Phosphorylation Of SHP-2 Research Articles

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.

Persistent activation of the D1R/Shp-2/Erk1/2 pathway in l-DOPA-induced dyskinesia in the 6-hydroxy-dopamine rat model of Parkinson's disease

Prolonged l-3,4-dihydroxyphenylalanine (l-DOPA) administration, the gold standard therapy for Parkinson's disease (PD) is associated with serious motor complications, known as l-DOPA-induced dyskinesia (LID). One of the major molecular changes associated with LID is the increased activity of the extracellular signal-regulated kinases 1/2 (Erk1/2) signaling in the medium spiny neurons of the striatum induced by malfunctioning in the dopamine D1 receptor (D1R)-mediated transmission.We have previously established that in the striatum, activation of Shp-2, an intracellular tyrosine phosphatase associated with the D1R, is a requisite for the D1R to activate Erk1/2.In this study, we investigated the role of striatal D1R/Shp-2 complex in the molecular event underlying LID in the 6-OHDA-lesioned rat model of PD.We found that in hemiparkinsonian rats experiencing LID, the physiological interaction between D1R and Shp-2 in the striatum was preserved. In these animals, the chronic activation of D1R either by l-DOPA or by the selective D1R agonist SKF 38393 induced both dyskinesia and Shp-2/Erk1/2 activation. These effects were prevented by the selective D1R-antagonist SCH23390 suggesting the involvement of striatal D1R/Shp-2 complex, via Erk1/2 activation, in the molecular events underlying LID. Interestingly, we found that D1R-mediated Shp-2-Erk1/2 activation was persistently detected in the striatum of dyskinetic rats during l-DOPA washout, with a close correlation between LID severity and the extent of long term activation of both Shp-2 and Erk1/2.Taken together, our data show that in hemiparkinsonian rats developing dyskinesia, the aberrant phosphorylation of Shp-2 by D1R activation, represents an upstream molecular event leading to the persistent phosphorylation of Erk1/2 and therefore a novel therapeutic target to counteract LID development and maintenance during l-DOPA therapy.

SHP-1 contributes to the CD40 signaling reciprocity and controls Leishmania major infection

Event Abstract Back to Event SHP-1 contributes to the CD40 signaling reciprocity and controls Leishmania major infection TABISH H. KHAN1* 1 National Centre for Cell Science, India CD40, a costimulatory molecule expressed on macrophages can induce counteractive immune responses depending on the intensity of CD40 crosslinking. A strong CD40 stimulation induces preferential phosphorylation of p38MAPK and pro-inflammatory cytokine IL-12 production whereas a weaker CD40 stimulation induces preferential phosphorylation of ERK-1/2 and anti-inflammatory cytokine IL-10 production. In the experimental infection with Leishmania major, CD40-induced p38MAPK activation and IL-12 expression are suppressed whereas ERK-1/2 activation and IL-10 expression are enhanced. We concluded that the observed reciprocity was due to differential phosphorylation of these two MAPKs and we propose that this observed reciprocity can be function of a phosphatase that dephosphorylates these MAPKs differentially. Leishmania infection activates a phosphotyrosine phosphatase SHP-1, which we propose to possibly play a role in MAPK dephosphorylation and thereby influences the reciprocity in CD40 signaling. Although CD40 plays important roles in L. major infection, whether SHP-1 influences CD40 signaling and affects L. major infection or vice versa have never been examined. In this report, we show that SHP-1 does contribute to the CD40 signaling reciprocity by differential p38MAPK and ERK-1/2 dephosphorylation. In L. major infection, SHP-1 phosphorylation and activity are increased causing reciprocal down-regulation of CD40-induced p38MAPK phosphorylation. SHP-1 overexpression renders the resistant C57BL/6 mice susceptible whereas its inhibition protects susceptible BALB/c mice to L. major infection. Thus, we demonstrate for the first time that SHP-1 contributes to the CD40 signaling reciprocity. These properties are exploited by the parasite to design the SHP-1-targeted immune evasion strategy ensuring their survival in a mammalian host.

Abstract 19603: Disruption of βPDGF Receptor-Dependent PI3k and Plcγ Signaling Protects from Hypoxia-Induced Pulmonary Hypertension

