Diverse injurious stimuli reduce protein tyrosine phosphatase-μ expression and enhance epidermal growth factor receptor signaling in human airway epithelia

In response to injury, airway epithelia utilize an epidermal growth factor (EGF) receptor (EGFR) signaling program to institute repair and restitution. Protein tyrosine phosphatases (PTPs) counterregulate EGFR autophosphorylation and downstream signaling. PTPμ is highly expressed in lung epithelia and can be localized to intercellular junctions where its ectodomain homophilically interacts with PTPμ ectodomain expressed on neighboring cells. We asked whether PTPμ expression might be altered in response to epithelial injury and whether altered PTPμ expression might influence EGFR signaling. In A549 cells, diverse injurious stimuli dramatically reduced PTPμ protein expression. Under basal conditions, small interfering RNA (siRNA)-induced silencing of PTPμ increased EGFR Y992 and Y1068 phosphorylation. In the presence of EGF, PTPμ knockdown increased EGFR Y845, Y992, Y1045, Y1068, Y1086, and Y1173 but not Y1148 phosphorylation. Reduced PTPμ expression increased EGF-stimulated phosphorylation of Y992, a docking site for phospholipase C (PLC)γ(1), activation of PLCγ(1) itself, and increased cell migration in both wounding and chemotaxis assays. In contrast, overexpression of PTPμ decreased EGF-stimulated EGFR Y992 and Y1068 phosphorylation. Therefore, airway epithelial injury profoundly reduces PTPμ expression, and PTPμ depletion selectively increases phosphorylation of specific EGFR tyrosine residues, PLCγ(1) activation, and cell migration, providing a novel mechanism through which epithelial integrity may be restored.     

Age-related decline in Ras/ERK mitogen-activated protein kinase cascade is linked to a reduced association between Shc and EGF receptor

Numerous studies have demonstrated that the proliferative capacity of cells declines with age. Using rat primary hepatocytes as a model system, we recently demonstrated that this age-related decline in the proliferative response to mitogenic stimulation is associated with decreased activities of both extracellular signal-regulated kinase (ERK) and p70 S6 kinase (p70(S6k)). To unravel the molecular basis for age-related defects in the ERK pathway, we have now characterized the upstream signaling events that occur after epidermal growth factor (EGF) stimulation in young and aged hepatocytes. As previously noted for ERK, the activities of both MEK (the kinase immediately upstream of ERK) and Ras following EGF stimulation were significantly lower in aged hepatocytes. An examination of the EGF receptor (EGFR) revealed a similar amount of EGFR in the two age groups. Likewise, EGFR and Shc, an adaptor protein that plays a crucial role in linking EGFR to Ras activation, underwent tyrosine phosphorylation to a similar degree in both young and aged hepatocytes. However, in aged cells Shc was unable to form stable complexes with EGFR after EGF stimulation. Our results suggest that a decrease in the association between Shc and EGFR in aged cells underlies the age-related declines in the ERK signaling cascade and in proliferative capacity.     

Advanced glycation end product (AGE) receptor 1 suppresses cell oxidant stress and activation signaling via EGF receptor

Advanced glycation end product receptors (AGERs) play distinct functional roles in both the toxicity and disposal of advanced glycation end products (AGEs), substances that are linked to diabetes and aging. Overexpression of AGER1 in murine mesangial cells (MCs) (MC-R1) inhibited AGE-induced MAPK1,2 phosphorylation and NF-kappaB activity and also increased AGE degradation. The mechanism of the inhibitory effects of AGER1, upstream of MAPK, was explored in MCs and HEK293 AGER1-expressing cells. AGE-induced Ras activation was found to be linked to Shc/Grb2 complex formation and Shc phosphorylation in MCs, responses that were markedly reduced in MC-R1 cells. AGE responses also included EGF receptor (EGFR) phosphorylation in MCs or HEK293 cells, but this link was blocked in both MC-R1 and HEK293-R1 cells. Coexpression of AGER1 and EGFR in HEK293 cells decreased AGE-mediated EGFR and p44/p42 phosphorylation but not EGF-induced p44/p42 activation. AGE, S100/calgranulin, or H(2)O(2) promoted MAPK phosphorylation in EGFR(+) cells in a manner that was inhibitable by an EGFR inhibitor, AG1478. Also, in AGER1 cells, AGE-induced H(2)O(2) formation and AGE- or S100-induced p44/p42 phosphorylation were suppressed, and these effects were restored by R1 siRNA. These data confirm that R1 negatively regulates AGE-mediated oxidant stress-dependent signaling via the EGFR and Shc/Grb2/Ras pathway. AGER1 could serve as a model for developing therapeutic targets against vascular and kidney disorders related to diabetes and aging.     

