its scaffolding role. By generating a mutation in mid1 that selectively disrupts Clp1/Flp1 tethering, we reveal the specific functional consequences of Clp1/Flp1 activity at the CR, including dephosphorylation of the essential CR component Cdc15, reductions in CR protein mobility, and CR resistance to mild perturbation. Our evidence indicates that Clp1/Flp1 must interact with the Mid1 scaffold to ensure the fidelity of Schizosaccharomyces pombe cytokinesis.