Tyrosine Phosphorylation Of SHP-2 Research Articles

SHP2 as a Potential Therapeutic Target in Diffuse-Type Gastric Carcinoma Addicted to Receptor Tyrosine Kinase Signaling.

Simple SummaryDiffuse-type gastric carcinoma (DGC) is characterized by rapid infiltrative growth associated with massive stroma and frequent peritoneal dissemination, which leads to poor patient outcomes. In this study, we found that the oncogenic tyrosine phosphatase SHP2 is tyrosine-phosphorylated downstream of the amplified receptor tyrosine kinases (RTKs) Met and fibroblast growth factor receptor 2 (FGFR2) in DGC cell lines. SHP2 knockdown or pharmacological inhibition selectively suppressed the growth of DGC addicted to amplified Met and FGFR2. Moreover, targeting SHP2 abrogated malignant phenotypes, including peritoneal dissemination, of Met-addicted DGC and could overcome acquired resistance to Met inhibitors. Our findings suggest that SHP2 is a potential target for the treatment of DGC addicted to amplified RTK signaling.Diffuse-type gastric carcinoma (DGC) exhibits aggressive progression associated with rapid infiltrative growth, massive fibrosis, and peritoneal dissemination. Gene amplification of Met and fibroblast growth factor receptor 2 (FGFR2) receptor tyrosine kinases (RTKs) has been observed in DGC. However, the signaling pathways that promote DGC progression downstream of these RTKs remain to be fully elucidated. We previously identified an oncogenic tyrosine phosphatase, SHP2, using phospho-proteomic analysis of DGC cells with Met gene amplification. In this study, we characterized SHP2 in the progression of DGC and assessed the therapeutic potential of targeting SHP2. Although SHP2 was expressed in all gastric carcinoma cell lines examined, its tyrosine phosphorylation preferentially occurred in several DGC cell lines with Met or FGFR2 gene amplification. Met or FGFR inhibitor treatment or knockdown markedly reduced SHP2 tyrosine phosphorylation. Knockdown or pharmacological inhibition of SHP2 selectively suppressed the growth of DGC cells addicted to Met or FGFR2, even when they acquired resistance to Met inhibitors. Moreover, SHP2 knockdown or pharmacological inhibition blocked the migration and invasion of Met-addicted DGC cells in vitro and their peritoneal dissemination in a mouse xenograft model. These results indicate that SHP2 is a critical regulator of the malignant progression of RTK-addicted DGC and may be a therapeutic target.

RMC-4550, an Allosteric Inhibitor of SHP2, Displays Therapeutic Efficacy in Pre-Clinical Models of Myeloproliferative Neoplasms

Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are JAK2-driven disorders resulting from mutations in JAK2, MPL, or CALR. Ruxolitinib, the only FDA-approved JAK2 inhibitor for MPNs, alleviates patient symptomology and improves quality of life, but has little effect on reducing mutant allele burden. This persistent survival of MPN cells in the face of ruxolitinib, as well as other JAK2 inhibitors that have been clinically tested, is a major clinical bottleneck to the development of an effective targeted therapy for MPN patients. Identifying new therapeutic targets which play critical roles in MPN cells and/or in JAK2 inhibitor persistence may lead to improved MPN therapies. SHP2 is an oncogenic tyrosine phosphatase that is an effector of growth factor and cytokine receptor signaling. SHP2 plays a critical role in the activation of the RAS-ERK pathway and regulates JAK-STAT signaling via numerous phosphatase-dependent mechanisms. Activating mutations of SHP2(PTPN11) have been identified in leukemia, including 8% of MPN patients whose disease progressed to acute myeloid leukemia (AML). In addition, SHP2 has been shown to mediate adaptive resistance to targeted therapies in several cancers. Given the role of SHP2 in cytokine and JAK-STAT signaling, we envisaged a potential role of SHP2 in MPN cell growth and/or survival and ruxolitinib persistence. Treatment of JAK2-V617F-driven MPN model cell lines (UKE1, SET2, and BaF3-JAK2-V617F) with ruxolitinib blocked constitutive tyrosine phosphorylation of SHP2, including phosphorylation of Y542, a marker for activated SHP2. This phosphorylation, however, was restored in ruxolitinib persistent cells. Combination treatment of the allosteric SHP2 inhibitor RMC-4550 (Revolution Medicines) with ruxolitinib prevented the development of ruxolitinib persistent cells and pre-established persistent cells remained sensitive to SHP2 inhibition. RMC-4550 treatment led to significantly reduced levels of pERK consistent with the role of SHP2 in RAS signaling. Interestingly, pERK levels in persistent cells were more sensitive to SHP2 inhibition compared to drug naïve cells suggesting pERK was more dependent on SHP2 in ruxolitinib persistent cells. SHP2 inhibitor treatment increased pSTAT5(Y694) in drug naïve cells but this increase was not observed in similarly treated persistent cells. Furthermore, while ruxolitinib inhibited pERK levels in UKE1 and SET2 cells, pERK levels recovered within 24 hrs of treatment. SHP2 inhibition prevented the recovery of pERK in the presence of ruxolitinib. Collectively, these data suggest that signaling pathways in MPN cells treated with ruxolitinib can become rewired, gaining greater dependence on SHP2, concomitant with sustained pERK and cell survival/growth. Interestingly, we identified a known activating SHP2 mutation (F71L) in UKE1 cells obtained from two independent sources - consistent with the presence of PTPN11 mutations in post-MPN AML. The persistent survival of UKE1 cells in ruxolitinib was antagonized by CRISPR-mediated reduction of SHP2 expression, providing further evidence that SHP2 contributes to ruxolitinib persistence. To assess the effects of a SHP2 inhibitor on MPN progression in vivo, we employed the MPLW515Lbone marrow transplant mouse model of MPN. Initial assessment of therapeutic treatment of mice with an established MPN phenotype indicated that once daily treatment of RMC-4550 (10 or 30 mg/kg) antagonized the MPN phenotype. Complete blood counts indicated a significant reduction in white blood cells, monocytes, and neutrophils compared to vehicle treated mice, while flow cytometry analysis indicated RMC-4550 diminished CD11b+ cell numbers to near that observed in mice transplanted with MPLWT-transduced bone marrow. RMC-4550 improved the overall health of diseased mice, as indicated by increased weight, and significantly reduced organomegaly of the spleen and liver compared to vehicle treated MPN mice. Finally, erythropoietin independent erythroid colony formation of JAK2V617F-positive MPN patient cells was suppressed following SHP2 inhibition, which synergized or enhanced the inhibition induced by ruxolitinib in this assay. In summary, our results suggest that SHP2 inhibition may represent a potential MPN therapy in both ruxolitinib naïve and resistant patients and is an attractive therapeutic target for future clinical investigation. Disclosures Epling-Burnette: Incyte Corporation: Research Funding; Forma Therapeutics: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding. Reuther:Incyte Corporation: Research Funding.

Amelioration of cisplatin-induced experimental peripheral neuropathy by a small molecule targeting p75NTR

Cisplatin is an effective and widely used first-line chemotherapeutic drug for treating cancers. However, many patients sustain cisplatin-induced peripheral neuropathy (CIPN), often leading to a reduction in drug dosages or complete cessation of treatment altogether. Therefore, it is important to understand cisplatin mechanisms in peripheral nerve tissue mediating its toxicity and identify signaling pathways for potential intervention. Rho GTPase activation is increased following trauma in several models of neuronal injury. Thus, we investigated whether components of the Rho signaling pathway represent important neuroprotective targets with the potential to ameliorate CIPN and thereby optimize current chemotherapy treatment regimens. We have developed a novel CIPN model in the mouse. Using this model and primary neuronal culture, we determined whether LM11A-31, a small-molecule, orally bioavailable ligand of the p75 neurotrophin receptor (p75NTR), can modulate Rho GTPase signaling and reduce CIPN. Von Frey filament analysis of sural nerve function showed that LM11A-31 treatment prevented decreases in peripheral nerve sensation seen with cisplatin treatment. Morphometric analysis of harvested sural nerves revealed that cisplatin-induced abnormal nerve fiber morphology and the decreases in fiber area were alleviated with concurrent LM11A-31 treatment. Cisplatin treatment increased RhoA activity accompanied by the reduced tyrosine phosphorylation of SHP2, which was reversed by LM11A-31. LM11A-31 also countered the effects of calpeptin, which activated RhoA by inhibiting SHP2 tyrosine phosphatase. Therefore, suppression of RhoA signaling by LM11A-31 that modulates p75NTR or activates SHP2 tyrosine phosphatase downstream of the NGF receptor enhances neuroprotection in experimental CIPN in mouse model.

