Isolation of a distinct class of gain-of-function SHP-2 mutants with oncogenic RAS-like transforming activity from solid tumors

SHP-2 protein tyrosine phosphatase plays an important role in activation of the RAS-dependent signaling. Gain-of-function mutations in the PTPN11 gene, which encodes SHP-2, have been found in the leukemia-prone developmental disorder Noonan syndrome as well as sporadic childhood leukemias, indicating that SHP-2 is a bona fide human oncoprotein. However, the role of SHP-2 mutations in non-hematological malignancies remains obscure. Here, we screened for PTPN11 mutations in primary solid tumors and identified a 1520C>A mutation that causes threonine-507 to lysine (T507K) substitution in the phosphatase domain of SHP-2 in a case of hepatocellular carcinoma. T507K SHP-2 exhibited altered substrate specificity with slightly elevated basal phosphatase activity. Upon expression in NIH3T3 cells, T507K SHP-2 induced transformed foci, which was not observed with wild type, Noonan-specific or leukemia-specific SHP-2. Furthermore, NIH3T3 cells transformed by T507K SHP-2 showed anchorage-independent growth and developed tumors in nude mice. These results indicate that quantitative and/or qualitative alteration in phosphatase activity determines the transforming potential as well as target cell/tissue spectrum of individual SHP-2 mutants as oncoproteins. Although rare in solid tumors, the identified T507K SHP-2 represents a distinct class of SHP-2 mutants with oncogenic RAS-like transforming activity, which could contribute to the development of solid tumors.     

Gab-family adapter molecules in signal transduction of cytokine and growth factor receptors, and T and B cell antigen receptors

Gab1 and Gab2 (Grb2 associated binder 1 and 2) are scaffolding adapter molecules that display sequence similarity with Drosophila DOS (daughter of sevenless), which is a potential substrate for the protein tyrosine phosphatase, Corkscrew, Both Gab1 and Gab2, like DOS, have a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab1 and Gab2 are phosphorylated on tyrosine upon the stimulation of various cytokines, growth factors, and antigen receptors, and interact with signaling molecules, such as Grb2, SHP-2, and PI-3 kinase. Overexpression of Gab1 or Gab2 mimics or enhances growth factor or cytokine-mediated biological processes and activates ERK MAP kinase. These data imply that Gab1 and Gab2 act downstream of a broad range of cytokine and growth factor receptors, as well as T and B antigen receptors, and link these receptors to ERK MAP kinase and biological actions.     

Blockade of epidermal growth factor- or heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has divergent downstream signaling and growth effects

Due to heterodimerization and a variety of stimulating ligands, the ErbB receptor system is both diverse and flexible, which proves particularly advantageous to the aberrant signaling of cancer cells. However, specific mechanisms of how a particular receptor contributes to generating the flexibility that leads to aberrant growth regulation have not been well described. We compared the utilization of ErbB2 in response to epidermal growth factor (EGF) and heregulin stimulation in colon carcinoma cells. Anti-ErbB2 monoclonal antibody 2C4 blocked heregulin-stimulated phosphorylation of ErbB2 and ErbB3; activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3'-kinase (PI3K), and Akt; proliferation; and anchorage-independent growth. 2C4 blocked EGF-mediated phosphorylation of ErbB2 and inhibited PI3K/Akt and anchorage-independent growth but did not affect ErbB1 or MAPK. Immunoprecipitations showed that ErbB3 and Grb2-associated binder (Gab) 1 were phosphorylated and associated with PI3K activity after heregulin treatment and that Gab1 and Gab2, but not ErbB3, were phosphorylated and associated with PI3K activity after EGF treatment. These data show that monoclonal antibody 2C4 inhibited all aspects of heregulin signaling as well as anchorage-independent and monolayer growth. Furthermore, we identify ErbB2 as a critical component of EGF signaling to the Gab1/Gab2-PI3K-Akt pathway and anchorage-independent growth, but EGF stimulation of MAPK and monolayer growth can occur efficiently without the contribution of ErbB2.     

