The protein tyrosine phosphatase SHP-1 is a critical regulator of macrophage biology, but its detailed mechanism of action remains largely undefined. SHP-1 associates with a 130-kDa tyrosyl-phosphorylated species (P130) in macrophages, suggesting that P130 might be an SHP-1 regulator and/or substrate. Here we show that P130 consists of two transmembrane glycoproteins, which we identify as PIR-B/p91A and the signal-regulatory protein (SIRP) family member BIT. These proteins also form separate complexes with SHP-2. BIT, but not PIR-B, is in a complex with the colony-stimulating factor 1 receptor (CSF-1R), suggesting that BIT may direct SHP-1 to the CSF-1R. BIT and PIR-B bind preferentially to substrate-trapping mutants of SHP-1 and are hyperphosphorylated in macrophages from motheaten viable mice, which express catalytically impaired forms of SHP-1, indicating that these proteins are SHP-1 substrates. However, BIT and PIR-B are hypophosphorylated in motheaten macrophages, which completely lack SHP-1 expression. These data suggest a model in which SHP-1 dephosphorylates specific sites on BIT and PIR-B while protecting other sites from dephosphorylation via its SH2 domains. Finally, BIT and PIR-B associate with two tyrosyl phosphoproteins and a tyrosine kinase activity. Tyrosyl phosphorylation of these proteins and the level of the associated kinase activity are increased in the absence of SHP-1. Our data suggest that BIT and PIR-B recruit multiple signaling molecules to receptor complexes, where they are regulated by SHP-1 and/or SHP-2.Protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) control the level of tyrosyl phosphorylation on cellular proteins (reviewed in references 38, 52, and 56). Although many substrates for PTKs have been identified, the specific targets of individual PTP family members, along with the consequences of protein dephosphorylation for cellular physiology, remain largely unknown. The finding that some PTPs possess SH2 domains suggested that these molecules might be recruited to specific phosphotyrosyl (pY) sites (via their SH2 domains), where their PTP domains could catalytically amplify or terminate phosphotyrosine-mediated signals (14, 37). Two vertebrate SH2-containing PTPs (SHPs), SHP-1 and SHP-2 (1), have been cloned and characterized (reviewed in reference 37). Despite their structural similarity, SHP-1 and SHP-2 appear to possess distinct biological functions (reviewed in references 3, 36 to 38, 52, and 56). SHP-2 is expressed ubiquitously and typically transmits positive (i.e., signal-enhancing) signals downstream of receptor tyrosine kinases, cytokine receptors, and multichain immune recognition receptors, although recent work suggests that it may have negative (i.e., signal-attenuating) effects in some pathways (33, 54). In contrast, SHP-1 is expressed predominantly in hematopoietic cells, where it negatively regulates multiple growth factor and cytokine signaling pathways (reviewed in references 36, 38 and 56).Our current understanding of the role played by S...

Previous reports indicate that the expression and͞or activity of the protein-tyrosine phosphatase (PTP) LAR are increased in insulinresponsive tissues of obese, insulin-resistant humans and rodents, but it is not known whether these alterations contribute to the pathogenesis of insulin resistance. To address this question, we generated transgenic mice that overexpress human LAR, specifically in muscle, to levels comparable to those reported in insulinresistant humans. In LAR-transgenic mice, fasting plasma insulin was increased 2.5-fold compared with wild-type controls, whereas fasting glucose was normal. Whole-body glucose disposal and glucose uptake into muscle in vivo were reduced by 39 -50%. Insulin injection resulted in normal tyrosyl phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) in muscle of transgenic mice. However, phosphorylation of IRS-2 was reduced by 62%, PI3 kinase activity associated with phosphotyrosine, IRS-1, or IRS-2 was reduced by 34 -57%, and association of p85␣ with both IRS proteins was reduced by 39 -52%. Thus, overexpression of LAR in muscle causes whole-body insulin resistance, most likely due to dephosphorylation of specific regulatory phosphotyrosines on IRS proteins. Our data suggest that increased expression and͞or activity of LAR or related PTPs in insulin target tissues of obese humans may contribute to the pathogenesis of insulin resistance.

Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway. In adipose tissue and muscle of obese humans and rodents, PTP1B expression is increased, which led to the hypothesis that PTP1B plays a role in the pathogenesis of insulin resistance. Consistent with this, mice in which the PTP1B gene was disrupted exhibit increased insulin sensitivity. To test whether increased expression of PTP1B in an insulin-sensitive cell type could contribute to insulin resistance, we overexpressed wild-type PTP1B in 3T3L1 adipocytes using adenovirusmediated gene delivery. PTP1B expression was increased ϳ3-5-fold above endogenous levels at 16 h, ϳ14-fold at 40 h, and ϳ20-fold at 72 h post-transduction. Total protein-tyrosine phosphatase activity was increased by 50% at 16 h, 3-4-fold at 40 h, and 5-6-fold at 72 h posttransduction. Compared with control cells, cells expressing high levels of PTP1B showed a 50 -60% decrease in maximally insulin-stimulated tyrosyl phosphorylation of IR and insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) activity associated with IRS-1 or with phosphotyrosine. Akt phosphorylation and activity were unchanged. Phosphorylation of p42 and p44 MAP kinase (MAPK) was reduced ϳ32%. Overexpression of PTP1B had no effect on basal, submaximally or maximally (100 nM) insulin-stimulated glucose transport or on the EC 50 for transport. Our results suggest that: 1) insulin stimulation of glucose transport in adipocytes requires <45% of maximal tyrosyl phosphorylation of IR or IRS-1 and <50% of maximal activation of PI3K, 2) a novel PI3K-independent pathway may play a role in insulin-induced glucose transport in adipocytes, and 3) overexpression of PTP1B alone in adipocytes does not impair glucose transport.