Substitution of two insulin receptor carboxy-terminal tyrosines with phenylalanine impairs the expression of MAP kinase phosphatase-1 (MKP-1) mRNA

Cells expressing mutant insulin receptors (Y/F2), in which tyrosines 1316 and 1322 have been replaced with phenylalanine, exhibit enhanced insulin-induced MAP kinase activity and DNA synthesis in comparison with cells expressing wild type insulin receptors (Hirc B). To elucidate the mechanism of enhanced responsiveness, the expression of MAP kinase phosphatase-1 (MKP-1), a negative regulator of MAP kinase activity, was measured in Hirc B and Y/F2 cells incubated in the absence and presence of insulin for various periods of time, and over increasing concentrations of the ligand. Treatment of both cell lines with insulin induced a time and concentration-dependent relative increase in MKP-1 mRNA expression. However, in Y/F2 cells both basal and insulin-stimulated MKP-1 mRNA levels were more than 60% lower than that observed in cells transfected with the wild-type receptors. Cyclic AMP analog (8-Br-cAMP)/inducer (Forskoline) increased MKP-1 mRNA levels in both cell lines, and to a lesser extent in Y/F2 cells. In contrast to insulin the relative increase in MKP-1 mRNA expression induced by 8-Br-cAMP or forskoline was similar in Y/F2 and Hirc B cells. The overexpression of MKP-1 in Y/F2 cells inhibited insulin stimulated DNA synthesis. Transfection of wild type insulin receptors into Y/F2 cells increased basal levels of MKP-1. These results suggest that insulin receptor tyrosine residues 1316 and 1322 play an important role in the regulation of MKP-1 expression both under basal and insulin stimulated conditions, and are not necessary for the induction of MKP-1 mRNA by cAMP. Furthermore, the enhanced insulin induced mitogenic signaling seen in Y/F2 cells is, at least in part, due to impaired MKP-1 expression.     

Endogenous macrophage migration inhibitory factor modulates glucocorticoid sensitivity in macrophages via effects on MAP kinase phosphatase-1 and p38 MAP kinase

The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is induced by glucocorticoids (GCs), but it was not previously known if MIF regulates cellular sensitivity to GC. Here we show in GC and LPS-treated peritoneal macrophages derived from MIF-/- and wt mice that the absence of endogenous MIF is associated with increased sensitivity to GC of TNF release. This is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), concomitant decreased phosphorylation of p38 MAPK, but no effect of MIF on nuclear factor kappaB (NF-kappaB). These results demonstrate that MIF regulates GC sensitivity by phosphorylation of p38, and provides a cellular mechanism for this observation, indicating that MKP-1 is a central target of this regulation.     

Identification by surface plasmon resonance of the mycobacterial lipomannan and lipoarabinomannan domains involved in binding to CD14 and LPS-binding protein

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), regulate host defence mechanisms through their interaction with pattern recognition receptors such as Toll-like receptors (TLRs). We have developed a surface plasmon resonance assay to analyse the molecular basis for the recognition of Mycobacterium kansasii LM or LAM, by immobilized CD14 and LPS-binding protein (LBP) both being capable to promote presentation of bacterial glycolipids to TLRs. The affinity of either LM/LAM was higher to CD14 than to LBP. Kinetic and Scatchard analyses were consistent with a model involving a single class of binding sites. These interactions required the lipidic anchor, but not the carbohydrate domains, of LM or LAM. We also provide evidence that addition of recombinant LBP enhanced the stimulatory effect of LM or LAM on matrix metalloproteinase-9 expression and secretion in macrophages, through a TLR1/TLR2-dependent mechanism.     

Identification of a novel Ser/Thr protein phosphatase Ppq1 as a negative regulator of mating MAP kinase pathway in Saccharomyces cerevisiae

The specificity and efficiency of cell signaling is largely governed by the complex formation of signaling proteins. The precise spatio-temporal control of the complex assembly is crucial for proper signaling and cell survival. Protein phosphorylation is a key mechanism of signal processing in most of cell signaling networks. Phosphatases, along with kinases, control the phosphorylation state of many proteins and thus play a critical role in the precise regulation of signaling at each stage such as activation, propagation, and adaptation. Identification and functional analysis of pathway-specific phosphatase is, therefore, crucial for the understanding of cell signaling mechanisms. Here, we have developed a novel screening strategy to identify pathway-specific phosphatases, in which the entire repertoire of cell’s phosphatases was tethered to a signaling complex and the changes in signaling response were monitored. As a model target, we have chosen the mating MAP kinase pathway in the budding yeast, which is composed of three kinases and Ste5 scaffold protein. Using this strategy, a putative Ser/Thr phosphatase, Ppq1, was identified to be mating-specific. Results show that Ppq1 down-regulates mating signaling by targeting at or upstream of the terminal MAP kinase Fus3 in the cascade. The catalytic activity of Ppq1 as a phosphatase was confirmed in vitro and is necessary for its function in the regulation of mating signaling. Overall, the data suggest that Ppq1 functions as a negative regulator of mating MAPK pathway by dephosphorylating target pathway protein(s) and plays a key role in the control of the background signaling noise.     