Gab2 and Src co-operate in human mammary epithelial cells to promote growth factor independence and disruption of acinar morphogenesis

The Gab2 docking protein is a target of several oncogenic protein tyrosine kinases and potentiates activation of the Ras/extracellular signal regulated kinase and phosphatidylinositol 3-kinase (PI3-kinase) pathways. Since Gab2 is phosphorylated by c-Src, and both proteins are overexpressed in breast cancers, we have determined the biological consequences of their co-expression in the immortalized human mammary epithelial cell line MCF-10A. While overexpression of c-Src did not affect acinar morphogenesis or growth factor dependence in three-dimensional culture, c-Src co-operated with Gab2 to promote epidermal growth factor (EGF)-independent acinar growth. In contrast, expression of v-Src or the activated mutant c-SrcY527F led to a spectrum of aberrant phenotypes ranging from spheroids with incomplete luminal clearance to highly disrupted, dispersed structures. Gab2 co-expression shifted the phenotypic distribution towards the dispersed phenotype, an effect not observed with a Gab2 mutant unable to bind the p85 subunit of PI3-kinase (Gab2Deltap85). In v-Src-expressing cells, Gab2, but not Gab2Deltap85, significantly decreased E-cadherin adhesive strength without altering its surface expression. Gab2 associated with E-cadherin in the presence and absence of v-Src, indicating that the ability of Gab2 to weaken the strength of cell-cell contacts may reflect enhanced activation of PI3-kinase at adherens junctions. Gab2 also increased migration and invasion of these cells in transwell assays, but these effects were p85-independent. Overall, these findings demonstrate a novel mechanism whereby Gab2 may promote metastatic spread and indicate that Gab2 may play several roles during breast cancer progression.     

The tyrosine phosphatase SHP-2 is required for mediating phosphatidylinositol 3-kinase/Akt activation by growth factors

SHP-2 is a ubiquitously expressed non-transmembrane tyrosine phosphatase with two SH2 domains. Multiple reverse-genetic studies have indicated that SHP-2 is a required component for organ and animal development. SHP-2 wild-type and homozygous mutant mouse fibroblast cells in which the N-terminal SH2 domain was target-deleted were used to examine the function of SHP-2 in regulating Phosphatidylinositol 3-Kinase (PI3K) activation by growth factors. In addition, SHP-2 and various mutants were introduced into human glioblastoma cells as well as SHP-2(-/-) mouse fibroblasts. We found that EGF stimulation and EGFR oncoprotein (DeltaEGFR) expression independently induced the co-immunoprecipitation of the p85 subunit of PI3K with SHP-2. Targeted deletion of the N-terminal SH2 domain of SHP-2 severely impaired PDGF- and IGF-induced Akt phosphorylation. Ectopic expression of SHP-2 in U87MG gliobastoma cells elevated EGF-induced Akt phosphorylation, and the effect was abolished by mutation of its N-terminal SH2 domain. Likewise, the reconstitution of SHP-2 expression in the SHP-2(-/-) cells enhanced Akt phosphorylation induced by EGF while rescuing that induced by PDGF and IGF. Further lipid kinase activity assays confirmed that SHP-2 modulation of Akt phosphorylation correlated with its regulation of PI3K activation. Based on these results, we conclude that SHP-2 is required for mediating PI3K/Akt activation, and the N-terminal SH2 domain is critically important for a "positive" role of SHP-2 in regulating PI3K pathway activation.     