Epidermal growth factor-receptor mutant lacking the autophosphorylation sites induces phosphorylation of Shc protein and Shc-Grb2/ASH association and retains mitogenic activity.

Epidermal growth factor (EGF) receptor (EGFR) can induce cell growth and transformation in a ligand-dependent manner. To examine whether the autophosphorylation of EGFR correlates with the capacity of the activated EGFR to induce cell growth and transformation, we truncated the human EGFR just after residue 1011, removing all three major autophosphorylation sites (DEL1011). Further, a point mutation was introduced at another autophosphorylation site, Tyr-992-->Phe (DEL1011+F992). The wild-type and mutant receptors were stably expressed in a NIH 3T3 variant cell line that expresses an extremely low level of endogenous EGFR and does not grow with EGF. As expected, DEL1011 and DEL1011+F992 were found to be severely impaired in EGF-induced autophosphorylation, due to the deletion of the appropriate target tyrosines. However, mutant receptors still could induce EGF-dependent DNA synthesis, morphological transformation, and anchorage-independent growth, although the extent of these was significantly reduced when compared with wild-type EGFR. EGF-induced tyrosine phosphorylation of Ras-GTPase activating protein-associated protein p62 and phospholipase C gamma 1 was dramatically reduced in the cells expressing DEL1011 and DEL1011+F992. On the other hand, tyrosine phosphorylation of Shc, complex formation of Shc-Grb2/Ash, and activation of microtubule-associated protein kinase were still fully induced upon EGF stimulation without binding of Shc or Grb2/Ash to the mutant receptor. Thus, tyrosine phosphorylation of Shc may play a crucial role for activating Ras and generating mitotic signals by the activated EGFR mutant.     

Fibronectin-induced proliferation in thyroid cells is mediated by alphavbeta3 integrin through Ras/Raf-1/MEK/ERK and calcium/CaMKII signals

We recently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium Ca2+/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FN-induced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2+ concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2+ concentration were mediated by FN binding to alphavbeta3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered.     

EGF potentiated oncogenesis requires a tissue transglutaminase-dependent signaling pathway leading to Src activation

EGF receptor (EGFR) signaling in human cancers elicits changes in protein-expression patterns that are crucial for potentiating tumor growth. Identifying those proteins with expression regulated by the EGFR and determining how they contribute0 to malignancy is fundamental for the development of more effective strategies to treat cancer. Here, we show that tissue transglutaminase (tTG) is one such protein. EGF up-regulates tTG expression in human breast-cancer cells, and knock-downs of tTG or the treatment of breast cancer cells with a tTG inhibitor blocks their EGF-stimulated anchorage-independent growth. We further show that the combined actions of Ras and Cdc42, leading to the activation of PI 3-kinase and NFκB, provide a mechanism by which EGF can up-regulate tTG in breast-cancer cells. Moreover, overexpression of wild-type tTG, but not its transamidation-defective counterpart, fully mimics the growth advantages afforded by EGF to these cancer cells. Surprisingly, the tTG-promoted growth of breast-cancer cells is dependent on its ability to activate the Src tyrosine kinase as an outcome of a complex formed between tTG and the breast-cancer marker and intermediate filament protein keratin-19. These findings identify tTG as a key participant in an EGFR/Src-signaling pathway in breast-cancer cells and a potential target for inhibiting EGFR-promoted tumor progression.     

