Protein tyrosine kinases and phosphatases cooperate to regulate normal immune cell function. We examined the role of PEST domain-enriched tyrosine phosphatase (PEP) in regulating T cell antigen-receptor function during thymocyte development and peripheral T cell differentiation. Although normal naïve T cell functions were retained in pep-deficient mice, effector/memory T cells demonstrated enhanced activation of Lck. In turn, this resulted in increased expansion and function of the effector/memory T cell pool, which was also associated with spontaneous development of germinal centers and elevated serum antibody levels. These results revealed a central role for PEP in negatively regulating specific aspects of T cell development and function.

SUMMARY Immune cells sense microbial products through Toll-like receptors (TLR), which trigger host defense responses including type 1 interferons (IFNs) secretion. A coding polymorphism in the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene is a susceptibility allele for human autoimmune and infectious disease. We report that Ptpn22 selectively regulated type 1 IFN production after TLR engagement in myeloid cells. Ptpn22 promoted host antiviral responses and was critical for TLR agonist-induced, type 1 IFN-dependent suppression of inflammation in colitis and arthritis. PTPN22 directly associated with TNF receptor-associated factor 3 (TRAF3) and promotes TRAF3 lysine 63-linked ubiquitination. The disease-associated PTPN22W variant failed to promote TRAF3 ubiquitination, type 1 IFN upregulation, and type 1 IFN-dependent suppression of arthritis. The findings establish a candidate innate immune mechanism of action for a human autoimmunity “risk” gene in the regulation of host defense and inflammation.

Pep and CD45 are tyrosine phosphatases whose targets include the Src-family kinases, critical mediators of Ag receptor signaling. A polymorphism in PTPN22, the gene that encodes the human Pep orthologue Lyp, confers susceptibility to multiple human autoimmune diseases in the context of complex genetic backgrounds. However, the functional significance of the R620W risk allele is not clear. We report that misexpression of wild-type or R620W Pep/Lyp in Jurkat cells, in the context of its binding partner Csk, unmasks the risk allele as a hypomorph. It has been shown previously that although Pep-deficient mice on the B6 background have hyperresponsive memory T cells, autoimmunity does not develop. Mice containing a point mutation in the CD45 juxtamembrane wedge domain (E613R) develop a B cell-driven, lupus-like disease on the mixed 129/B6 background, but not on the B6 background. We studied the ability of Pep deficiency to act as a genetic modifier of the CD45 E613R mutation on the nonautoimmune B6 background to understand how complex susceptibility loci might interact in autoimmunity. In this study we report that double mutant mice develop a lupus-like disease as well as lymphadenopathy, polyclonal lymphocyte activation, and accelerated memory T cell formation. Following Ag receptor stimulation, peripheral B cells in the double mutant mice phenocopy hyperresponsive CD45 E613R B cells, whereas peripheral T cells respond like Pep−/− T cells. These studies suggest that Pep−/− T cells in the context of a susceptible microenvironment can drive hyperresponsive CD45 E613R B cells to break tolerance.

Protein tyrosine phosphatases (PTPs), together with protein tyrosine kinases (PTKs), are involved in the regulation of cell activation, growth, and differentiation. To further elucidate the fine tuning of cell growth and differentiation through tyrosine phosphorylation, we tried to isolate mouse receptor-type PTP (RPTP) cDNA clones by screening mouse brain cDNA libraries with mouse CD45 PTP domain probes under reduced-stringency conditions. Characterization of isolated cDNA clones for RPTP showed that the cytoplasmic region contains two tandem repeats of PTP domain of about 230 amino acids with intrinsic phosphatase activity. The extracellular region was composed of immunoglobulin (Ig)-like domains and fibronectin type III (FN-III)-like domains. The gene was highly homologous to human PTP delta (HPTP delta) and thus was named MPTP delta (murine counterpart of HPTP delta). The MPTP delta gene appeared to generate at least three species of mRNA, which differ in the composition of the extracellular domain: type A, one Ig-like and four FN-III-like domains; type B, one Ig-like and eight FN-III-like domains; and type C, three Ig-like and eight FN-III-like domains. Interestingly, the 5' untranslated region and the leader peptide of types A and B were completely different from those of type C. Northern (RNA) blot analysis demonstrated that brain, kidney, and heart cells express three mRNA species of about 7 kb. Antibody directed against part of the extracellular domain of type A MPTP delta recognized a 210-kDa protein in brain and kidney lysates. In situ hybridization of brain samples revealed that MPTP delta mRNA is present in the hippocampus, thalamic reticular nucleus, and piriform cortex, where some Src family PTKs have been also demonstrated to exist. Although MPTP delta mRNA was not detected in lymphoid tissues, all of the pre-B-cell lines tested and one of three B-cell lines tested expressed MPTP delta mRNA, whereas antibody-producing B-cell hybridomas and T-cell and macrophage lines did not. Finally, the MPTP delta locus was tightly linked to the brown (b) locus on mouse chromosome 4.