A negative role of SHP-2 tyrosine phosphatase in growth factor-dependent hematopoietic cell survival

SHP-2 tyrosine phosphatase is highly expressed in hematopoietic cells; however, the function of SHP-2 in hematopoietic cell processes is not fully understood. Recent identification of SHP-2 mutations in childhood leukemia further emphasizes the importance of SHP-2 regulation in hematopoietic cells. We previously reported that SHP-2 played a positive role in IL-3-induced activation of Jak2 kinase in a catalytic-dependent manner. Interestingly, enforced expression of wild-type (WT) SHP-2 in Ba/F3 cells enhanced growth factor deprivation-induced apoptosis. Biochemical analyses revealed that although IL-3 activation of Jak2 kinase was increased, tyrosyl phosphorylation of its downstream substrate STAT5 was disproportionately decreased by the overexpression of SHP-2. Following IL-3 deprivation, the tyrosyl phosphorylation of STAT5 that is required for its antiapoptotic activity was rapidly diminished in SHP-2 overexpressing cells. As a result, reduction of the putative downstream targets of STAT5-Bcl-X(L) and pim-1 was accelerated by overexpression of SHP-2. Further investigation showed that SHP-2 associated with STAT5, and that it was indeed able to dephosphorylate STAT5. Finally, overexpression of SHP-2 in primary bone marrow hematopoietic progenitor cells compromised their differentiative and proliferative potential, and enhanced growth factor withdrawal-induced cell death. And, the effect of SHP-2 overexpression on growth factor-dependent survival was diminished in STAT5-deficient hematopoietic cells. Taken together, these results suggest that SHP-2 tyrosine phosphatase negatively regulates hematopoietic cell survival by dephosphorylation of STAT5.     

Epidermal growth factor protects epithelial-derived cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by inhibiting cytochrome c release.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in combination with chemotherapeutic drugs induces a synergistic apoptotic response in cancer cells. TRAIL death receptors have also been implicated in chemotherapeutic drug-induced apoptosis. This has lead to TRAIL being proposed as a potential cancer treatment. In nude mice injected with human tumors, TRAIL reduces the size of these tumors without toxic side effects. We demonstrate that the epidermal growth factor (EGF) stimulation of human embryonic kidney cells HEK 293 and breast cancer cell line MDA MB 231 effectively protects these cells from TRAIL-induced apoptosis in a dose-dependent manner. This stimulation blocks apoptosis by inhibiting TRAIL-mediated cytochrome c release from the mitochondria and caspase 3-like activation. EGF survival response involves the activation of AKT. Expression of activated AKT was sufficient to block TRAIL-induced apoptosis, and kinase-inactive AKT expression blocked EGF-protective response. In contrast, inhibition of EGF stimulation of extracellular-regulated kinase (ERK) activity did not affect EGF protection. These findings indicate that EGF receptor activation provides a survival response against TRAIL-induced apoptosis by inhibiting mitochondrial cytochrome c release that is mediated by AKT activation in epithelial-derived cells.     

Shp2 SUMOylation promotes ERK activation and hepatocellular carcinoma development

Shp2, an ubiquitously expressed protein tyrosine phosphatase, is essential for regulation of Ras/ERK signaling pathway and tumorigenesis. Here we report that Shp2 is modified by SUMO1 at lysine residue 590 (K⁵⁹⁰) in its C-terminus, which is reduced by SUMO1-specific protease SENP1. Analysis of wild-type Shp2 and SUMOylation-defective Shp2^K590R mutant reveals that SUMOylation of Shp2 promotes EGF-stimulated ERK signaling pathway and increases anchorage-independent cell growth and xenografted tumor growth of hepatocellular carcinoma (HCC) cell lines. Furthermore, we find that mutant Shp2^K590R reduces its binding with the scaffolding protein Gab1, and consistent with this, knockdown of SENP1 increased the interaction between Shp2 and Gab1. More surprisingly, we show that human Shp2 (hShp2) and mouse Shp2 (mShp2) have differential effects on ERK activation as a result of different SUMOylation level, which is due to the event of K⁵⁹⁰ at hShp2 substituted by R⁵⁹⁴ at mShp2. In summary, our data demonstrate that SUMOylation of Shp2 promotes ERK activation via facilitating the formation of Shp2-Gab1 complex and thereby accelerates HCC cell and tumor growth, which presents a novel regulatory mechanism underlying Shp2 in regulation of HCC development.     