Background: Vascular remodeling is critical during the pathogenesis of pulmonary hypertension (PH). Growth factors, such as platelet-derived growth factor (PDGF), appear to be involved in these processes. With pharmacological inhibitors, we demonstrated previously that phosphatidyl inositol-3 kinase (PI3K) and phospholipase Cγ (PLCγ) are the critical mediators of βPDGF receptor (βPDGFR)-dependent proliferation and migration in pulmonary arterial smooth muscle cells (PASMC). Whereas expression data and data with the tyrosine kinase inhibitor imatinib suggest a role for PDGF in PH, data from genetic models is lacking. Methods and results: We utilized βPDGFR F3/F3 (“F3”) mice that were generated by introducing point mutations of the PI3K and PLCγ binding sites into the βPDGFR locus by gene targeting. We isolated PASMC from F3 and wild-type (WT) mice. Co-immunoprecipitation experiments after PDGF treatment showed that PI3K and PLCγ did not bind to the activated F3 receptor, whereas overall phosphorylation and binding of RasGap and SHP-2 were unaffected. In line with these observations, PDGF-mediated phosphorylation of Akt and Erk 1/2 was inhibited in F3-PASMC compared to WT. To address, if cellular responses were altered, we analyzed βPDGFR-dependent proliferation (as BrdU incorporation) and migration (modified Boyden chamber). These experiments revealed that these responses were essentially completely blocked in F3-PASMC compared to WT. To analyze the development and severity of PH in vivo, we exposed F3 and WT mice to hypoxia for 3 weeks. Following this treatment, right ventricular systolic pressure (RVSP) was measured by right heart catheterization, and right ventricular (RV) hypertrophy was assessed as RV weight/left ventricular + septal weight (RV/LV+S). These analyses revealed that RVSP and RV/LV+S were significantly reduced in F3 compared to WT mice. Conclusions: These data show that disruption of βPDGFR-dependent PI3K and PLCγ signaling blocks PDGF-dependent proliferation and migration of PASMC in vitro and protects from the development of hypoxia-induced PH in vivo. Consequently, these data support a critical role of PDGF during the pathogenesis of PH.

Reduced C/EBPα Expression Favors Monopoiesis Over Granulopoiesis

Abstract 1230C/EBPα is required for the formation of the granulocyte-monocyte progenitor; however, its role in subsequent lineage specification remains uncertain. We previously compared G-CSF with M-CSF signaling in murine marrow, finding that G-CSF more potently induces phosphorylation of SHP2 whereas M-CSF more potently activates ERK. Knockdown (KD) of SHP2 impaired granulopoiesis relative to monopoiesis and unexpectedly led to reduced C/EBPα RNA. A. Cantor and colleagues recently reported that SHP2 dephosphorylates Src kinase-modified RUNX1 and increases the ability of RUNX1 to induce megakaryopoiesis, and we demonstrated that in myeloid cells RUNX1 binds and activates the CEBPA gene, via its promoter and via a conserved 450 bp enhancer located at +37 kb containing 4 RUNX1-binding sites. Thus, G-CSF or other cytokines potentially direct SHP2 to activate RUNX1 which then increases C/EBPα transcription in myeloid progenitors to favor granulopoiesis. As a test of this model, we have now transduced lineage-depleted murine marrow cells, cultured in SCF, TPO, and Flt3L to minimize myeloid differentiation, with shRNAs targeting CEBPA or with a control lentiviral vector. Western blotting indicated 2-fold KD by shRNAs B9 or B11 after 2 days of transduction followed by 2 days of puromycin selection. As this analysis was done on a mixed population, greater KD may occur in rare progenitors. Upon transfer to IL3, IL6, and SCF to favor myeloid maturation, vector-transduced cells produced 4-fold more CFU-G than CFU-M in methylcellulose, whereas CEBPA KD resulted in 4- or 5-fold more CFU-M than CFU-G. FACS analysis of pooled CFUs demonstrated significant reduction of Mac-1+Gr-1+ granulocytes and an increase in Mac-1+Gr-1− monocytes, and examination of cell morphology after Wright’s-Giemsa staining further confirmed a shift towards monopoiesis in response to reduced C/EBPα. Similar findings were obtained for cells placed in liquid culture. In addition, on day 3 of liquid culture Q-RTPCR analysis showed that CEBPA shRNAs induced 2- or 3-fold CEBPA RNA KD, reduced granulocytic MPO, NE, PR3, and GCSFR RNAs, increased monocytic MCSFR and CD14 RNAs, and increased expression of EGR1, IRF8, and c-Jun RNAs, encoding transcription factors that help direct monopoiesis. RUNX1 RNA was unchanged, and PU.1 RNA was reduced <2-fold. To determine the effect of CEBPA KD in vivo, transduced, puromycin-selected CD45.2+ marrow cells were transplanted into lethally irradiated, syngeneic CD45.1+ recipients together with CD45.1+ carrier cells. At day 28, FACS analysis indicated a shift towards monopoiesis amongst shRNA B9- or B11-transduced marrow cells, and CFU assay of sorted CD45.2+ cells demonstrated significantly reduced CFU-G with increased CFU-M. Besides affecting myeloid lineage determination, CEBPA KD increased the rate of cell proliferation in liquid culture, associated with blasts evident upon morphologic analysis and an increase in Mac-1−Gr-1− cells, which mainly consisted of c-Kit+Sca-1− myeloid progenitors. In addition, CEBPA KD by shRNA B9, which impaired granulopoiesis more effectively than shRNA B11, increased CFU replating to 8 generations, compared with 4 generations for control cells. Analysis on day 3 of liquid culture did not reveal an alteration in the proportion of cells in G1, S, or G2/M or increased apoptosis, and c-Myc and Bcl-2 RNA levels were unaffected. We propose that CEBPA KD not only strongly impairs granulopoiesis, but also modestly impedes monopoiesis to produce a transient accumulation of cells with blast morphology and the ability to proliferate. Overall, our findings suggest that relative to monopoiesis, granulopoiesis requires increased level or increased activity of C/EBPα. Offering a potential mechanism, we previously found that C/EBPα :AP-1 leucine zipper heterodimers are more abundant than C/EBPα homodimers during monopoiesis compared with granulopoiesis, in part due to c-Fos, c-Jun, and JunB induction, and that C/EBPα :AP-1 heterodimers can direct marrow monopoiesis. Perhaps higher levels of C/EBPα favor homodimer formation, with reduced levels still able to heterodimerize with AP-1 proteins. ERK modification of C/EBPα on S21, as occurs downstream of M-CSF, reduces its ability to direct granulopoiesis; phospho-C/EBPα (S21) potentially retains the ability to direct AP-1 proteins to hybrid DNA elements recently identified in numerous monocytic enhancers. Disclosures:No relevant conflicts of interest to declare.