Cross-linking of membrane IgM receptor on B cells induces tyrosine phosphorylation within 1 min. This biochemical alteration triggers a cascade of signaling events which ultimately leads to activation in mature B cells but growth arrest and cell death by apoptosis in immature B cells. To study the mechanisms underlying the bifurcation of signals, we chose to examine the role of receptor-type protein tyrosine phosphatase (PTP) CD45 using CD45- clones isolated from an immature B cell line WEHI-231. Here we report that in CD45- clones, tyrosine phosphorylation was constitutively induced but not enhanced by anti-IgM stimulation and anti-IgM-induced Ca2+ flux was slightly delayed but evidently prolonged. Further, the degree of growth arrest and DNA fragmentation induced by anti-IgM antibody was more evident in CD45- clones than the parental cells. These results indicate that initial alterations in signaling are effectively transduced into effector signals and that IgM receptor-mediated growth arrest and apoptosis in immature B cells are negatively regulated by CD45.

Activation of the tumor suppressor protein p53 is a critical cellular response to various stress stimuli and to inappropriate activity of growth-promoting proteins, such as Myc, Ras, E2F, and B-catenin. Protein stability and transcriptional activity of p53 are modulated by protein-protein interactions and post-translational modifications, including acetylation. Here, we show that inappropriate activity of prothymosin A (PTMA), an oncoprotein overexpressed in human cancers, triggers a p53 response. Overexpression of PTMA enhanced p53 transcriptional activity in reporter gene assays for p53 target gene promoters hdm2, p21, and cyclin G. Overexpressed PTMA resulted in increased mRNA and protein levels for endogenous p53 target genes, hdm2 and p21, and in growth suppression. In contrast, reduction of endogenous PTMA through RNA interference decreased p53 transcriptional activity. Histone acetyltransferases (HATs) act as p53 coactivators and acetylate p53. PTMA, known to interact with HATs, led to increased levels of acetylated p53. PTMA did not increase the transcriptional activity of an acetylation-deficient p53 mutant, suggesting that p53 acetylation is an indispensable part of the p53 response to PTMA. Chromatin immunoprecipitation assays showed that excess PTMA associates with the p21 promoter and results in increased levels of acetylated p53 at the p21 promoter. Our findings indicate that overexpressed PTMA elicits a p53 response that involves p53 acetylation. (Cancer Res 2006; 66(6): 3137-44)

Role of CD45 in B cell antigen receptor (BcR)-mediated signaling events in mature B cells was examined using BAL-17 and its CD45-negative clones. In the CD45-negative clones, BcR stimulation induced tyrosine phosphorylation almost identical to the parental cells, with a few exceptions of reduced phosphorylation, especially of a protein of about 60 kDa. BcR-induced calcium responses were reduced in the CD45-negative clones, but the kinetics were similar to the parent. BcR stimulation led to growth inhibition in the parental cells, but signals for growth inhibition were completely blocked in the CD45-negative clones. Interestingly, the same stimulation induced low, but significant levels of apoptosis both in the parent and in the CD45-negative clones. Thus, in mature BAL-17 cells, CD45 subtly mediate early signaling events (tyrosine phosphorylation and Ca2+ mobilization), and is absolutely required for the signaling pathway leading to growth regulation, but has limited effects on apoptosis.

SummarySrc homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1; previously named HCP, PTP1C, SH-PTP1, and SHP) is a cytosolic protein tyrosine phosphatase that contains two SH2 domains. Recent data have demonstrated that the gene encoding SHP-1 is mutated in motheaten (me) and viable motheaten (me v) mice resulting in autoimmune disease. More recently, SHP-1 has been shown to negatively regulate B cell antigen receptor (BCR.)-initiated signaling. To elucidate potential mechanisms of SHP-1 action in BCR signal transduction, we studied proteins that interact with' SHP-1 in B cells. Both anti-SHP-1 antibody and the two SH2 domains of SHP-1 expressed as glutathione S-transferase fusion proteins precipitated at least three phosphoproteins of ~-'75, 110, and 150 kD upon anti-immunoglobulin M stimulation of the WEHI-231 immature B cell line. Binding of SHP-1 to the 75-and 110-kD proteins appeared to be mediated mainly by the NH2-terminal SH2 domain of SHP-1, whereas both the NH 2-and COOH-terminal SH2 domains are required for maximal binding to the 150-kD protein, hnmunoprecipitation and Western blot analysis revealed that the SHP-l-associated 75-kD protein is the hematopoietic cell-specific, SH2-containing protein SLP-76. Further, this protein-protein association was constitutively observed and stable during the early phase of BCl< signaling. However, significant tyrosine phosphorylation of SLP-76 as well as of SHP-1 was observed after BCR. ligation. Constitutive association of SHP-1 with SLP-76 could also be detected in normal splenic B cells. Collectively, these results suggest possible mechanisms by which SHP-1 may modulate signals delivered by BCR engagement.