Src Kinase Can Activate RUNX1 Activity Via Phosphorylation Of C-Terminal Tyrosines and Activated RUNX1 Stimulates Granulopoiesis

We demonstrated that G-CSF signaling in lineage-negative marrow myeloid progenitors induces C-terminal SHP2 tyrosine phosphorylation more potently than does M-CSF signaling and that SHP2 knockdown in marrow or in the 32Dcl3 cell line reduces Cebpa gene transcription and impairs granulopoiesis.1,2 We also found that Runx1 directly activates Cebpa transcription via promoter elements and via a +37 kb enhancer and that Runx1 gene deletion reduces Cebpa mRNA and impairs granulopoiesis.3 Runx1 is phosphorylated by Src kinase on five tyrosines and dephosphorylated by SHP2, and RUNX1(5F) enhances megakaryopoiesis and rescues CD8 T cell development, whereas RUNX1(5D) is ineffective.4 Mutation to F prevents while change to D or E mimics phosphorylation. These findings suggested that in myeloid cells there exists a G-CSF to SHP2 to RUNX1 to Cebpa activation pathway directing granulopoiesis. We compared wild-type (WT) murine RUNX1b and a series of variants, Y260/375/378/379/386F (5F), Y260F (1F), Y375/378/379/386F (4F), 5D, 1D, and 4D for activation in 293T cells of a reporter with four RUNX1-binding sites linked to the TK promoter and the luciferase cDNA. Expression was compared by Western blotting. WT stimulated the reporter 5-fold on average, relative to empty CMV vector, 5D activated the reporter 15-fold, while 5F was nearly inactive. 4F was also nearly inactive, while 4D had WT activity, as did the 1F and 1D variants. Both 4F and 5F were expressed at or above WT levels; interestingly, 5D, but not 4D or 1D, was expressed at much higher levels than the other variants. A similar pattern was obtained with the MCSFR-Luc reporter. To further query the activity of the four C-terminal tyrosines, we evaluated the activity of Y375/378F (2F*), Y379/386F (2F), 2E, Y375F, Y378F, Y379F, and Y386F. The single residue mutations retained WT activity, whereas both the 2F* and 2F variants demonstrated significant reduction despite expression at or above WT levels. The 2E variant expressed at levels below WT but had increased activity. We then transfected the RUNX1 reporter with activated Src, Runx1, or both – in several experiments Src stimulated the reporter approximately 3-fold, on average, RUNX1 4-fold, and the combination 35-fold. In the same experiments, Src + 5F led to only 6.6-fold activation, and synergy was also lost if the reporter lacked the RUNX1-binding sites. These findings, together with the observation that SHP2 directly activates Src family kinases downstream of G-CSF receptor signaling,5 raises the possibility that in myeloid cells there actually is a G-CSF to SHP2 to Src to Runx1 to Cebpa activation pathway. Consistent with this idea, we find that the 5D variant has reduced interaction with co-expressed HDAC3 relative to WT, as assessed by co-IP, whereas the 5F variant has increased interaction. We previously found that RUNX1-ER stimulates endogenous Cebpa transcription when activated in 32Dcl3 cells. As the ER segment has trans-activating activity, to evaluate RUNX1 variants we utilizing the zinc-inducible MT promoter. Preliminary analysis indicates that both WT RUNX1b and its 5D variant can induce C/EBPa protein and RNA in this context. We previously found that exogenous RUNX1-ER can rescue granulopoiesis when transduced into marrow from Runx1(flox/flox);Mx1-Cre mice subjected 4 weeks earlier to pIpC injections. We now have constructed MIGC retroviral vectors expressing RUNX1, 5F, or 5D and GFP-Cre. These, or the empty MIG or MIGC vectors, were packaged and transduced into marrow isolated from RUNX1(flox/flox) mice. Three days later, lineage-depleted, GFP+ cells were cultured in methylcellulose with IL-3, IL-6, and SCF. Compared with MIG, MIGC reduced the percent CFU-G amongst CFU-G+CFU-M, from 55+/-1% to 10+/-2%; WT RUNX1 or 5D partially rescued granulopoiesis, to 31+/- 8% or 23+/-9% respectively, whereas 5F transduction led to only 4+/-1% CFU-G. In summary, in 293T cells and potentially in myeloid cells Src kinase activates Runx1 and this may favor granulopoiesis.1. Jack et al. Blood 114:2172, 2009. 2. Zhang and Friedman. Blood 118:2266, 2011. 3. Guo et al. Blood 119:4408, 2012. 4. Huang et al. Genes Dev. 26:1587, 2012. 5. Futami et al. Blood 118:1077, 2011. Disclosures:Cantor:AMGEN: Membership on an entity's Board of Directors or advisory committees.

Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants

Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

Involvement of EphA2-mediated tyrosine phosphorylation of Shp2 in Shp2-regulated activation of extracellular signal-regulated kinase

Shp2 is a positive regulator for Erk activation downstream of receptor tyrosine kinases for growth factors. It has been controversial how Shp2 induces Erk activation. We here demonstrate that EphA2 is responsible for Shp2-mediated Erk activation by phosphorylating Tyr542 and Tyr580 of Shp2 in the cells stimulated with growth factors. In NMuMG mammary epithelial cells stimulated with hepatocyte growth factor (HGF), HGF-dependent Erk phosphorylation was prolonged only in the presence of EphA2. This Erk activation paralleled the phosphorylation of Tyr542/580 of Shp2 and the association of Grb2 with Shp2, suggesting the positive signal involving Grb2 signal to activate Ras-Erk pathway. Immunohistochemical studies of mammary cancer specimens revealed that the cancer progression was associated with both Tyr580 phosphorylation of Shp2 and increased expression of EphA2, which were also correlated with increased Erk phosphorylation. Overexpression of either Shp2Thr468Met (a phosphatase-defective mutant found in Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormal genitalia, Retardation of growth and sensorineural Deafness (LEOPARD) syndrome) or Shp2Asn308Asp (a phosphatase-active mutant found in Noonan syndrome) with EphA2 exhibited comparable activation of Erk and stronger activation than wild-type Shp2, suggesting the phosphatase-independent Erk activation. Expression of Shp2Thr468Met with Tyr542/580Phe mutations resulted in the suppression of Erk activation. Phosphatase-active and -inactive, and wild-type Shp2s bound equally to Grb2, suggesting that phosphorylation of Tyr542/580 of Shp2 was essential but not sufficient for Shp2-mediated Erk activation. We found that Gab1 (Grb2-associated binder 1) was involved in the mutant Shp2-mediated Erk activation. Zebrafish injected with Shp2Thr468Met mRNA showed cardiac edema, whereas those depleted of EphA2b showed less phenotype, suggesting that EphA2 might partly account for the phenotype of LEOPARD syndrome. Collectively, tyrosine phosphorylation of Shp2 by EphA2 contributes to the phosphatase-independent Shp2-mediated activation of Erk and might be involved in Shp2-associated diseases.

SHP2 tyrosine phosphatase stimulates CEBPA gene expression to mediate cytokine-dependent granulopoiesis

AbstractG-CSF signals contribute to granulocyte lineage specification. We previously found that G-CSF induces SHP2 tyrosine phosphorylation and that chemical inhibition of SHP1/SHP2 reduces CFU-G and prevents G-CSF but not M-CSF activation of ERK. We now find that SHP2 shRNA knockdown in the 32Dcl3 granulocytic line reduces ERK activation, diminishes CEBPA protein and RNA expression and promoter histone acetylation, and inhibits granulopoiesis. Exogenous, shRNA-resistant SHP2 rescues these effects of SHP2 knockdown, exogenous C/EBPα rescues granulocytic markers, and exogenous RUNX1 rescues C/EBPα. 32Dcl3 lines with knockdown of ERK1 and ERK2 retain normal levels of C/EBPα and differentiate normally in G-CSF despite also having reduced proliferation. SHP2 knockdown reduces CEBPA levels in lineage-negative murine marrow cells cultured in TPO, Flt3 ligand, and SCF, without affecting the rate of cell expansion. On transfer to IL-3, IL-6, and SCF to induce myelopoiesis, levels of granulocytic RNAs are reduced and monocyte-specific RNAs are increased by SHP2 knockdown, and there is a reduction in the percentage of CFU-G that form in methylcellulose and of granulocytes that develop in liquid culture. In summary, SHP2 is required for induction of C/EBPα expression and granulopoiesis in response to G-CSF or other cytokines independent of SHP2-mediated ERK activation.

The tyrosine phosphatase Shp‐2 interacts with the dopamine D1 receptor and triggers D1‐mediated Erk signaling in striatal neurons

We report a novel mechanism for dopamine D(1) receptor (D(1) R)-mediated extracellular signal-regulated kinases (Erk) activation in rat striatum. Erk signaling depends on phosphorylation and dephosphorylation events mediated by specific kinases and phosphatases. The tyrosine phosphatase Shp-2, that is required for Erk activation by tyrosine kinase receptors, has been recently shown to regulate signaling downstream of few G protein-coupled receptors. We show that the D(1) R interacts with Shp-2, that D(1) R stimulation results in Shp-2 tyrosine phosphorylation and activation in primary striatal neuronal cultures and that D(1) R/Shp-2 interaction is required for transmitting D(1) R-dependent signaling to Erk1/2 activation. D(1) R-mediated Erk1/2 phosphorylation in cultured striatal neurons is in fact abolished by over-expression of the inactive Shp-2(C/S) mutant and by small interfering RNA-induced Shp-2 silencing. Moreover, by using selective inhibitors we show that both D(1) R-induced Shp-2 activation and Erk1/2 phosphorylation are dependent on the cyclic AMP/protein kinase A pathway and require Src. These results, which were substantiated also in transfected human embryonic kidney 293 cells, provide a novel mechanism by which to converge D(1) R signaling to the Erk pathway and suggest that Shp-2 or the D(1) R/Shp-2 interface could represent a potential drug target for disorders of dopamine transmission involving malfunctioning of D(1) R signaling.