Regulation of apoptosis in HL‐1 cardiomyocytes by phosphorylation of the receptor tyrosine kinase EphA2 and protection by lithocholic acid

Heart failure and atrial fibrillation are associated with apoptosis of cardiomyocytes, suggesting common abnormalities in pro-apoptotic cardiac molecules. Activation of the receptor tyrosine kinase EphA2 causes apoptosis in vitro, and dysregulation of EphA2-dependent signalling is implicated in LEOPARD and Noonan syndromes associated with cardiomyopathy. Molecular pathways and regulation of EphA2 signalling in the heart are poorly understood. Here we elucidated the pathways of EphA2-dependent apoptosis and evaluated a therapeutic strategy to prevent EphA2 activation and cardiac cell death. EphA2 signalling was studied in an established model of doxazosin-induced apoptosis in HL-1 cells. Apoptosis was measured with TUNEL assays and as cell viability using a formazan method. Western blotting and siRNA for EphA2 were also used. Apoptosis induced by doxazosin (EC(50) = 17.3 μM) was associated with EphA2 activation through enhanced phosphorylation (2.2-fold). Activation of pro-apoptotic downstream factors, phospho-SHP-2 (3.9-fold), phospho-p38 MAPK (2.3-fold) and GADD153 (1.6-fold) resulted in cleavage of caspase 3. Furthermore, two anti-apoptotic enzymes were suppressed (focal adhesion kinase, by 41%; phospho-Akt, by 78%). Inactivation of EphA2 with appropriate siRNA mimicked pro-apoptotic effects of doxazosin. Finally, administration of lithocholic acid (LCA) protected against apoptosis by increasing EphA2 protein levels and decreasing EphA2 phosphorylation. EphA2 phosphorylation and activation of SHP-2 are critical steps in apoptosis. Reduction of EphA2 phosphorylation by LCA may represent a novel approach for future anti-apoptotic treatment of heart failure and atrial fibrillation.

Src Homology 2 Domain-containing Phosphatase 2 (Shp2) Is a Component of the A-kinase-anchoring Protein (AKAP)-Lbc Complex and Is Inhibited by Protein Kinase A (PKA) under Pathological Hypertrophic Conditions in the Heart

AKAP-Lbc is a scaffold protein that coordinates cardiac hypertrophic signaling. AKAP-Lbc interacts with Shp2, facilitating its regulation by PKA. AKAP-Lbc integrates PKA and Shp2 signaling in the heart. Under pathological hypertrophic conditions Shp2 is phosphorylated by PKA, and phosphatase activity is inhibited. Inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote pathological cardiac hypertrophy. Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Our results identify a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that the tyrosine phosphatase, Shp2, is a component of the A-kinase-anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits its protein-tyrosine phosphatase activity. Given the important cardiac roles of both AKAP-Lbc and Shp2, we investigated the AKAP-Lbc-Shp2 interaction in the heart. AKAP-Lbc-tethered PKA is implicated in cardiac hypertrophic signaling; however, mechanism of PKA action is unknown. Mutations resulting in loss of Shp2 catalytic activity are also associated with cardiac hypertrophy and congenital heart defects. Our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Thus, while induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote compensatory cardiac hypertrophy.

Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and p38 regulate the expression of chemokine CXCL8 in human astrocytes.