The docking protein Gab2 is overexpressed and estrogen regulated in human breast cancer

Grb2-associated binder 2 (Gab2) is a recently identified member of the Gab/Daughter of sevenless family of docking proteins, which localize, amplify and integrate signaling pathways activated by various receptors including receptor tyrosine kinases (RTKs). To date, Gab2 signaling has been primarily investigated in hematopoietic cells. Here we report marked overexpression of Gab2 in a subset of breast cancer cell lines relative to normal breast epithelial strains and a trend for increased Gab2 expression in estrogen receptor (ER)-positive lines. Overexpression relative to normal ductal epithelium was also observed in some primary breast cancers. In MCF-7 breast cancer cells Gab2 was markedly tyrosine phosphorylated in response to heregulin and also following EGF, insulin or bFGF administration, indicating that a variety of RTKs implicated in breast cancer development or progression couple to this docking protein. In hormone-responsive breast cancer cells, GAB2 mRNA and protein expression were induced by estradiol in a manner sensitive to the pure anti-estrogen ICI 182780, indicating that this regulation is mediated via the ER. Gab2 therefore represents a novel link between steroid and growth factor signaling in breast cancer, and when overexpressed, may modulate the sensitivity of breast cancer cells to these important growth regulators.     

Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/erbB2 and downstream Erk1/2 and AKT pathways

Dual EGFR/erbB2 inhibition is an attractive therapeutic strategy for epithelial tumors, as ligand-induced erbB2/EGFR heterodimerization triggers potent proliferative and survival signals. Here we show that a small molecule, GW572016, potently inhibits both EGFR and erbB2 tyrosine kinases leading to growth arrest and/or apoptosis in EGFR and erbB2-dependent tumor cell lines. GW572016 markedly reduced tyrosine phosphorylation of EGFR and erbB2, and inhibited activation of Erk1/2 and AKT, downstream effectors of proliferation and cell survival, respectively. Complete inhibition of activated AKT in erbB2 overexpressing cells correlated with a 23-fold increase in apoptosis compared with vehicle controls. EGF, often elevated in cancer patients, did not reverse the inhibitory effects of GW572016. These observations were reproduced in vivo, where GW572016 treatment inhibited activation of EGFR, erbB2, Erk1/2 and AKT in human tumor xenografts. Erk1/2 and AKT represent potential biomarkers to assess the clinical activity of GW572016. Inhibition of activated AKT in EGFR or erbB2-dependent tumors by GW572016 may lead to tumor regressions when used as a monotherapy, or may enhance the anti-tumor activity of chemotherapeutics, since constitutive activation of AKT has been linked to chemo-resistance.     

Role of Gab proteins in phosphatidylinositol 3-kinase activation by thrombopoietin (Tpo)

In this study, we show that upon thrombopoietin (Tpo) stimulation the two adapter proteins Gab1 and Gab2 are strongly tyrosine phosphorylated and associated with Shc, SHP2, PI 3-kinase and Grb2 in mpl-expressing UT7 cells. Although Gab1 and Gab2 seem to mediate overlapping biological signals in many cells, only Gab1 is expressed and phosphorylated in response to Tpo in primary human megakaryocytic progenitors; furthermore, it associates with the same proteins. Although a low level of tyrosine phosphorylated IRS-2 protein is also detected in PI 3-kinase immunoprecipitates, Gab proteins are the essential proteins associated with PI 3-kinase after Tpo stimulation. We demonstrate that, albeit no association is detected between the Tpo receptor mpl and Gab proteins, Y112 located in the C-terminal cytoplasmic domain of mpl is required for Gab1/2 tyrosine phosphorylation. Gab proteins are not tyrosine phosphorylated after Tpo stimulation of UT-7 and Ba/F3 cells expressing a mpl mutant lacking Y112. Moreover, no activation of the PI 3-kinase/Akt pathway is observed in cells expressing this mpl mutant. Finally, we show that this mutant does not allow cell proliferation, thereby confirming that PI 3-kinase activation is required for Tpo-induced cell proliferation.     

