The Sts Proteins: Modulators of Host Immunity.

The suppressor of TCR signaling (Sts) proteins, Sts-1 and Sts-2, are a pair of closely related signaling molecules that belong to the histidine phosphatase (HP) family of enzymes by virtue of an evolutionarily conserved C-terminal phosphatase domain. HPs derive their name from a conserved histidine that is important for catalytic activity and the current evidence indicates that the Sts HP domain plays a critical functional role. Sts-1HP has been shown to possess a readily measurable protein tyrosine phosphatase activity that regulates a number of important tyrosine-kinase-mediated signaling pathways. The in vitro catalytic activity of Sts-2HP is significantly lower than that of Sts-1HP, and its signaling role is less characterized. The highly conserved unique structure of the Sts proteins, in which additional domains, including one that exhibits a novel phosphodiesterase activity, are juxtaposed together with the phosphatase domain, suggesting that Sts-1 and -2 occupy a specialized intracellular signaling niche. To date, the analysis of Sts function has centered predominately around the role of Sts-1 and -2 in regulating host immunity and other responses associated with cells of hematopoietic origin. This includes their negative regulatory role in T cells, platelets, mast cells and other cell types, as well as their less defined roles in regulating host responses to microbial infection. Regarding the latter, the use of a mouse model lacking Sts expression has been used to demonstrate that Sts contributes non-redundantly to the regulation of host immunity toward a fungal pathogen (C. albicans) and a Gram-negative bacterial pathogen (F. tularensis). In particular, Sts-/- animals demonstrate significant resistance to lethal infections of both pathogens, a phenotype that is correlated with some heightened anti-microbial responses of phagocytes derived from mutant mice. Altogether, the past several years have seen steady progress in our understanding of Sts biology.

STS-1 and STS-2, Multi-Enzyme Proteins Equipped to Mediate Protein-Protein Interactions.

STS-1 and STS-2 form a small family of proteins that are involved in the regulation of signal transduction by protein-tyrosine kinases. Both proteins are composed of a UBA domain, an esterase domain, an SH3 domain, and a PGM domain. They use their UBA and SH3 domains to modify or rearrange protein-protein interactions and their PGM domain to catalyze protein-tyrosine dephosphorylation. In this manuscript, we discuss the various proteins that have been found to interact with STS-1 or STS-2 and describe the experiments used to uncover their interactions.

TULA Proteins in Men, Mice, Hens, and Lice: Welcome to the Family.

The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key biological functions, including immunity and hemostasis, in mammalian biological systems. Negative regulation of signaling through immune receptor tyrosine-based activation motif (ITAM)- and hemITAM-bearing receptors mediated by Syk-family protein tyrosine kinases appears to be a major molecular mechanism of the down-regulatory effect of TULA-family proteins, which possess protein tyrosine phosphatase (PTP) activity. However, these proteins are likely to carry out some PTP-independent functions as well. Whereas the effects of TULA-family proteins overlap, their characteristics and their individual contributions to cellular regulation also demonstrate clearly distinct features. Protein structure, enzymatic activity, molecular mechanisms of regulation, and biological functions of TULA-family proteins are discussed in this review. In particular, the usefulness of the comparative analysis of TULA proteins in various metazoan taxa, for identifying potential roles of TULA-family proteins outside of their functions already established in mammalian systems, is examined.

STS1 and STS2 Phosphatase Inhibitor Baicalein Enhances the Expansion of Hematopoietic and Progenitor Stem Cells and Alleviates 5-Fluorouracil-Induced Myelosuppression.