Abnormalities of insulin-like growth factor-I signaling and impaired cell proliferation in osteoblasts from subjects with osteoporosis

IGF-I regulates bone acquisition and maintenance, even though the cellular targets and signaling pathways responsible for its action in human bone cells are poorly understood. Whether abnormalities in IGF-I action and signaling occur in human osteoblasts under conditions of net bone loss has not been determined. Herein we carried out a comparative analysis of IGF-I signaling in primary cultures of human osteoblasts from osteoporotic and control donors. In comparison with control cells, osteoporotic osteoblasts showed increased tyrosine phosphorylation of the IGF-I receptor in the basal state and blunted stimulation of receptor phosphorylation by IGF-I. Augmentation of basal IGF-I receptor phosphorylation was associated with coordinate increases in basal tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and activation of Erk, which were also minimally responsive to IGF-I stimulation. By contrast, phosphorylation levels of IRS-1, Akt, and glycogen synthase kinase-3 were similar in the basal state in control and osteoporotic osteoblasts and showed marked increases after IGF-I stimulation in both cell populations, even though these responses were significantly lower in the osteoporotic osteoblasts. The IGF-I signaling abnormalities in osteoporotic osteoblasts were associated with reduced DNA synthesis both under basal conditions and after stimulation with IGF-I. Interestingly, treatment of the osteoporotic osteoblasts with the MAPK kinase inhibitor PD098059 reduced the elevated levels of Erk phosphorylation and increased basal DNA synthesis. Collectively, our data show that altered osteoblast proliferation in human osteoporosis may result from dysregulation of IGF-I receptor signaling, including constitutive activation of the IRS-2/Erk signaling pathway, which becomes unresponsive to IGF-I, and defective induction of the IRS-1/Akt signaling pathway.     

Tyrosine phosphorylation-dependent and -independent role of Shc in the regulation of IGF-1-induced mitogenesis and glycogen synthesis.

To examine the functional role of Shc tyrosine phosphorylation in IGF-1 signaling, wild-type (WT)-Shc and Y239,240,317F (3F)-Shc were transiently transfected into L6 myoblasts. IGF-1 signaling was compared among the transfected cells. IGF-1-induced tyrosine phosphorylation of Shc and its subsequent association with Grb2 were increased in WT-Shc cells, whereas they were decreased in 3F-Shc cells compared with those in parental L6 cells. Consistent with their changes, IGF-1-induced MAPK activation and thymidine incorporation were enhanced in WT-Shc cells, whereas they were again decreased in 3F-Shc cells. It is possible that Shc and insulin receptor substrate (IRS)-1 can interact competitively, via their phosphotyrosine binding (PTB) domains, with the activated IGF-1 receptor. In this regard, IGF-1-induced tyrosine phosphorylation of IRS-1 was decreased by overexpressing both WT-Shc and 3F-Shc cells. Consistent with the decrease, IGF-1-induced IRS-1 association with the p85 subunit of PI3K and activation of PI3K and Akt were reduced in both WT-Shc and 3F-Shc cells. As a result, IGF-1-induced glycogen synthesis was also decreased in both cells. Furthermore, expression of Shc PTB domain alone inhibited IGF-1 stimulation of Akt and glycogen synthesis. These results indicate that tyrosine phosphorylation of Shc is important for IGF-1 stimulation of MAPK leading to mitogenesis and that Shc, via its PTB domain, negatively regulates IGF-1-induced glycogen synthesis by competing with IRS-1, which is not relevant to Shc tyrosine phosphorylation.     

Growth hormone alters epidermal growth factor receptor binding affinity via activation of extracellular signal-regulated kinases in 3T3-F442A cells