Gas6-mediated survival in NIH3T3 cells activates stress signalling cascade and is independent of Ras.

Gas6 is a growth factor membrane of the vitamin K-dependent family of proteins which is preferentially expressed in quiescent cells. Gas6 was identified as the ligand for Axl tyrosine kinase receptor family. Consistent with this, Gas6 was previously reported to induce cell cycle re-entry of serum-starved NIH3T3 cells and to prevent cell death after complete growth factor withdrawal, the survival effect being uncoupled from Gas6-induced mitogenesis. We have previously demonstrated that both Gas6 mitogenic and survival effects are mediated by Src and the phosphatidylinositol3-OH kinase (PI3K). Here we report that Ras is required for Gas6 mitogenesis but is dispensable for its survival effect. Gas6-induced survival requires the activity of the small GTPases of the Rho family, Rac and Rho, together with the downstream kinase Pak. Overexpression of the respective dominant negative constructs abrogates Gas6-mediated survival functions. Addition of Gas6 to serum starved cells results in the activation of AKT/PKB and in the phosphorylation of the Bcl-2 family member, Bad. By ectopic expression of a catalytically inactive form of AKT/PKB, we demonstrate that AKT/PKB is necessary for Gas6-mediated survival functions. We further show evidence that Gas6 stimulation of serum starved NIH3T3 cells results in a transient ERK, JNK/SAPK and p38 MAPK activation. Blocking ERK activation did not influence Gas6-induced survival, suggesting that such pathway is not involved in Gas6 protection from cell death. On the contrary we found that the late constitutive increase of p38 MAPK activity associated with cell death was downregulated in Gas6-treated NIH3T3 cells thus suggesting that Gas6 might promote survival by interfering with this pathway. Taken together the evidence here provided identity elements involved in Gas6 signalling more specifically elucidating the pathway responsible for Gas6-induced cell survival under conditions that do not allow cell proliferation.     

三氧化二砷对淋巴瘤细胞酪氨酸磷酸酶1基因的去甲基化作用

目的 探讨淋巴瘤细胞系T2和非霍奇金淋巴瘤(NHL)组织中酪氨酸磷酸酶1基因启动子区甲基化状态,三氧化二砷(As2O3)对T2细胞中SHP-1的去甲基化作用及对T2细胞生长增殖的生物学影响.方法 以不同浓度As2O3处理淋巴瘤细胞株T2,采用二苯基溴化四氮唑蓝(MTT)法检测T2细胞的生长变化,采用流式细胞术检测细胞凋亡率的变化,采用实时定量聚合酶链反应(FQ-PCR)和Western blot方法检测T2细胞SHP-1基因mRNA和蛋白表达及c-kit蛋白表达变化.采用甲基化特异性聚合酶链反应(MSP)检测T2细胞和32例NHL组织SHP-1基因启动子甲基化状态.结果 As2O3使T2细胞增殖受抑,凋亡增加,效应均具有时间和剂量依赖性.As2O3能逆转T2细胞SHP-1基因启动子甲基化,SHP-1基因恢复表达,同时c-kit蛋白磷酸化水平降低.SHP-1基因在对照组淋巴组织中呈完全性非甲基化状态,而在NHL组织中启动子甲基化出现率为87.5%(28/32).结论 在淋巴瘤细胞和NHL组织中,SHP-1基因启动子区域存在高度甲基化.As2O3能逆转T2细胞DNA异常甲基化,诱导SHP-1基因表达,并可能通过抑制c-kit受体及其信号传导路径的活化,抑制肿瘤细胞增殖.     