STS1 and STS2, as the protein phosphatases that dephosphorylate FLT3 and cKIT, negatively regulate the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs). To obtain the small molecule inhibitors of STS1/STS2 phosphatase activity used to expand HSPCs both in vitro and in vivo, we establish an in vitro phosphatase assay using the recombinant proteins of the STS1/STS2 histidine phosphatase (HP) domain, by which we screened out baicalein (BC) as one of the effective inhibitors targeting STS1 and STS2. Then, we further demonstrate the direct binding of BC with STS1/STS2 using molecular docking and capillary electrophoresis and verify that BC can restore the phosphorylation of FLT3 and cKIT from STS1/STS2 inhibition. In a short-term in vitro culture, BC promotes profound expansion and enhances the colony-forming capacity of both human and mouse HSPCs along with the elevation of phospho-FLT3 and phospho-cKIT levels. Likewise, in vivo administration with BC significantly increases the proportions of short-term hematopoietic stem cells (ST-HSCs), multipotent progenitors (MPPs) and especially long-term HSCs (LT-HSCs) in healthy mouse bone marrow and increases the numbers of colony-forming units (CFU) formed by HSPCs as well. More importantly, pre-administration of BC significantly enhances the survival of mice with lethal 5-fluorouracil (5-FU) injection due to the alleviation of 5-FU-induced myelosuppression, as evidenced by the recovery of bone marrow histologic injury, the increased proportions of LT-HSCs, ST-HSCs and MPPs, and enhanced colony-forming capacity. Collectively, our study not only suggests BC as one of the small molecule candidates to stimulate HSPC expansion both in vitro and in vivo when needed in either physiologic or pathologic conditions, but also supports STS1/STS2 as potential therapeutic drug targets for HSPC expansion and hematopoietic injury recovery.

The Sts1 nuclear import adapter uses a non-canonical bipartite nuclear localization signal and is directly degraded by the proteasome.

The proteasome is an essential regulator of protein homeostasis. In yeast and many mammalian cells, proteasomes strongly concentrate in the nucleus. Sts1 from the yeast Saccharomyces cerevisiae is an essential protein linked to proteasome nuclear localization. Here, we show that Sts1 contains a non-canonical bipartite nuclear localization signal (NLS) important for both nuclear localization of Sts1 itself and the proteasome. Sts1 binds the karyopherin-α import receptor (Srp1) stoichiometrically, and this requires the NLS. The NLS is essential for viability, and over-expressed Sts1 with an inactive NLS interferes with 26S proteasome import. The Sts1-Srp1 complex binds preferentially to fully assembled 26S proteasomes in vitro Sts1 is itself a rapidly degraded 26S proteasome substrate; notably, this degradation is ubiquitin independent in cells and in vitro and is inhibited by Srp1 binding. Mutants of Sts1 are stabilized, suggesting that its degradation is tightly linked to its role in localizing proteasomes to the nucleus. We propose that Sts1 normally promotes nuclear import of fully assembled proteasomes and is directly degraded by proteasomes without prior ubiquitylation following karyopherin-α release in the nucleus.

An unexpected 2-histidine phosphoesterase activity of suppressor of T-cell receptor signaling protein 1 contributes to the suppression of cell signaling.

The suppressor of T-cell receptor (TCR) signaling (Sts) proteins Sts-1 and Sts-2 suppress receptor-mediated signaling pathways in various immune cells, including the TCR pathway in T cells and the Dectin-1 signaling pathway in phagocytes. As multidomain enzymes, they contain an N-terminal ubiquitin-association domain, a central Src homology 3 domain, and a C-terminal histidine phosphatase domain. Recently, a 2-histidine (2H) phosphoesterase motif was identified within the N-terminal portion of Sts. The 2H phosphoesterase motif defines an evolutionarily ancient protein domain present in several enzymes that hydrolyze cyclic phosphate bonds on different substrates, including cyclic nucleotides. It is characterized by two invariant histidine residues that play a critical role in catalytic activity. Consistent with its assignment as a phosphoesterase, we demonstrate here that the Sts-1 2H phosphoesterase domain displays catalytic, saturable phosphodiesterase activity toward the dinucleotide 2',3'-cyclic NADP. The enzyme exhibited a high degree of substrate specificity and selectively generated the 3'-nucleotide as the sole product. Sts-1 also had phosphodiesterase catalytic activity toward a 5-mer RNA oligonucleotide containing a 2',3'-cyclic phosphate group at its 3' terminus. To investigate the functional significance of Sts-1 2H phosphoesterase activity, we generated His-to-Ala variants and examined their ability to negatively regulate cellular signaling pathways. Substitution of either conserved histidine compromised the ability of Sts-1 to suppress signaling pathways downstream of both the TCR and the Dectin-1 receptor. Our results identify a heretofore unknown cellular enzyme activity associated with Sts-1 and indicate that this catalytic activity is linked to specific cell-signaling outcomes.

Probing the settlement signals of Amphibalanus amphitrite.

