FLT3/ITD mutation signaling includes suppression of SHP-1

Mutations in the FLT3 gene are the most common genetic alteration found in AML patients. FLT3 internal tandem duplication (ITD) mutations result in constitutive activation of FLT3 tyrosine kinase activity. The consequences of this activation are an increase in total phosphotyrosine content, persistent downstream signaling, and ultimately transformation of hematopoietic cells to factor-independent growth. The Src homology (SH)2 domain-containing protein-tyrosine phosphatase (SHP)-1 is involved in the down-regulation of a broad range of growth factor and cytokine-driven signaling cascades. Loss-of-function or deficiency of SHP-1 activity results in a hyperproliferative response of myelomonocytic cell populations to growth factor stimulation. In this study, we examined the possible role of SHP-1 in regulating FLT3 signaling. We found that transformation of TF-1 cells with FLT3/ITD mutations suppressed the activity of SHP-1 by approximately 3-fold. Suppression was caused by decreased SHP-1 protein expression, as analyzed at both the protein and RNA levels. In contrast, protein levels of SHP-2, a phosphatase that plays a stimulatory role in signaling through a variety of receptors, did not change significantly in FLT3 mutant cells. Suppressed SHP-1 protein levels in TF-1/ITD cells were partially overcome after cells were exposed to CEP-701, a selective FLT3 inhibitor. SHP-1 protein levels also increased in naturally occurring FLT3/ITD expressing AML cell lines and in primary FLT3/ITD AML samples after CEP-701 treatment. Furthermore, a small but reproducible growth/survival advantage was observed in both TF-1 and TF-1/ITD cells when SHP-1 expression was knocked down by RNAi. Taken together, these data provide the first evidence that suppression of SHP-1 by FLT3/ITD signaling may be another mechanism contributing to the transformation by FLT3/ITD mutations.     

The SHP—2 tyrosine phosphatase：Signaling mechanisms and biological functions

Cellular biological avtivities are tightly controlled by intracellular signaling processes initiated by extracellular signals.Protein tyrosine phosphatases,which remove phosphate groups from phosphorylated signaling molecules,play equally important tyrosine roles as protein tyrosine kinases in signal transduction.SHP-2 a cytoplasmic SH2 domain containing protein tyrosine phosphatase,is involved in the signaling pathways of a variety of growth factors and cytokines.Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signal relay from the cell surface to the nucleus,and is a critical intracellular regulator in mediating cell proliferation and differentiation.     

Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2

The nontransmembrane protein tyrosine phosphatase SHP-2 plays a critical role in growth factor and cytokine signaling pathways. Previous studies revealed that a fraction of SHP-2 moves to focal contacts upon integrin engagement and that SHP-2 binds to SHP substrate 1 (SHPS-1)/SIRP-1alpha, a transmembrane glycoprotein with adhesion molecule characteristics (Y. Fujioka et al., Mol. Cell. Biol. 16:6887-6899, 1996; M. Tsuda et al., J. Biol. Chem. 273:13223-13229). Therefore, we asked whether SHP2-SHPS-1 complexes participate in integrin signaling. SHPS-1 tyrosyl phosphorylation increased upon plating of murine fibroblasts onto specific extracellular matrices. Both in vitro and in vivo studies indicate that SHPS-1 tyrosyl phosphorylation is catalyzed by Src family protein tyrosine kinases (PTKs). Overexpression of SHPS-1 in 293 cells potentiated integrin-induced mitogen-activated protein kinase (MAPK) activation, and potentiation required functional SHP-2. To further explore the role of SHP-2 in integrin signaling, we analyzed the responses of SHP-2 exon 3(-/-) and wild-type cell lines to being plated on fibronectin. Integrin-induced activation of Src family PTKs, tyrosyl phosphorylation of several focal adhesion proteins, MAPK activation, and the ability to spread on fibronectin were defective in SHP-2 mutant fibroblasts but were restored upon SHP-2 expression. Our data suggest a positive-feedback model in which, upon integrin engagement, basal levels of c-Src activity catalyze the tyrosyl phosphorylation of SHPS-1, thereby recruiting SHP-2 to the plasma membrane, where, perhaps by further activating Src PTKs, SHP-2 transduces positive signals for downstream events such as MAPK activation and cell shape changes.     