Pellino proteins are more than scaffold proteins in TLR/IL-1R signalling: a role as novel RING E3-ubiquitin-ligases

Members of the Toll-like receptor (TLR) and IL-1 receptor (IL-1R) family initiate signalling pathways that shape innate immunity. Pellino proteins have recently been implicated as evolutionary conserved scaffold proteins in TLR/IL-1R signalling leading to nuclear factor-kappaB and mitogen activated protein kinase-dependent gene expression. We found that Pellino proteins contain a new RING-like motif. Because RING motifs are a feature of a subclass of E3-ubiquitin-ligases that target specific proteins for ubiquitination, we suggest that Pellino proteins are involved in TLR/IL-1R signalling not only as scaffold proteins but also as RING E3-ubiquitin-ligases. In support of this hypothesis we show that Pellino proteins induce IRAK-1 polyubiquitination in a RING-dependent manner. We further propose a model in which Pellino-mediated IRAK-1 polyubiquitination regulates TLR/IL-1R signalling.     

Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.     

PTEN, a negative regulator of PI3 kinase signalling, alters tau phosphorylation in cells by mechanisms independent of GSK-3

Deregulation of PTEN/Akt signalling has been recently implicated in the pathogenesis of Alzheimer's disease (AD), but the effects on the molecular processes underlying AD pathology have not yet been fully described. Here we report that overexpression of PTEN reduces tau phosphorylation in CHO cells. This effect was abrogated by mutant PTEN constructs with either a catalytically inactive point mutation (C124S) or with only inactive lipid phosphatase activity (G129E), suggesting an indirect, lipid phosphatase-dependent process. The predominant effects of PTEN on tau appeared to be mediated by reducing ERK1/2 activity, but were independent of Akt, GSK-3, JNK and the tau phosphatases PP1 and PP2A. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis, and provide some insight into the mechanisms through which deregulation of PTEN may contribute towards the progression of tauopathy.     

Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced

Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.     

Microarray and phosphokinase screenings leading to studies on ERK and JNK regulation of connective tissue growth factor expression by angiotensin II 1a and bradykinin B2 receptors in Rat1 fibroblasts

Rat1 fibroblasts stably transfected with the rat angiotensin II (AngII) AT1a and bradykinin (BK) B2 receptor cDNAs gained the ability to bind Ang II and BK. Wild-type Rat1 cells bound neither ligand. Exposure to either effector led to characteristic Galphai and Galphaq signal cascades, the release of arachidonic acid (ARA), and the intracellular accumulation of inositol phosphates (IP). Microarray analyses in response to BK or AngII showed that both receptors markedly induce the CCN family genes, CTGF (CCN2) and Cyr61 (CCN1), as well as the vasculature-related genes, Cnn1 and Egr1. Real time PCR confirmed the increased expression of connective tissue growth factor (CTGF) mRNA. Combined sequence-based analysis of gene promoter regions with statistical prevalence analyses identified CREB, SRF, and ATF-1, downstream targets of ERK, and JNK, as prominent products of genes that are regulated by ligand binding to the BK or AngII receptors. The binding of AngII or BK markedly stimulated the phosphorylation and thus the activation of ERK2, JNK, and p38MAPK. A BKB2R and an AT1aR chimera which displayed only negligible G-protein-related signaling were constructed. Both mutant receptors continued to activate these kinases and stimulate CTGF expression. Inhibitors of ERK1/2 and JNK but not p38MAPK inhibited the BK- and AngII-stimulated expression of CTGF in cells expressing either the WT or mutant receptors, illustrating that ERK and JNK participate in the control of CTGF expression in a manner that appears to be independent of G-protein. Conversely, addition of BK or AngII to the cell line expressing WT AT1aR and BKB2R downregulated the expression of collagen alpha1(I) (COL1A1) mRNA. However, these effectors did not have this effect in cells expressing the mutant receptors. Thus, a robust G-protein related response is necessary for BK or AngII to affect COL1A1 expression.     

Augmented oxygen-mediated transcriptional activation of cytochrome P450 (CYP)1A expression and increased susceptibilities to hyperoxic lung injury in transgenic mice carrying the human CYP1A1 or mouse 1A2 promoter in vivo

Background/aims

TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involved in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation.

Methods

We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC^KM) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by ³H-TdR incorporation.

Results

TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk.

Conclusions

TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.     

The two faces of PTP1B in cancer

PTP1B is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and is a promising drug target for type 2 diabetes and obesity. Accumulating evidence also indicates that PTP1B is involved in cancer, but contrasting findings suggest that it can exert both tumor suppressing and tumor promoting effects depending on the substrate involved and the cellular context. In this review, we will discuss the diverse mechanisms by which PTP1B may influence tumorigenesis as well as recent in vivo data on the impact of PTP1B deficiency in murine cancer models. Together, these results highlight not only the great potential of PTP1B inhibitors in cancer therapy but also the need for a better understanding of PTP1B function prior to use of these compounds in human patients.     

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E₂ (PGE₂) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE₂ formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β).     

Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-β-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G_q/11 protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP₃) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G_q/11 protein and inositol-1,4,5-trisphosphate-induced Ca²⁺ mobilization in human ASMCs.