To achieve their reproductive potential, barnacles combine tactile exploration of surface structural properties and integration of cellular signals originating from their antennular sensory setae within a developmentally defined, temporally narrow window of settlement opportunity. Behavioural assays with cyprids coupled with biometric analysis of scanning electron microscopy-acquired images in the presence of specific chemical compounds were used to investigate how settlement on a substratum is altered in response to the presence of these compounds. It is shown that impeding tactile exploration, altering cellular signalling and/or inducing malformations of anatomical features of the antennular sensory setae can disrupt the settlement behaviour of the model barnacle species Amphibalanus amphitrite. It is concluded that surface exploration by the cyprids relies on mechanical and nociception-related and calcium-mediated signals while a protein kinase C signalling cascade controls the timely metamorphosis of the cyprids to sessile juveniles.

STS-1 promotes IFN-α induced autophagy by activating the JAK1-STAT1 signaling pathway in B cells.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the overexpression of IFN-α. IFN-α induces autophagy via the JAK1-STAT1 signaling pathway, contributing to the pathogenesis of SLE. Recent studies reported that B cells from patients with SLE and NZB/W F1 mice had enhanced autophagy activity; however, the mechanism still remains unknown. Here, we show that the protein tyrosine phosphatase STS-1 (suppressor of T-cell receptor signaling 1) was significantly overexpressed in B cells from patients with SLE and MRL/lpr mice. Notably, STS-1 promoted IFN-α-induced autophagy in B cells by enhancing the JAK1-STAT1 signaling activation. STS-1 inhibited the phosphorylation of the E3 ubiquitin protein ligase c-cbl, and subsequently promoted IFN-α-induced phosphorylation of tyrosine kinase 2, leading to JAK1-STAT1 signaling activation. Furthermore, STAT1 and JAK1 inhibitors blocked the IFN-α-induced autophagy promoted by STS-1, indicating that STS-1 promotes IFN-α-induced autophagy via the JAK1-STAT1 signaling. Our results demonstrate the importance of STS-1 in regulating IFN-α-induced autophagy in B cells, and this could be used as a therapeutic approach to treat SLE.

TULA proteins as signaling regulators.

Two members of the UBASH3/STS/TULA family exhibit a unique protein domain structure, which includes a histidine phosphatase domain, and play a key role in regulating cellular signaling. UBASH3A/STS-2/TULA is mostly a lymphoid protein, while UBASH3B/STS-1/TULA-2 is expressed ubiquitously. Dephosphorylation of tyrosine-phosphorylated proteins by TULA-2 and, probably to a lesser extent, by TULA critically contribute to the molecular basis of their regulatory effect. The notable differences between the effects of the two family members on cellular signaling and activation are likely to be linked to the difference between their specific enzymatic activities. However, these differences might also be related to the functions of their domains other than the phosphatase domain and independent of their phosphatase activity. The down-regulation of the Syk/Zap-70-mediated signaling, which to-date appears to be the best-studied regulatory effect of TULA family, is discussed in detail in this publication.

Modulating Host Signaling Pathways to Promote Resistance to Infection by Candida albicans.

Candida albicans is a common human fungal pathogen capable of causing serious systemic infections that can progress to become lethal. Current therapeutic approaches have limited effectiveness, especially once a systemic infection is established, in part due to the lack of an effective immune response. Boosting the immune response to C. albicans has been the goal of immunotherapy, but it has to be done selectively to prevent deleterious hyperinflammation (sepsis). Although an efficient inflammatory response is necessary to fight infection, the typical response to C. albicans results in collateral damage to tissues thereby exacerbating the pathological effects of infection. For this reason, identifying specific ways of modulating the immune system holds promise for development of new improved therapeutic approaches. This review will focus on recent studies that provide insight using mutant strains of mice that are more resistant to bloodstream infection by C. albicans. These mice are deficient in signal transduction proteins including the Jnk1 MAP kinase, the Cbl-b E3 ubiquitin ligase, or the Sts phosphatases. Interestingly, the mutant mice display a different response to C. albicans that results in faster clearance of infection without hyper-inflammation and collateral damage. A common underlying theme between the resistant mouse strains is loss of negative regulatory proteins that are known to restrain activation of cell surface receptor-initiated signaling cascades. Understanding the cellular and molecular mechanisms that promote resistance to C. albicans in mice will help to identify new approaches for improving antifungal therapy.