Abstract 4097: Protein tyrosine phosphatase SHP-2 regulates human pancreatic cancer cell biology on collagen I

Abstract With a five year survival rate of less than 5%, pancreatic cancer stands as one of the deadliest human malignancies. There is a great need for novel therapeutic strategies for this illness, and improving our understanding of pancreatic cancer biology will be a key step in developing these therapies. The purpose of these studies is to investigate the regulation and function of the Src Homology 2 (SH2) domain containing protein tyrosine phosphatase SHP-2 in human pancreatic cancer cells and to determine its potential value as a therapeutic target for this disease. In other cellular systems, SHP-2 is crucial for integrin and receptor tyrosine kinase-mediated activation of diverse signaling pathways, regulating many of the cellular events necessary for oncogenesis, including cell division, motility, adhesion, and survival. We hypothesized that inhibition of SHP-2 function would negatively impact integrin-mediated cell signaling and biology in response to the extracellular matrix protein collagen I, an abundant component of pancreatic tumor stroma in vivo. Initial results demonstrate that SHP-2 expression and tyrosine phosphorylation/activity are elevated in pancreatic cancer cells, relative to immortalized normal human pancreatic cells. Phosphorylation of SHP-2 is further elevated in the presence of collagen I. SHP-2 tyrosine phosphorylation in pancreatic cancer cells occurrs in a Src family kinase-dependent fashion. Initial results using miRNA-mediated knock-down of SHP-2 expression indicate the importance of SHP-2 for the ability of pancreatic cancer cells to proliferate, migrate and heal cell culture wounds on collagen I. Additionally, SHP-2 knockdown cells grown on collagen I demonstate altered tyrosine phosphorylation patterns of several signal transduction proteins, including focal adhesion kinase (FAK), in comparison to controls. These data suggest, for the first time, an importance for SHP-2 in pancreatic cancer cell biology and the potential value of SHP-2 as a therapeutic target for this disease. Ongoing and future experiments will compare the effects of SHP-2 inhibition on pancreatic tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4097.

A Novel Role of Vascular Endothelial Cadherin in Modulating c-Src Activation and Downstream Signaling of Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis and vascular permeability, in which c-Src tyrosine kinase plays an essential role. However, the mechanisms by which VEGF stimulates c-Src activation have remained unclear. Here, we demonstrate that vascular endothelial cadherin (VE-cadherin) plays a critical role in regulating c-Src activation in response to VEGF. In vascular endothelial cells, VE-cadherin was basally associated with c-Src and Csk (C-terminal Src kinase), a negative regulator of Src activation. VEGF stimulated Csk release from VE-cadherin by recruiting the protein tyrosine phosphatase SHP2 to VE-cadherin signaling complex, leading to an increase in c-Src activation. Silencing VE-cadherin with small interference RNA significantly reduced VEGF-stimulated c-Src activation. Disrupting the association of VE-cadherin and Csk through the reconstitution of Csk binding-defective mutant of VE-cadherin also diminished Src activation. Moreover, inhibiting SHP2 by small interference RNA and adenovirus-mediated expression of a catalytically inactive mutant of SHP2 attenuated c-Src activation by blocking the disassociation of Csk from VE-cadherin. Furthermore, VE-cadherin and SHP2 differentially regulates VEGF downstream signaling. The inhibition of c-Src, VE-cadherin, and SHP2 diminished VEGF-mediated activation of Akt and endothelial nitric-oxide synthase. In contrast, inhibiting VE-cadherin and SHP2 enhanced ERK1/2 activation in response to VEGF. These findings reveal a novel role for VE-cadherin in modulating c-Src activation in VEGF signaling, thus providing new insights into the importance of VE-cadherin in VEGF signaling and vascular function.