Sustained recruitment of phospholipase C-gamma to Gab1 is required for HGF-induced branching tubulogenesis

A distinctive property of Hepatocyte Growth Factor (HGF) is its ability to induce differentiation of tubular structures from epithelial and endothelial cells (branching tubulogenesis). The HGF receptor directly activates PI3 kinase, Ras and STAT signalling pathways and phosphorylates the adaptator GRB2 Associated Binder-1 (Gab1). Gab1 is also phosphorylated in response to Epidermal Growth Factor (EGF) but is unable to induce tubule formation. Comparison of 32P-peptide maps of Gab1 from EGF- versus HGF-treated cells, demonstrates that the same sites are phosphorylated in vivo. However, while both EGF and HGF induce rapid tyrosine phosphorylation of Gab1 with a peak at 15 min, the phosphorylation persists for over 1 h, only in response to HGF. Nine tyrosines are phosphorylated by both receptors. Three of them (Y307, Y373, Y407) bind phospholipase C-gamma (PLC-gamma). Interestingly, the overexpression of a Gab1 mutant unable to bind PLC-gamma (Gab1 Y307/373/407F) did not alter HGF-stimulated cell scattering, only partially reduced the growth stimulation but completely abolished HGF-mediated tubulogenesis. It is concluded that sustained recruitment of PLCgamma to Gab1 plays an important role in branching tubulogenesis.     

Bioinformatics-based discovery and characterization of an AKT-selective inhibitor 9-chloro-2-methylellipticinium acetate (CMEP) in breast cancer cells

AKT is a promising target for anticancer drug development. In this work, a bioinformatics approach was applied to search for AKT inhibitors based on the correlation analysis between phospho-Ser473 AKT expression level and the antiproliferative data of NCI small molecule compounds against NCI 60 cancer cell lines, the candidate compounds were then subject to AKT kinase assay. The possible effects of potent compound on PI3K/AKT, PDK1, and MAPK, its antiproliferative and apoptosis-inducing effects on breast cancer cells which have high-levels of AKT activation were assessed by Western blot analysis, cell viability assay, and apoptosis assay. One compound, CMEP (NSC632855, 9-chloro-2-methylellipticinium acetate) was identified with all three correlation algorithm, Pearson's, Sperman's, and Kendall's, showing a high-ranked correlation coefficient. CMEP inhibits only AKT, but does not inhibit PI3K, PDK1, or MAPK. CMEP also inhibits heregulin-induced AKT activation, does not inhibit heregulin-induced MAPK activation in MCF-7 breast cancer cells. Increased concentrations of ATP reverse the AKT inhibitory effect of CMEP. CMEP inhibits growth and induces apoptosis in breast cancer cells which have high-levels of AKT activation and lack functional PTEN; however, CMEP only shows a minimal activity in NIH3T3 cells which do not have AKT activation. In conclusion, a lead compound CMEP, as an AKT selective inhibitor has been identified started with a bioinformatics-based approach. CMEP inhibits growth and induces apoptosis in cancer cells which have high-levels of AKT activation and lack PTEN or harbor PTEN mutation.     

Catalytic-dependent and -independent roles of SHP-2 tyrosine phosphatase in interleukin-3 signaling

SHP-2 tyrosine phosphatase is highly expressed in hematopoietic cells, however, the function of SHP-2 in hematopoietic cell signaling is not well understood. Here we focus on the role of SHP-2 phosphatase in the signal transduction of interleukin (IL)-3, a cytokine involved in hematopoietic cell survival, proliferation, and differentiation. We established immortalized SHP-2(-/-) hematopoietic cell pools and showed that IL-3-induced proliferative response was diminished in SHP-2(-/-) cells. Moreover, inhibition of the catalytic activity of SHP-2 in wild-type (WT) bone marrow hematopoietic progenitor cells and Ba/F3 cells by overexpression of catalytically inactive SHP-2 mutant suppressed their differentiative and proliferative responses to IL-3, demonstrating an important positive role for SHP-2 in IL-3 signal transduction. Further biochemical analyses revealed that IL-3-induced Jak/Stat, Erk, and PI3 kinase pathways in SHP-2(-/-) cells were impaired and reintroduction of WT SHP-2 into mutant cells partially restored IL-3 signaling. Interestingly, in catalytically inactive SHP-2-overexpressing Ba/F3 cells, although IL-3-induced activation of Jak2 and Erk kinases was reduced and shortened, PI3 kinase activation remained unaltered. Taken together, these results suggest that SHP-2 tyrosine phosphatase plays multiple roles in IL-3 signal transduction, functioning in both catalytic-dependent and -independent manners in the Jak/Stat, Erk, and PI3 kinase pathways.     