CXCL8, one of the first chemokines found in the brain, is upregulated in the brains and cerebrospinal fluid of HIV-1 infected individuals suggesting its potential role in human immune deficiency virus (HIV)-associated neuroinflammation. Astrocytes are known to be the major contributors to the CXCL8 pool. Interleukin (IL)-1β activated astrocytes exhibit significant upregulation of CXCL8. In order to determine the signaling pathways involved in CXCL8 regulation in astrocytes, we employed pharmacological inhibitors for non-receptor Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and mitogen-activated protein kinases (MAPK) pathway and observed reduced expression of CXCL8 following IL-1β stimulation. Overexpression of SHP2 and p38 enzymes in astrocytes led to elevated CXCL8 expression; however, inactivating SHP2 and p38 with dominant negative mutants abrogated CXCL8 induction. Furthermore, SHP2 overexpression resulted in higher SHP2 and p38 enzyme activity whereas p38 overexpression resulted in higher p38 but not SHP2 enzyme activity. Phosphorylation of SHP2 was important for phosphorylation of p38, which in turn was critical for phosphorylation of extracellular signal regulated kinase (ERK). Thus, our findings suggest an important role for SHP2 in CXCL8 expression in astrocytes during inflammation, as SHP2, directly or indirectly, modulates p38 and ERK MAPK in the signaling cascade leading to CXCL8 production. This study provides detailed understanding of the mechanisms involved in CXCL8 production during neuroinflammation.

Diminished functional role and altered localization of SHP2 in non-small cell lung cancer cells with EGFR-activating mutations

Non-small cell lung cancer (NSCLC) cells harboring activating mutations of the epidermal growth factor receptor (EGFR) tend to display elevated activity of several survival signaling pathways. Surprisingly, these mutations also correlate with reduced phosphorylation of ERK and SHP2, a protein tyrosine phosphatase required for complete ERK activation downstream of most receptor tyrosine kinases. Since ERK activity influences cellular response to EGFR inhibition, altered SHP2 function could play a role in the striking response to gefitinib witnessed with EGFR mutation. Here, we demonstrate that impaired SHP2 phosphorylation correlates with diminished SHP2 function in NSCLC cells expressing mutant, versus wild-type, EGFR. In NSCLC cells expressing wild-type EGFR, SHP2 knockdown decreased ERK phosphorylation, basally and in response to gefitinib, and increased cellular sensitivity to gefitinib. In cells expressing EGFR mutants, these effects of SHP2 knockdown were less substantial, but expression of constitutively active SHP2 reduced cellular sensitivity to gefitinib. In cells expressing EGFR mutants, which do not undergo efficient ligand-mediated endocytosis, SHP2 was basally associated with GAB1 and EGFR, and SHP2′s presence in membrane fractions was dependent on EGFR activity. Whereas EGF promoted a more uniform intracellular distribution of initially centrally localized SHP2 in cells expressing wild-type EGFR, SHP2 was basally evenly distributed and did not redistribute in response to EGF in cells with EGFR mutation. Thus, EGFR mutation may promote association of a fraction of SHP2 at the plasma membrane with adapters which promote SHP2 activity. Consistent with this, SHP2 immunoprecipitated from cells with EGFR mutation was active, and EGF treatment did not change this activity. Overall, our data suggest that a fraction of SHP2 is sequestered at the plasma membrane in cells with EGFR mutation in a way that impedes SHP2′s ability to promote ERK activity and identify SHP2 as a potential target for co-inhibition with EGFR in NSCLC.

SHPS‐1 deficiency induces robust neuroprotection against experimental stroke by attenuating oxidative stress

Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), also known as Signal-regulatory protein alpha (SIRPα) or SIRPA is a transmembrane protein that is predominantly expressed in neurons, dendritic cells, and macrophages. This study was conducted to investigate the role of SHPS-1 in the oxidative stress and brain damage induced by acute focal cerebral ischemia. Wild-type (WT) and SHPS-1 mutant (MT) mice were subjected to middle cerebral artery occlusion (60 min) followed by reperfusion. SHPS-1 MT mice had significantly reduced infarct volumes and improved neurological function after brain ischemia. In addition, neural injury and oxidative stress were inhibited in SHPS-1 MT mice. The mRNA and protein levels of the antioxidant genes nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase 1 were up-regulated in SHPS-1 MT mice. The SHPS-1 mutation suppressed the phosphorylation of SHP-1 and SHP-2 and increased the phosphorylation of Akt and GSK3β. These results provide the first demonstration that SHPS-1 plays an important role in the oxidative stress and brain injury induced by acute cerebral ischemia. The activation of Akt signaling and the up-regulation of Nrf2 and heme oxygenase 1 likely account for the protective effects that were observed in the SHPS-1 MT mice.

The Helicobacter pylori Virulence Effector CagA Abrogates Human β-Defensin 3 Expression via Inactivation of EGFR Signaling

Antimicrobial peptides are constituents of the first-line innate mucosal defense system that acts as a barrier to establishment of infection. The highly successful human gastric pathogen, Helicobacter pylori, is able to persistently colonize its host despite inducing expression of several antimicrobial peptides, including human β-defensin 3 (hBD3). We find that hBD3 is highly active against H.pylori invitro and is rapidly induced during early infection via EGFR-dependent activation of MAP kinase and JAK/STAT signaling. However, during prolonged infection, hBD3 was subsequently downregulated by the H.pylori virulence determinant CagA. Upon translocation into host cells, CagA activated the cellular tyrosine phosphatase, SHP-2, terminating EGFR activation and downstream signaling and increasing bacterial viability. Chemical inhibition and knockdown of SHP-2 expression rescued hBD3 synthesis and bactericidal activity. Thus, we reveal how cagPAI-positive H.pylori strains use CagA to evade a key innate mucosal defense pathway to support the establishment of persistent infection.