Role of the SHP-2 tyrosine phosphatase in cytokine-induced signaling and cellular response

Cytokines and growth factors are important extracellular regulatory proteins. They exert their biological functions through binding to their cognate receptors on the cell surface and triggering intracellular signaling cascades. However, the intracellular signaling mechanisms of cytokines and growth factors are not well understood. Accumulating evidence has shown that protein phosphorylation and dephosphorylation carried out by protein kinases and protein phosphatases are fundamental biochemical events in intracellular signal transduction. SHP-2, a Src homology (SH) 2 domain-containing protein tyrosine phosphatase (PTP), is widely involved in a variety of signaling pathways triggered by cytokines and growth factors, including the MAP kinase, Jak-Stat, and PI3 kinase pathways. Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signals relayed from the cell surface to the nucleus, and is a critical intracellular regulator in cytokine and growth factor-induced cell survival, proliferation, and differentiation.     

Tyrosine phosphatase SHP-2 regulates IL-1 signaling in fibroblasts through focal adhesions

Interleukin-1beta (IL-1beta) mediates destruction of matrix collagens in diverse inflammatory diseases including arthritis, periodontitis, and pulmonary fibrosis by activating fibroblasts, cells that interact with matrix proteins through integrin-based adhesions. In vitro, IL-1beta signaling is modulated by focal adhesions, supramolecular protein complexes that are enriched with tyrosine kinases and phosphatases. We assessed the importance of tyrosine phosphatases in regulating cell-matrix interactions and IL-1beta signaling. In human gingival fibroblasts plated on fibronectin, IL-1beta enhanced the maturation of focal adhesions as defined by morphology and enrichment with paxillin and alpha-actinin. IL-1beta also induced activation of ERK and recruitment of phospho-ERK to focal complexes/adhesions. Treatment with the potent tyrosine phosphatase inhibitor pervanadate, in the absence of IL-1beta, recapitulated many of these responses indicating the importance of tyrosine phosphatases. Immunoblotting of collagen bead-associated complexes revealed that the tyrosine phosphatase, SHP-2, was also enriched in focal complexes/adhesions. Depletion of SHP-2 by siRNA or by homologous recombination markedly altered IL-1beta-induced ERK activation and maturation of focal adhesions. IL-1beta-induced tyrosine phosphorylation of SHP-2 on residue Y542 promoted focal adhesion maturation. Association of Gab1 with SHP-2 in focal adhesions correlated temporally with activation of ERK and was abrogated in cells expressing mutant (Y542F) SHP-2. We conclude that IL-1beta mediated maturation of focal adhesions is dependent on tyrosine phosphorylation of SHP-2 at Y542, leading to recruitment of Gab1, a process that may influence the downstream activation of ERK.     

The protein tyrosine phosphatase SHP-2 regulates interleukin-1-induced ERK activation in fibroblasts

Focal adhesion complexes are actin-rich, cytoskeletal structures that mediate cell adhesion to the substratum and also selectively regulate signal transduction pathways required for interleukin (IL)-1beta signaling to the MAP kinase, ERK. IL-1-induced ERK activation is markedly diminished in fibroblasts deprived of focal adhesions whereas activation of p38 and JNK is unaffected. While IL-1 signaling is known to involve the activity of protein and lipid kinases including MAP kinases, FAK, and PI3K, little is known about the role of phosphatases in the regulation of IL-1 signal generation and attenuation. Here we demonstrate that SHP-2, a protein tyrosine phosphatase present in focal adhesions, modulates IL-1-induced ERK activation and the transient actin stress fiber disorganization that occurs following IL-1 treatment in human gingival fibroblasts. Using a combination of immunoblotting, immunoprecipitation, and immunostaining we show that SHP-2 is present in nascent focal adhesions and undergoes phosphorylation on tyrosine 542 in response to IL-1 stimulation. Blocking anti-SHP-2 antibodies, electoporated into the cytosol of fibroblasts, inhibited IL-1-induced ERK activation, actin filament assembly, and cell contraction, indicating a role for SHP-2 in these processes. In summary, our data indicate that SHP-2, a focal adhesion-associated protein, participates in IL-1-induced ERK activation likely via an adaptor function.     