Gab1 signaling is regulated by EGF receptor sorting in early endosomes

Although combinatorial signaling through the ErbB network is implicated in certain types of human cancer, the specifics of how particular receptors contribute to the transformed phenotype are not well understood. The goal of this study was to identify epidermal growth factor (EGF) receptor-dependent cell signaling abnormalities specifically associated with mutations in a previously described 679-LL lysosomal sorting signal, which restrict ligand-dependent receptor downregulation by promoting recycling. Importantly, the 679-LL signal is not conserved in any of the other members of the ErbB receptor family suggesting its physiological function may be tightly regulated during EGF receptor-dependent signaling. Our data indicate that cells expressing receptors with an inactive 679-AA signal are rapidly transported to Rab4+ early endosomes after they are internalized in contrast to wild-type receptors that are localized to early endocytic antigen 1 (EEA1)+ early endosomes. Divergent trafficking in early endosomes is associated with prolonged activation of p44/42 mitogen-activated protein kinases (MAPK) but not Akt. Gab1 appears to be the critical signaling molecule facilitating prolonged MAPK signaling, and activated Gab1 is recruited to early endosomes in 679-AA receptor-expressing cells. Activated Gab1 is also recruited to early endosomes in breast cancer cells characterized by high levels of EGF receptor-ErbB2 heterodimers, suggesting 679-AA expressing cells recapitulate certain aspects of EGF receptor signaling and transformation by activated ErbB2. Phosphatidylinositol 3-kinase (PI3K)-dependent membrane translocation known to be important for maintaining Gab1 activity in other settings was dispensable. We conclude that 679-LL has dual functions in EGF receptor trafficking and threshold signaling through a subset of signaling molecules including p44/42 MAPK and Gab1.     

SH2-containing phosphotyrosine phosphatase SHP-1 is involved in BCR-ABL signal transduction pathways

The BCR-ABL fusion protein is strongly implicated in the malignant process of Philadelphia (Ph-1)-positive leukemia. The BCR-ABL fusion protein exhibits a deregulated tyrosine kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. SHP-1 (SHPTP1, PTP1C, HCP, SHP) is an SH2 domain-containing tyrosine phosphatase expressed predominantly in hematopoietic cells. The association of the murine motheaten phenotype of severe hematopoietic dysregulation with loss of SHP-1 tyrosine phosphatase activity indicates a critical role of SHP-1 in the regulation of hematopoietic cell growth and differentiation. Experiments were performed to determine whether SHP-1 might form specific complexes with BCR-ABL signaling pathway. We found that SHP-1 was highly and constitutively tyrosine phosphorylated in 32DCl3 and TF-1 cells transfected with BCR-ABL expression vector. Furthermore, SHP-1 and BCR-ABL formed stable complexes in BCR-ABL expressing cells. Direct binding between SHP-1 and Grb2 was observed in vitro. Expression of BCR-ABL in TF-1 cells resulted in a two-fold increase in SHP-1 phosphatase activity. BCR-ABL tyrosine kinase was able to phosphorylate recombinant SHP-1 protein in vitro. SHP-1 therefore has the capacity to bind and potentially modulate the signaling effectors involved in activation of Ras, and accordingly, regulation of cellular transformation of BCR-ABL.