235 Pseudokinase Tribbles Homolog 3 is Induced in Helicobacter pylori Infected Gastric Mucosa and Contributes to Gastric Carcinogenesis Through Phosphorylation of Oncogenic Protein SHP-2

235 Pseudokinase Tribbles Homolog 3 is Induced in Helicobacter pylori Infected Gastric Mucosa and Contributes to Gastric Carcinogenesis Through Phosphorylation of Oncogenic Protein SHP-2

Abstract 2859: Dual targeting of protein tyrosine kinase c-Src and protein tyrosine phosphatase SHP-2 is a novel therapeutic strategy that induces potent inhibition of pancreatic cancer cell viability in vitro and tumor progression in vivo

Abstract Pancreatic adenocarcinoma is a deadly human malignancy, with over 95% of patients succumbing within five years. The majority of pancreatic cancer deaths are caused by metastatic dissemination of the primary tumor. The only curative treatment is surgical resection, which is limited by tumor stage, and the standard chemotherapeutic option, gemcitabine, offers only modest survival and quality of life benefits. There is a need for a greater understanding of pancreatic cancer biology and development of novel therapies to inhibit tumor progression and metastasis. The non-receptor protein tyrosine kinase, c-Src (Src) has emerged as a potential target for the treatment of pancreatic cancer, having been demonstrated to promote a number of tumorigenic processes. Src is overexpressed and/or aberrantly activated in greater than 70% of human pancreatic cancers. In addition, SHP-2, a non-receptor protein tyrosine phosphatase, regulates Src family kinase activity. Evidence has established clinical relevance for SHP-2 in human diseases. A recent study demonstrated that inhibition of SHP-2 abrogates in vitro and in vivo angiogenesis and inhibits activity of the MAPK/Erk1/2 and PI3K/Akt survival pathways. SHP-2 and Src function as key signaling intermediates downstream of growth factor receptors, cytokine receptors, and integrins, and are crucial for activation of downstream cascades of tumor progression and metastasis. The purpose of this study was to determine the importance of SHP-2 and Src, for pancreatic cancer signaling, cell biology, tumorigenicity, and metastasis. We have demonstrated elevated SHP-2 expression and phosphorylation in pancreatic cancer cells, relative to normal pancreatic cells. Phosphorylation of SHP-2 in pancreatic cancer cells occurs in a Src kinase-dependent fashion. Functional inhibition of SHP-2 and Src, individually or in combination, resulted in reduced ERK-1/2 phosphorylation, increased cleavage of caspase 3 and PARP, increased expression of the pro-apoptotic protein Bax, and decreased levels of the anti-apoptotic protein Bcl-xL. These results were mirrored by decreased viability and increased caspase activity in the presence of Src and SHP-2 inhibition. Finally, treatment of mice with small molecule inhibitors of Src or SHP-2, produced markedly smaller pancreatic tumors relative to controls in an orthotopic nude mouse model and dual treatment exacerbated this effect. The potential use for the Src and SHP-2 signaling axes as therapeutic targets remains largely untapped. These data suggest the importance of SHP-2 in pancreatic cancer cell biology and its potential value as a therapeutic target. Our studies suggest that the anti-tumor/anti-metastatic effects of Src inhibition may be improved through simultaneous inhibition of SHP-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2859. doi:1538-7445.AM2012-2859

Requirements of Gαi1 and Gαi3 for BDNF and NGF induced PI3K/AKT and MAPK signaling, neuronal survival and migration

Our previous study has indicated an unexpected and critical role of Gαi proteins in mediating receptor tyrosine kinase signaling (Cao et al., Science Signaling, 2009). Here we discovered a novel function of Gαi proteins in BDNF/NGF signaling. Gαi proteins are required for Brain‐Derived Neurotrophic Factor (BDNF) and Nerve Growth Factor (NGF) to transduce signals from Trk receptor to stimulate MAPK (p38, JNK and ERK) and PI3K/AKT/mTORC1 signaling. Knockout of Gαi1 and Gαi3 completely blocked BDNF and NGF induced PI3K/AKT/mTORC1 and MAPK activation in MEFs (mouse embryonic fibroblasts). Both Gαi1 and Gαi3 associated with the Grb2‐associated binding protein 1 (Gab1), and were required for the interaction of Gab1 with SHP2 as well as the interactions of Grb2 with SHP2, Shc, MEKK1 and MEKK2, in response to BDNF and NGF. Loss of Gαi1 and Gαi3 impaired the phosphorylation of Gab1, SHP2 and Shc in response to BDNF and NGF. Importantly, siRNA knockdown of both Gαi1 and Gαi3 inhibited BDNF and NGF induced AKT/mTORC1 and MAPK activation in cerebellar granule neuron, and BDNF induced neuronal survival, neuronal migration and neurite outgrowth were also largely impaired in cerebellar neurons with Gαi1 and Gαi3 deficiency. Overall, these results showed that Gαi1 and Gαi3 lie downstream of Trk receptor, but upstream of Gab1‐Shc‐Grb2‐Shp2, mediating MAPK and PI3K/AKT/mTORC1 signaling as well neuronal survival and migration.