An ITIM-like motif within the CCK2 receptor sequence required for interaction with SHP-2 and the activation of the AKT pathway

SHP-2 is a tyrosine phosphatase which functions as a positive regulator downstream of RTKs, activating growth-stimulatory signalling pathways. To date, very few G protein-coupled receptors (GPCRs) have been shown to be connected to SHP-2 and very little is known about the positive role of SHP-2 in GPCR signalling. The CCK2 receptor (CCK2R), a GPCR, is now recognized to mediate mitogenic effects of gastrin on gastrointestinal cells. In the present study, we demonstrate the role of SHP-2 in the activation of the AKT pathway by the CCK2R in COS-7 cells transfected with the CCK2R and in a pancreatic cancer cell line expressing the endogenous receptor. Using surface plasmon resonance analysis, we identified a highly conserved ITIM motif, containing the tyrosine residue 438, located in the C-terminal intracellular tail of the CCK2R which directly interacts with the SHP-2 SH2 domains. The interaction was confirmed by pull down assays and co-immunoprecipitation of the receptor with SHP-2. This interaction was transiently increased following gastrin stimulation of the CCK2R and correlated with the tyrosine phosphorylation of SHP-2. Mutational analysis of the key ITIM residue 438 confirmed that the CCK2R ITIM sequence is required for interaction with SHP-2 and the activation of the AKT pathway.

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.

Flow Activates ERK1/2 and Endothelial Nitric Oxide Synthase via a Pathway Involving PECAM1, SHP2, and Tie2

Blood flow modulates endothelial cell (EC) functions through specific signaling events. Previous data show that flow stimulates SHP2 translocation to cell membranes and binding to phosphotyrosine proteins. Flow-induced ERK1/2 phosphorylation depends on SHP2 phosphatase activity and SHP2 binding to phospho-PECAM1 (platelet endothelial adhesion molecule 1), suggesting that SHP2 forms a signaling module with PECAM1. We hypothesized that flow induces assembly of the multi-protein complexes with SHP2 that are required for downstream signaling. ECs were exposed to flow for 10 min, and endogenous SHP2 was immunoprecipitated. SHP2-associated proteins were analyzed by SDS-PAGE and identified by mass spectrometry. Tie2 and several known SHP2-binding proteins were identified in flow-induced SHP2 complexes. Flow significantly increased tyrosine phosphorylation of both Tie2 and PECAM1 and their association with SHP2. To evaluate their functional roles, ECs were treated with Tie2 or PECAM1 small interfering RNA (siRNA). Tie2 and PECAM1 expression decreased >80% after siRNA treatment, and flow-stimulated phosphorylation of ERK1/2, Akt, and endothelial nitric oxide synthase was significantly inhibited by Tie2 and PECAM1 siRNA. Tie2 phosphorylation by flow was significantly inhibited by PECAM1 siRNA treatment. These results establish Tie2 transactivation via PECAM1 as an early event in flow-mediated mechanotransduction and suggest an important role for a PECAM1-SHP2-Tie2 pathway in flow-mediated signal transduction.

SHP2 and MKP5 in P2Y purinergic receptor-mediated prostate cancer invasion

To investigate the effects of protein tyrosine phosphatase-SHP2 and dual-specificity MAPK phosphatase-MKP5 on the activation of MAPKs and cell invasion induced by P2Y purinergic receptor in human prostate cancer cell lines with different metastatic potentials. The wide type (-wt) SHP2, mutant type (-cs) SHP2 and wide type (-wt) MKP5 cDNA expression vectors were constructed and stably transfected into 1E8 cells (highly metastatic) and/or 2B4 cells (non-metastatic). The tyrosine phosphorylation of SHP2 was examined by immunoprecipitation. The activation of ERK1/2 and p38 induced by P2Y receptor agonist ATP was analyzed by Western blot with phospho-specific antibodies against the dually phosphorylated, active forms of ERK1/2 and p38. The in-vitro invasive ability through Matrigel was measured by boyden-chamber assay. ATP induced significant SHP2 phosphorylation, which was stronger and lasted longer in 1E8 than in 2B4. SHP2-wt enhanced the ERK1/2 activation induced by ATP in 2B4 cells, while SHP2-cs delayed and decreased this effect in 1E8 cells. Both SHP2-wt and SHP2-cs had no obvious influence on p38 activation. ATP stimulated cell invasion of both 1E8 and 2B4, while transfection of SHP2-wt into 2B4 cells further increased the invasive-stimulating ability of ATP (18.7% increase compared with ATP treatment alone). Transfection of SHP2-cs into 1E8 cells, however, antagonized the invasive-stimulating ability of ATP (40.9% decrease compared with ATP treated group). Up-regulation of MKP5-wt inhibited phosphorylation of p38 by ATP and reduced cell invasion stimulated by ATP (22.4% and 28.7% decrease compared with ATP treated group of 1E8 and 2B4, respectively). Both SHP2 and MKP5 play some roles in P2Y receptor-mediated activation of MEK/ERK, p38 signaling pathways and prostate cancer invasion. SHP2 positively regulates ERK activation and prostate cancer invasion, whereas MKP5 inhibits the invasion by suppressing p38 activation.