Tyrosine phosphatase SHP-2 is a mediator of activity-dependent neuronal excitotoxicity

Calcium influx can promote neuronal differentiation and survival, at least in part by activating Ras and its downstream targets, including the Erk pathway. However, excessive calcium influx can initiate molecular signals leading to neuronal death during excitotoxicity or in neurodegenerative diseases. Here we describe a new signaling pathway associated with calcium influx that contributes to neuronal cell death in cerebellar neurons. Influx of calcium, mediated either by L-type voltage-sensitive calcium channels or glutamate receptors, is associated with the suppression of brain-derived neurotrophic factor (BDNF) activation of Ras and its effectors Erk and Akt. This is the result of enhanced association of the tyrosine phosphatase Shp-2 with TrkB receptors, which inhibits BDNF-induced TrkB autophosphorylation and activation. Deletion of the Shp2 gene in neuronal cultures reverses inhibition of TrkB function and increases neuronal survival after extended depolarization or glutamate treatment. These findings implicate Shp-2 in a feedback system initiated by calcium that negatively regulates neurotrophin signaling and sensitizes neurons to excitotoxicity.     

The tyrosine phosphatase SHP-2 controls urokinase-dependent signaling and functions in human vascular smooth muscle cells

The urokinase (uPA)/urokinase receptor (uPAR) multifunctional system is an important mediator of functional behaviour of human vascular smooth muscle cells (VSMC). uPAR associates with platelet-derived growth factor receptor β (PDGFR-β), which serves as a transmembrane adaptor for uPAR in VSMC, to transduce intracellular signaling and initiate functional changes. The precise and rapid propagation of these signaling cascades demands both strict and flexible regulatory mechanisms that remain unexplored. We provide evidence that the tyrosine phosphatase SHP-2 mediates these processes. uPA regulated SHP-2 phosphorylation, catalytic activity, and its co-localization and association with the PDGFR-β. Active PDGFR-β was required for the uPA-induced SHP-2 phosphorylation. uPAR-directed STAT1 pathway was disturbed in cells expressing SHP-2 inactive mutant. Both, cell proliferation and migration were impaired in VSMC with downregulated SHP-2. Elucidating the underlying mechanisms, we found that uPA induced SHP-2 recruitment to lipid rafts. Disruption of rafts abolished uPA-related control of SHP-2 phosphorylation, its association with PDGFR-β and finally the VSMC functional responses. Our results demonstrate that SHP-2 plays an important role in uPA-directed signaling and functional control of human VSMC and suggest that this phosphatase might contribute to the pathogenesis of the uPA-related vascular remodeling.     

SHP-2激活突变促进小鼠MEFs细胞粘附迁移及增殖能力

目的探讨SHP-2D61G/＋和SHP-2D61G/D61G激活突变对小鼠胚胎成纤维细胞（MEFs）粘附迁移及增殖能力的影响,并研究其发生的机制。方法雌雄小鼠合笼交配建立SHP-2D61G/＋、SHP-2D61G/D61G激活突变的小鼠MEFs细胞,并以SV40T抗原进行永生化;细胞粘附实验检测SHP-2D61G/＋、SHP-2D61G/D61G激活突变对MEFs细胞粘附能力的影响;Transwell体外迁移实验检测SHP-2D61G/＋、SHP-2D61G/D61G激活突变对MEFs细胞的迁移能力的影响;MTT法检测SHP-2D61G/＋、SHP-2D61G/D61G激活突变对MEFs细胞增殖能力的影响;Western Blot法检测p-ERK的表达水平。结果 （1）与对照组相比,SHP-2D61G/＋、SHP-2D61G/D61G激活突变组小鼠MEFs细胞粘附的细胞数明显增多,差异具有统计学意义;（2）与对照组相比SHP-2D61G/＋、SHP-2D61G/D61G激活突变组MEFs细胞迁移的细胞数增加,差异具有统计学意义;（3）MTT结果显示,SHP-2D61G/＋、SHP-2D61G/D61G激活突变的小鼠MEFs细胞增殖能力较对照组强,差异具有统计学意义;（4）Western Blot结果显示与对照组相比,无论是刚刚贴壁还是贴壁后30 min和60 min SHP-2D61G/＋、SHP-2D61G/D61G激活突变组其p-ERK的表达水平都增加。结论 SHP-2D61G/＋、SHP-2D61G/D61G激活突变促进小鼠MEFs细胞粘附迁移及增殖能力,其发生机制主要与p-ERK的表达水平增加有关。     

Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia encountered in clinical practice. We first reported an S140G mutation of KCNQ1, an alpha subunit of potassium channels, in one Chinese kindred with AF. However, the molecular defects and cellular mechanisms in most patients with AF remain to be identified. We evaluated 28 unrelated Chinese kindreds with AF and sequenced eight genes of potassium channels (KCNQ1, HERG, KCNE1, KCNE2, KCNE3, KCNE4, KCNE5, and KCNJ2). An arginine-to-cysteine mutation at position 27 (R27C) of KCNE2, the beta subunit of the KCNQ1-KCNE2 channel responsible for a background potassium current, was found in 2 of the 28 probands. The mutation was present in all affected members in the two kindreds and was absent in 462 healthy unrelated Chinese subjects. Similar to KCNQ1 S140G, the mutation had a gain-of-function effect on the KCNQ1-KCNE2 channel; unlike long QT syndrome-associated KCNE2 mutations, it did not alter HERG-KCNE2 current. The mutation did not alter the functions of the HCN channel family either. Thus, KCNE2 R27C is a gain-of-function mutation associated with the initiation and/or maintenance of AF.     

FGFR2功能增强对小鼠下颌骨髁突发育影响

成纤维细胞生长因子受体2(Fibroblast growth factor receptor, FGFR2)是参与调控骨骼发育的重要分子,在调控软骨内成骨过程中发挥着重要作用。为了探讨FGFR2功能增强对小鼠下颌骨髁突生长发育的影响,文章以FGFR2功能增强型点突变(Fgfr2^+/S252W)小鼠为研究对象,采用番红固绿染色研究Fgfr2^+/S252W小鼠下颌骨髁突不同生长发育阶段的组织形态;利用免疫细胞化学染色和实时荧光定量PCR方法检测X型胶原(Col X)在3周龄小鼠髁突肥大软骨细胞中的表达。结果显示,1周龄、3周龄和6周龄突变型小鼠下颌骨髁突的软骨细胞层宽度都比同窝野生型窄,钙化软骨细胞层退化时间早,骨小梁钙化绿染程度深;Col X在突变型小鼠下颌骨髁突的表达高于同窝野生型小鼠(P<0.001)。结果表明,FGFR2功能增强可导致小鼠下颌骨髁突软骨层组织形态异常,抑制髁突软骨内成骨,从而导致下颌骨髁突发育畸形。     

Tyrosine phosphatase SHP-2 is a regulator of p27Kip1 tyrosine phosphorylation

Tyrosine phosphorylation of the cell cycle regulator p27^Kip1 plays a crucial role in its binding to cyclin dependent kinases and its subcellular localization. While Src and Bcr-Abl were shown to be responsible for tyrosine phosphorylation, no data are available on the dephosphorylation of p27^Kip1 and the phosphatase involved. Considering the associated dephosphorylation as a pivotal event in the regulation of cell cycle proteins, we focused on the tyrosine phosphatase SHP-2, which is regulated in promyelocytic leukemia cells on G-CSF stimulation. SHP-2 was thus found in association with p27^Kip1 and the G-CSF receptor, and we observed a nuclear translocation of SHP-2 on G-CSF stimulation. Using a catalytically inactive form of SHP-2 and siRNA directed against SHP-2, we could demonstrate the involvement of SHP-2 in tyrosine dephosphorylation of p27^Kip1. Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27^Kip1 in vitro. Most importantly, we could illustrate that SHP-2 modulates p27^Kip1 stability and contributes to p27^Kip1-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27^Kip1 tyrosine phosphorylation.     

A687V EZH2 is a gain-of-function mutation found in lymphoma patients

Heterozygous point mutations at Y641 and A677 in the EZH2 SET domain are prevalent in about 10-24% of Non-Hodgkin lymphomas (NHL). Previous studies indicate that these are gain-of-function mutations leading to the hypertrimethylation of H3K27. These EZH2 mutations may drive the proliferation of lymphoma and make EZH2 a molecular target for patients harboring these mutations. Here, another EZH2 SET domain point mutation, A687V, occurring in about 1-2% of lymphoma patients, is also shown to be a gain-of-function mutation that greatly enhances its ability to perform dimethylation relative to wild-type EZH2 and is equally proficient at catalyzing trimethylation. We propose that A687V EZH2 also leads to hypertrimethylation of H3K27 and may thus be a driver mutation in NHL.     