An osteoclastic protein-tyrosine phosphatase regulates the β3-integrin, syk, and shp1 signaling through respective src-dependent phosphorylation in osteoclasts

This study utilized the glutathione transferase (GST) pull-down assay to identify novel substrates of an osteoclastic protein-tyrosine phosphatase, PTP-oc. Consistent with the previous findings that the phosphorylated tyr-527 (pY527) of Src is a substrate of PTP-oc, the major protein pulled down with the phosphatase-deficient (PD)-PTP-oc-GST trapping mutant in RAW264.7 cells was Src. The GST-PD-PTP-oc also pulled down pY-Syk and pY-β(3)-integrin, but not after PP2 pretreatment. However, PTP-oc transgenic osteoclasts or PTP-oc-overexpressing RAW264.7 cells had elevated, and not reduced, levels of pY525/526-Syk and pY759-β(3) integrin, and the PTP-oc siRNA treatment drastically reduced levels of pY525/526 Syk and pY759-β(3)-integrin in RAW264.7 cells. These findings are incompatible with the premise that they are substrates of PTP-oc. The PTP-oc-dependent increases in pY525/526-Syk and pY759-β(3)-integrin levels were completely blocked by PP2, indicating that these effects are secondary to PTP-oc-mediated activation of the Src protein-tyrosine kinase (PTK). Overexpression of PTP-oc increased, and siRNA-mediated suppression of PTP-oc reduced, pY160-Vav1, pY173-Vav3, and pY783-PLCγ levels, and Rac1 activation, which are downstream mediators of the ITAM/Syk signaling. Overexpression of PTP-oc also increased, and PTP-oc siRNA treatment decreased, the pY-Shp1 levels, which were blocked by PP2. Since Shp1 is a negative regulator of osteoclast activity and is a key mediator of the ITIM signaling, these findings suggest that PTP-oc is an upstream suppressor of the ITIM/Shp1 signaling through PTP-oc-induced Src-dependent Shp1 phosphorylation. In summary, PTP-oc plays a central regulatory role in the concerted regulation of the β(3)-integrin, the ITAM/Syk, and the ITIM/Shp1 signaling indirectly through activation of Src PTK.

Spinal SIRPα1-SHP2 interaction regulates spinal nerve ligation-induced neuropathic pain via PSD-95-dependent NR2B activation in rats

The fact that neuropathic pain mechanisms are not well understood is a major impediment in the development of effective clinical treatments. We examined whether the interaction between signal regulatory protein alpha 1 (SIRPα1) and Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2), and the downstream spinal SHP2/postsynaptic density 95 (PSD-95)/N-methyl-d-aspartate receptor NR2B subunit signaling cascade play a role in neuropathic pain. Following spinal nerve ligation (L5), we assessed tactile allodynia using the von Frey filament test and analyzed dorsal horn samples (L4-5) by Western blotting, reverse transcription polymerase chain reaction, coimmunoprecipitation, and immunofluorescence. Nerve ligation induced allodynia, SIRPα1, SHP2, phosphorylated SHP2 (pSHP2), and phosphorylated NR2B (pNR2B) expression, and SHP2-PSD-95, pSHP2-PSD-95, PSD-95-NR2B, and PSD-95-pNR2B coimmunoprecipitation in the ipsilateral dorsal horn. In allodynic rats, injury-induced SHP2 immunoreactivity was localized in the ipsilateral dorsal horn neurons and coincident with PSD-95 and NR2B immunoreactivity. SIRPα1 silencing using small interfering RNA (siRNA; 1, 3, or 5μg/rat for 7days) prevented injury-induced allodynia and the associated changes in protein expression, phosphorylation, and coimmunoprecipitation. Intrathecal administration of NSC-87877 (an SHP2 antagonist; 1, 10, or 100μM/rat) and SIRPα1-neutralizing antibodies (1, 10, or 30μg/rat) suppressed spinal nerve ligation-induced allodynia, spinal SHP2 and NR2B phosphorylation, and SHP2/phosphorylated SHP2-PSD-95 and PSD-95-NR2B/phosphorylated NR2B coprecipitation. SHP2 siRNA led to similar effects as the NSC-87877 and SIRPα1 antibody treatments, except it prevented the allodynia-associated spinal SHP2 expression. In conclusion, our results suggest that a spinal SIRPα1-SHP2 interaction exists that subsequently triggers SHP2/PSD-95/NR2B signaling, thereby playing a role in neuropathic pain development.