Phosphorylation of Grb2-associated binder 2 on serine 623 by ERK MAPK regulates its association with the phosphatase SHP-2 and decreases STAT5 activation.

IL-2 stimulation of T lymphocytes induces the tyrosine phosphorylation and adaptor function of the insulin receptor substrate/Grb2-associated binder (Gab) family member, Gab2. In addition, Gab2 undergoes a marked decrease in its mobility in SDS-PAGE, characteristic of migration shifts induced by serine/threonine phosphorylations in many proteins. This migration shift was strongly diminished by treating cells with the MEK inhibitor U0126, indicating a possible role for ERK in Gab2 phosphorylation. Indeed, ERK phosphorylated Gab2 on a consensus phosphorylation site at serine 623, a residue located between tyrosine 614 and tyrosine 643 that are responsible for Gab2/Src homology 2 domain-containing tyrosine phosphatase (SHP)-2 interaction. We report that pretreatment of Kit 225 cells with U0126 increased Gab2/SHP-2 association and tyrosine phosphorylation of SHP-2 in response to IL-2, suggesting that ERK phosphorylation of serine 623 regulates the interaction between Gab2 and SHP-2, and consequently the activity of SHP-2. This hypothesis was confirmed by biochemical analysis of cells expressing Gab2 WT, Gab2 serine 623A or Gab2 tyrosine 614F, a mutant that cannot interact with SHP-2 in response to IL-2. Activation of the ERK pathway was indeed blocked by Gab2 tyrosine 614F and slightly increased by Gab2 serine 623A. In contrast, STAT5 activation was strongly enhanced by Gab2 tyrosine 614F, slightly reduced by Gab2 WT and strongly inhibited by Gab2 serine 623A. Analysis of the rate of proliferation of cells expressing these mutants of Gab2 demonstrated that tyrosine 614F mutation enhanced proliferation whereas serine 623A diminished it. These results demonstrate that ERK-mediated phosphorylation of Gab2 serine 623 is involved in fine tuning the proliferative response of T lymphocytes to IL-2.

Adenosine acts through A2 receptors to inhibit IL-2-induced tyrosine phosphorylation of STAT5 in T lymphocytes: role of cyclic adenosine 3',5'-monophosphate and phosphatases.

Adenosine is a purine nucleoside with immunosuppressive activity that acts through cell surface receptors (A(1), A(2a), A(2b), A(3)) on responsive cells such as T lymphocytes. IL-2 is a major T cell growth and survival factor that is responsible for inducing Jak1, Jak3, and STAT5 phosphorylation, as well as causing STAT5 to translocate to the nucleus and bind regulatory elements in the genome. In this study, we show that adenosine suppressed IL-2-dependent proliferation of CTLL-2 T cells by inhibiting STAT5a/b tyrosine phosphorylation that is associated with IL-2R signaling without affecting IL-2-induced phosphorylation of Jak1 or Jak3. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reversed by the protein tyrosine phosphatase inhibitors sodium orthovanadate and bpV(phen). Adenosine dramatically increased Src homology region 2 domain-containing phosphatase-2 (SHP-2) tyrosine phosphorylation and its association with STAT5 in IL-2-stimulated CTLL-2 T cells, implicating SHP-2 in adenosine-induced STAT5a/b dephosphorylation. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reproduced by A(2) receptor agonists and was blocked by selective A(2a) and A(2b) receptor antagonists, indicating that adenosine was mediating its effect through A(2) receptors. Inhibition of STAT5a/b phosphorylation was reproduced with cell-permeable 8-bromo-cAMP or forskolin-induced activation of adenylyl cyclase, and blocked by the cAMP/protein kinase A inhibitor Rp-cAMP. Forskolin and 8-bromo-cAMP also induced SHP-2 tyrosine phosphorylation. Collectively, these findings suggest that adenosine acts through A(2) receptors and associated cAMP/protein kinase A-dependent signaling pathways to activate SHP-2 and cause STAT5 dephosphorylation that results in reduced IL-2R signaling in T cells.

TNF-alpha induces tyrosine phosphorylation and recruitment of the Src homology protein-tyrosine phosphatase 2 to the gp130 signal-transducing subunit of the IL-6 receptor complex.