SHP-2-Erk signaling regulates concanavalin A-dependent production of TIMP-2

To search for the signaling critical for the production of tissue inhibitor of metalloproteinase-2 (TIMP-2), we investigated the role of SHP-2 in TIMP-2 production with Concanavalin A (Con A)-treated cells. In wild-type fibroblasts, Con A-treatment dramatically activated TIMP-2 production. In contrast, production of TIMP-2 in response to Con A-treatment was severely impaired in cells expressing mutant SHP-2 whose 65 amino acids in the SH2-N domain were deleted. Con A-treatment activated dual signaling pathways, Erk and p38, in a SHP-2-dependent manner. Pretreatment of wild-type cells with U0126, a potent inhibitor of MEK1, significantly inhibited the production of TIMP-2, whereas SB203580, a specific inhibitor for p38, could not. Finally, expression of exogenous wild-type SHP-2 in SHP-2 mutant cells clearly rescued Erk activation and TIMP-2 production in response to Con A-treatment. Taken together, our results strongly suggest that SHP-2 plays a critical role as a positive modulator for the production of TIMP-2 via MEK1-Erk signaling in fibroblasts.     

Hereditary pancreatitis caused by a double gain-of-function trypsinogen mutation

Hereditary pancreatitis, an autosomal dominant disease with ∼80% penetrance, can be caused by both ‘gain-of-function’ missense and copy number mutations in the cationic trypsinogen gene (PRSS1). Here we demonstrate a heterozygous hybrid PRSS2 (encoding anionic trypsinogen)/PRSS1 gene in a French white family with hereditary pancreatitis, by means of quantitative fluorescent multiplex PCR and RT-PCR analyses. The hybrid gene, in which exons 1 and 2 are derived from PRSS2 and exons 3–5 from PRSS1, apparently resulted from a non-allelic homologous recombination (NAHR) event between the chromosome 7 homologs or sister chromatids during meiosis. Interestingly, this hybrid gene causes the disease through a combination of its inherent ‘double gain-of-function’ effect, acting simultaneously as a ‘quantitative’ copy number mutation and a ‘qualitative’ missense mutation (i.e. the known disease-causing p.N29I mutation). Our finding reveals a previously unknown mechanism causing human inherited disease, enriches the lexicon of human genetic variation and goes beyond the known interaction between copy number variations (CNVs) and single nucleotide substitutions in health and disease. Our finding should also stimulate more interest in analyzing both types of genetic variation whenever one tries to determine the contribution of a specific locus to a given disease phenotype. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The SHP-1 protein tyrosine phosphatase negatively modulates Akt signaling in the ghrelin/GHSR1a system

The aim of the present study was to identify the signaling mechanism(s) responsible for the modulation of growth hormone secretagogue receptor type 1a (GHSR1a)-associated Akt activity. Ghrelin leads to the activation of Akt through the interplay of distinct signaling mechanisms: an early G(i/o) protein-dependent pathway and a late pathway mediated by β-arrestins. We found that the Src homology 2-containing protein tyrosine phosphatase (SHP-1) was an essential molecule in both G(i/o) protein-dependent and β-arrestin-mediated pathways. More specifically, the role of SHP-1 in the G(i/o) protein-dependent pathway was demonstrated by the fact that the overexpression of a catalytically defective SHP-1 augments tyrosine phosphorylation of the PI3K regulatory subunit p85, leading to an increase in the phosphorylation of cSrc and phosphoinositide-dependent protein kinase 1, and finally activating Akt. The presence of SHP-1 in the β-arrestin-scaffolded complex and its attenuating effect on the cSrc and Akt activities verified that SHP-1 regulates not only the G(i/o) protein-dependent pathway but also the β-arrestin-mediated pathway. Assays performed in preadipocyte and adipocyte 3T3-L1 cells showed SHP-1 expression. According to our results in HEK-GHSR1a cells, ghrelin stimulated SHP-1 phosphorylation in 3T3-L1 cells. The increase in ghrelin-induced Akt activity was enhanced by small interfering RNA of SHP-1 in preadipocyte 3T3-L1 cells. These results were reproduced in white adipose tissue obtained from mice, in which SHP-1 exhibited higher expression in omental than in subcutaneous tissue. Furthermore, this pattern of expression was inverted in mice fed a high-fat diet, suggesting a role for SHP-1 in controlling ghrelin sensitivity in adipose tissue. Indeed, SHP-1 deficiency was associated with augmented ghrelin-evoked Akt phosphorylation in omental tissue, as well as decreased phosphorylation under overexpression of SHP-1 in subcutaneous tissue. These findings showed a novel role for SHP-1 in the regulation of Akt activity through the modulation of the ghrelin/GHSR1a system signaling.