A newly identified complex of spinophilin and the tyrosine phosphatase, SHP-1, modulates platelet activation by regulating G protein–dependent signaling

AbstractPlatelets are essential for normal hemostasis, but close regulation is required to avoid the destructive effects of either inappropriate platelet activation or excessive responses to injury. Here, we describe a novel complex comprising the scaffold protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1, and show that it can modulate platelet activation by sequestering RGS10 and RGS18, 2 members of the regulator of G protein signaling family. We also show that SPL/RGS/SHP1 complexes are present in resting platelets where constitutive phosphorylation of SPL(Y398) creates an atypical binding site for SHP-1. Activation of the SHP-1 occurs on agonist-induced phosphorylation of SHP-1(Y536), triggering dephosphorylation and decay of the SPL/RGS/SHP1 complex. Preventing SHP-1 activation blocks decay of the complex and produces a gain of function. Conversely, deleting spinophilin in mice inhibits platelet activation. It also attenuates the rise in platelet cAMP normally caused by endothelial prostacyclin (PGI2). Thus, we propose that the role of the SPL/RGS/SHP1 complex in platelets is time and context dependent. Before injury, the complex helps maintain the quiescence of circulating platelets by maximizing the impact of PGI2. After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs.

Mechanisms underlying somatostatin receptor 2 down‐regulation of vascular endothelial growth factor expression in response to hypoxia in mouse retinal explants

Hypoxia is a trigger of VEGF expression, the primary cause of retinal pathologies characterized by neovascularization. During hypoxia, transcription factors such as STAT3 and HIF-1 promote the increase in VEGF expression. Octreotide, a somatostatin receptor 2 (sst(2) )-preferring agonist, reduces retinal VEGF expression and neovascularization. To investigate the intracellular pathways linking sst(2) activation to the inhibition of hypoxia-induced VEGF up-regulation, we used pharmacological approaches and siRNA in mouse retinal explants cultured in normoxia or hypoxia. In hypoxic explants in which STAT3 or HIF-1 was inhibited, we observed the existence of reciprocal interactions between STAT3 and HIF-1, which synergistically induced VEGF expression. Octreotide prevented hypoxia-induced activation of STAT3 and HIF-1, and the downstream increase in VEGF expression, as evaluated in hypoxic explants treated with pharmacological inhibitors of STAT3 or HIF-1 and in normoxic explants in which pharmacological activators of STAT3 or HIF-1 were used to mimic a hypoxia-like response. The effect of octreotide on STAT3 activation is in part indirect, through the blockade of VEGFR-2 phosphorylation. The effect of octreotide on STAT3, HIF-1, VEGFR-2, and VEGF required Src homology region 2 domain-containing phosphatase 1 (SHP-1). In hypoxic extracts, octreotide induced SHP-1 phosphorylation and activation, and inhibiting SHP-1 abolished the octreotide effect on STAT3, HIF-1, VEGFR-2, and VEGF. The central role of SHP-1 in the modulation of STAT3 and HIF-1 was confirmed in normoxic explants in which pharmacologically activated SHP-1 prevented the effect of STAT3 or HIF-1 activation. Immunohistochemical studies showed that under hypoxia sst(2) and VEGF are expressed by retinal vessels, thus indicating a possible direct effect of octreotide on VEGF-containing endothelial cells. These data clarify the mechanism by which octreotide prevents hypoxia-induced VEGF up-regulation and support the effectiveness of octreotide in treatment of oxygen-induced retinopathies. These results may have implications in designing therapies targeting STAT3 and/or HIF-1 aimed at preventing retinal neovascularization.

OSU-2S, A Novel FTY720 derivative, Mediates Cytotoxicity In Chronic Lymphocytic Leukemia Through PKC Dependent Modulation of SHP1 Tyrosine Phosphatase