Recently, it has been demonstrated that TNF-alpha and LPS induce the expression of suppressor of cytokine signaling 3 (SOCS3) and inhibit IL-6-induced STAT3 activation in macrophages. Inhibitor studies suggested that both induction of SOCS3 and inhibition of IL-6-induced STAT3 activation depend on the activation of p38 mitogen-activated protein kinase. Since recruitment of the tyrosine phosphatase Src homology protein tyrosine phosphatase 2 (SHP2) to the signal-transducing receptor subunit gp130 attenuates IL-6-mediated STAT-activation, we were interested in whether TNF-alpha also induces the association of SHP2 to the gp130 receptor subunit. In this study we demonstrate that stimulation of macrophages and fibroblast cell lines with TNF-alpha causes the recruitment of SHP2 to the gp130 signal-transducing subunit and leads to tyrosine phosphorylation of SHP2 and gp130. In this context the cytoplasmic SHP2/SOCS3 recruitment site of gp130 tyrosine 759 is shown to be important for the inhibitory effects of TNF-alpha, since mutation of this residue completely restores IL-6-stimulated activation of STAT3 and, consequently, of a STAT3-dependent promoter. In this respect murine fibroblasts lacking exon 3 of SHP2 are not sensitive to TNF-alpha, indicating that functional SHP2 and its recruitment to gp130 are key events in inhibition of IL-6-dependent STAT activation by TNF-alpha. Furthermore, activation of p38 mitogen-activated protein kinase is shown to be essential for the inhibitory effect of TNF-alpha on IL-6 signaling and TNF-alpha-dependent recruitment of SHP2 to gp130.

Src Family Kinase-independent Signal Transduction and Gene Induction by Leukemia Inhibitory Factor

Members of the interleukin-6 (IL-6) family of cytokines exert their biological effects via binding to their cognate ligand-binding receptor subunit on a target cell. The subsequent recruitment of the common signal transducer glycoprotein 130 and activation of the JAK/STAT and SHP-2/Ras/mitogen-activated protein kinase (MAPK) pathways are responsible for the majority of cellular responses elicited by IL-6 cytokines. Several types of experiments suggest that the Src family of kinases (SFK) also participates in IL-6 family cytokine-mediated signaling events. SYF cells, which lack expression of SFKs Src, Yes, and Fyn, were used to determine the role of SFKs in IL-6 family cytokine signaling and gene induction. SYF and wild type (WT) control fibroblasts displayed similar activation of signaling intermediates following stimulation with leukemia inhibitory factor (LIF). LIF-stimulated tyrosine phosphorylation of SHP-2 and subsequent activation of MAPK in SYF cells were identical to that seen in LIF-stimulated WT cells. Both LIF-stimulated tyrosine phosphorylation of STAT1 and STAT3, as well as LIF-stimulated DNA binding activity of STAT-containing nuclear complexes were indistinguishable when compared in SYF and WT cells. In addition, the phosphatidylinositol 3-kinase-sensitive Akt kinase and p38 MAPK were activated by LIF in both SYF and WT cells. Furthermore, LIF-stimulated expression of c-fos, egr-1, and suppressor of cytokine signaling-3 was retained in SYF cells. The IL-6 family cytokine oncostatin M was also capable of activating MAPK, STAT3, STAT1, Akt, and p38 in both WT and SYF cells. These results demonstrate that IL-6 family cytokines can activate a full repertoire of signaling pathways and induce gene expression independent of SFKs.

Identification of Fer tyrosine kinase localized on microtubules as a platelet endothelial cell adhesion molecule-1 phosphorylating kinase in vascular endothelial cells.

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.

Chemical dissection of the effects of tyrosine phosphorylation of SHP-2.

The regulation of the protein tyrosine phosphatase (PTPase) SHP-2 by tyrosine phosphorylation has been difficult to elucidate because of the intrinsic instability of the phosphoprotein. In the past, expressed protein ligation has been used to site-specifically incorporate the phosphotyrosine mimic Pmp (phosphonomethylene phenylalanine) into the two tyrosine phosphorylation sites (542, 580) of SHP-2 one at a time to analyze the effects on catalytic behavior. In this study, we have incorporated two Pmps into the phosphorylation sites simultaneously and examined the effects of double SHP-2 tyrosine phosphorylation. We have found that the Pmp groups show close to additive effects on PTPase stimulation, suggesting dual SH2 domain occupancy. The relative effects of the phosphotyrosine analogue difluoromethylene phosphonophenylalanine (F(2)Pmp) compared to those of Pmp were also examined. It was found that the F(2)Pmp analogue showed slightly enhanced PTPase stimulation compared with the Pmp analogue, consistent with its higher affinity for SH2 domains. Taken together with the bis-Pmp studies, these data suggest that double phosphorylation of the SHP-2 C-terminus could give rise to a 9-fold overall PTPase activation, 30-50% of the value associated with deletion of the SH2 domains. Catalytically inactive forms of phosphorylated SHP-2 proteins were also produced by expressed protein ligation. This allowed for a systematic analysis of intermolecular autodephosphorylation of SHP-2, which revealed how conformational plasticity can modulate phosphotyrosine stability.