Abstract 2499Despite development of numerous chemotherapeutic agents against Chronic Lymphocytic Leukemia (CLL), drug resistance remains impediment in the successful treatment of CLL. Currently several kinases including SYK, BTK, Pim and PI3K are being targeted with inhibitors for therapy in CLL. However, no phosphatase activation directed therapeutics has been described for CLL. We have recently reported FTY720, a potent immunosuppressive agent derived from fungal sphingosine analog ISP-1 to exhibit potent in-vitro and in-vivo preclinical activity against CLL and Mantle cell Lymphoma through protein phosphatase 2A (PP2A) activation dependent mechanisms. However, the immunosuppressive nature of this drug hinders its further development for clinical therapy in cancer. To overcome this limitation we have developed several derivatives of FTY720 by structure-activity relationship analysis. Here we demonstrate OSU-2S as an agent that modulates SHP1 and PP2A protein phosphatases in CLL with potential therapeutic options. OSU-2S is a novel FTY720 derivative that does not interact with sphingosine 1 phosphate receptor 1, thus lacking immunosuppressive property without compromising the cytotoxic potential. Here we have shown preclinical activity of OSU-2S in cell lines representing CLL (MEC-1), ALL (697), lymphoblastic lymphoma cell (Raji, Ramos), Mantle cell Lymphoma (Mino, Jeko), T-cells (Jurkat) and primary CLL and T cell leukemia cells. In primary CLL B-cells, OSU-2S induces activation of major caspases including caspase 3, 8, and 9 resulting in Poly (ADP-ribose) polymerase cleavage. It also induces reactive oxygen species in CLL primary cells with no modulation of Bcl-2 or Mcl-1 levels. Interestingly, OSU-2S induces phosphorylation of Ser 591 of the SHP1 phosphatase in time dependent manner. Consistent with a previously described role for pSer-591 to serve as the nuclear localization signal for SHP1, OSU-2S induced accumulation of pSer-591 SHP1 in the nuclear fraction. Interestingly, OSU-2S induced phosphorylation of Ser-591 SHP1 appears to be mediated through PKC dependent mechanism as activation of PKC with PMA induced phosphorylation of Ser-591 SHP1 and PKC inhibitor Bisindolylmaleimide inhibited OSU-2S induced Serine 591 phosphorylation of SHP1. Moreover, concentrations of Bisindolylmaleimide that prevented the SHP1 Ser-591 phosphorylation also partially rescued OSU-2S induced apoptosis of primary CLL B cells. Co-immunoprecipitation experiments revealed association of PP2A with pSer-591 SHP1 in CLL cells treated with OSU-2S indicating a potential crosstalk between these two phosphatases. Consistent with this hypothesis OSU-2S treatment resulted in activation of PP2A in primary CLL B cells. Further, administration of OSU-2S into Eμ-Tcl-1 transgenic mice with high circulating peripheral blood leukemic cells resulted in significant reduction in circulating CD19+CD5+ leukemic B cells indicating therapeutic efficacy. Ongoing studies on the biochemical basis of cross-talk between the Serine threonine (PP2A) and protein tyrosine (SHP1) phosphatases in response to OSU-2S treatment in CLL will be presented.This work was supported by LLS-Specialized Center of Research in Leukemia and NCI-Leukemia SPORE Grants Disclosures:No relevant conflicts of interest to declare.

Molecular Mechanisms by Which the Novel Tumor Suppressor Phospholipase C-b3 Inhibits Hematopoietic Stem Cell Proliferation and Myeloid Differentiation

Abstract 2446Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate, phospholipase C (PLC) is implicated in promoting cell growth. However, we recently found that PLC-b3-deficient mice develop myeloproliferative neoplasm (MPN), lymphoma, and other tumors (Xiao et al., Cancer Cell, 2009). The mutant mice had increased numbers of hematopoietic stem cells (HSC) with increased proliferative, survival, and myeloid-differentiative abilities. These properties were dependent on the transcription factor Stat5 and could be antagonized by the SH2 domain-containing protein tyrosine phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-b3 deficiency was shown for mouse lymphomas in PLC-b3−/− and in Em-myc;PLC-b3+/− mice and human Burkitt’s lymphoma cells. The same mechanism for malignant transformation seemed to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-b3 was shown to be a novel tumor suppressor (Xiao et al., Cancer Cell, 2009). Similar to PLC-b3-deficient mice, Lyn (Src family kinase)-deficient mice develop MPN. Lyn/PLC-b3 doubly deficient lyn−/−;PLC-β3−/− mice developed a fatal myelodysplastic/myeloproliferative neoplasm (MDS/MPN), which was similar to human chronic myelomonocytic leukemia (CMML). In HSCs of lyn−/−;PLC-β3−/− mice that caused the CMML-like disease, phosphorylation of SHP-1 at Tyr536 and Tyr564 was abrogated, resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore, SHP-1 phosphorylation at Tyr564 by Lyn was indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand, Tyr536 in SHP-1 could be phosphorylated by Lyn and another kinase(s) and was necessary for efficient interaction with Stat5. Therefore, we identified a novel Lyn/PLC-b3-mediated regulatory mechanism of SHP-1 and Stat5 activities (Xiao et al., Blood 2010).PLC-b3 could form the multimolecular SPS complex together with SHP-1, Stat5, and Lyn (Xiao et al., Cancer Cell, 2009; Xiao et al., Blood 2010). The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-b3 (PLC-b3-CT) accelerated SHP-1-mediated dephosphorylation of Stat5. More recently, we identified the minimal sequences within PLC-b3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also bound SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988–998) suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-b3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-b3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies.This study is supported in part by a grant from the MPN Foundation. Disclosures:No relevant conflicts of interest to declare.