Epidermal growth factor receptor (EGFR) is a transmembrane protein that binds EGF in its extracellular domain and initiates signaling via intrinsic tyrosine kinase activity in its cytoplasmic domain. EGFR is important in development, cellular proliferation, and cancer. GH is a critical growthpromoting and metabolic regulatory hormone that binds the GH receptor, thereby engaging various signaling pathways, including ERKs. Prior studies suggest cross-talk between the GH receptor and EGFR signaling systems. Using the GH- and EGF-responsive 3T3-F442A preadipocyte, we previously observed that GH, in addition to causing EGFR tyrosine phosphorylation, also induced EGFR phosphorylation that was detected by PTP101, an antibody reactive with ERK consensus phosphorylation sites. This latter phosphorylation was prevented by pretreatment with MAPK kinase (MEK)1 inhibitors, suggesting ERK pathway dependence. Furthermore, GH cotreatment with EGF markedly slowed EGF-induced EGFR degradation and down-regulation, thereby potentiating EGF-induced EGFR signaling. These effects were also MEK1 dependent and suggested ERK pathway-dependent influence of GH on EGF-induced EGFR postendocytic trafficking and signaling. We now explore the impact of GH on cell surface binding of EGF in 3T3-F442A cells. We found that GH pretreatment caused transient, but substantial, lessening of (125)I-EGF binding. Competitive binding experiments revealed that the decreased binding was primarily due to decreased affinity, rather than a change in the number of EGF binding sites. The effect of GH on EGF binding was concentration dependent and temporally correlated with GH-induced ERK activation and EGFR PTP101-reactive phosphorylation. Blockade of the MEK1/ERK but not the protein kinase C pathway, prevented GH's effects on EGF binding, and our results indicate that the mechanisms of GH- and phorbol-12-myristate-13-acetateinduced inhibition of EGF binding differ substantially. Overall, our findings suggest that GH can modulate both EGF binding kinetics and the EGFR's postbinding signaling itinerary in a MEK1/ERK pathway-dependent fashion.     

The tethered configuration of the EGF receptor extracellular domain exerts only a limited control of receptor function

Quantitative epidermal growth factor (EGF)-binding experiments have shown that the EGF-receptor (EGFR) is displayed on the surface of intact cells in two forms, a minority of high-affinity and a majority of low-affinity EGFRs. On the basis of the three-dimensional structure of the extracellular ligand binding domain of the EGFR, it was proposed that the intramolecularly tethered and autoinhibited configuration corresponds to the low-affinity receptor, whereas the extended configuration accounts for the high-affinity EGFRs on intact cells. Here we test this model by analyzing the properties of EGFRs mutated in the specific regions responsible for receptor autoinhibition and dimerization, respectively. Our results show that mutagenic disruption of the autoinhibitory tether in EGFR results in a decrease in the dissociation rate of EGF without a detectable change in EGFR activation and signaling through EGFR even in response to stimulation with low concentrations of EGF. Mutagenic disruption of the dimerization arm, on the other hand, increased the rate of EGF dissociation and impaired EGFR activation and signaling via the EGFR. This study demonstrates that the extended configuration of EGFR does not account for the apparent high-affinity EGF-binding to EGFR on intact cells. Furthermore, the autoinhibition conferred by the tethered configuration of the extracellular ligand-binding domain provides only a limited control of EGFR function.     

Galphaq expression activates EGFR and induces Akt mediated cardiomyocyte survival: dissociation from Galphaq mediated hypertrophy

Our laboratory has previously shown that adenoviral-mediated overexpression of Galphaq in neonatal rat ventricular cardiomyocytes increases the phosphorylation of Akt, a well-established anti-apoptotic effector. As demonstrated here, Galphaq expression protects cardiomyocytes against apoptosis induced by treatment with 2-deoxyglucose (2DOG) and this protection is lost when Akt activation is prevented by treatment with LY294002 (an inhibitor of PI3K). Galphaq-induced Akt phosphorylation is not caused by increased Gbetagamma signaling and does not appear to involve PKC activation. Rather studies using the EGF receptor inhibitor AG1478 and the Src inhibitor PP2 implicate these tyrosine kinases in the pathway inducing Akt phosphorylation. EGFR phosphorylation is increased in cells expressing Galphaq and this effect is inhibited by PP2, placing Src upstream of EGFR phosphorylation. EGFR activation appears to be required for Galphaq-mediated protection since inhibition of Src or EGFR rendered cells susceptible to 2DOG-induced apoptosis. In contrast to the requirement for EGFR mediated Akt activation in cardioprotection, neither EGFR nor Akt activation are necessary for the hypertrophic increases in cell size or ANF content elicited by Galphaq overexpression. These data demonstrate that increased Galphaq activity can provide anti-apoptotic signals by eliciting EGFR phosphorylation and subsequent Akt activation, independent of the well-known ability of Galphaq signaling to elicit hypertrophy.     

Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.     

Heparin blockade of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor (EGF) receptor transactivation by heparin-binding EGF-like growth factor

Agonists of G protein-coupled receptors, such as thrombin, act in part by transactivating the epidermal growth factor (EGF) receptor (EGFR). Although at first a ligand-independent mechanism for EGFR transactivation was postulated, it has recently been shown that this transactivation by various G protein-coupled receptor agonists can involve heparin-binding EGF-like growth factor (HB-EGF). Because thrombin stimulation of vascular smooth muscle cell migration is blocked by heparin and because heparin can displace HB-EGF, we investigated the possibility that thrombin stimulation of smooth muscle cells (SMCs) depends on EGFR activation by HB-EGF. In rat SMCs, EGFR phosphorylation and extracellular signal-regulated kinase (ERK) activation in response to thrombin are inhibited not only by the EGFR inhibitor AG1478 and by EGFR blocking antibody but also by heparin and by neutralizing HB-EGF antibody. HB-EGF-dependent signaling induced by thrombin is inhibited by batimastat, which suggests a requirement for pro-HB-EGF shedding by a metalloproteinase. We further demonstrate that this novel pathway is required for the migration of rat and baboon SMCs in response to thrombin. We conclude from these data that the inhibitory effect of heparin on SMC migration induced by thrombin relies, at least in part, on a blockade of HB-EGF-mediated EGFR transactivation.     

Betacellulin and amphiregulin induce upregulation of cyclin D1 and DNA synthesis activity through differential signaling pathways in vascular smooth muscle cells

Activation of EGF receptors is closely involved in vascular proliferative diseases. The signaling mechanisms of EGF ligands, including betacellulin (BTC) and amphiregulin (AR), are poorly understood. We examined how BTC and AR induced DNA synthesis activity in primary cultures of human thoracic aortic smooth muscle cells (HTASMCs). BTC induced phosphorylation of all four EGF receptors present on HTASMCs: ErbB1, ErbB2, ErbB3, and ErbB4. BTC rapidly induced the phosphorylation of Akt, GSK3alpha/beta, and two FoxO factors, FKHR and AFX, in a dose- and time-dependent manner. BTC increased nuclear beta-catenin accumulation. BTC increased cyclin D1 mRNA, cyclin D1 protein, and DNA synthesis activity. Pretreatment with the phosphatidylinositol 3'-kinase (PI 3'-kinase) inhibitor wortmannin suppressed BTC-induced cyclin D1 mRNA and protein and DNA synthesis activity. In contrast, AR, a specific ErbB1 ligand, induced sustained ERK1/2 and Elk1 phosphorylation, increased cyclin D1 mRNA and protein, and increased DNA synthesis activity. AR did not produce any changes in Akt phosphorylation. Pretreatment with PD98059 suppressed AR-induced cyclin D1 mRNA and protein. Thus, the PI 3'-kinase/Akt/GSK/FoxO/beta-catenin pathway could be the major signaling cascade for BTC-induced upregulation of cyclin D1 protein, whereas a sustained ERK/Elk1 activation could be the major signaling cascade for AR-induced upregulation of cyclin D1 protein in HTASMCs. Moreover, immunohistochemical staining revealed that that BTC, ErbB1, and ErbB4 are upregulated in the plaques of human atherosclerotic coronary arteries. Taken together, BTC and AR could be potent growth factors in proliferative vascular diseases.     

Compartmentalization of signaling-competent epidermal growth factor receptors in endosomes

In this study, the preparation of detergent-resistant membranes (DRMs) and the immunoisolation of intracellular vesicles enriched in raft markers were used to investigate the effect of physiological doses of epidermal growth factor (EGF) in vivo on the compartmentalization and activation of EGF receptor (EGFR) in rat liver endosomes. Both of these techniques show that after EGF administration, a distinctive population of intracellular EGFR, which was characterized by a high level of tyrosine phosphorylation, accumulated in endosomes. EGFR recruited to early endosomes were more tyrosine phosphorylated than those from late endosomes. However, the level of tyrosine phosphorylation of EGFR in DRMs isolated from early and late endosomes was comparable, suggesting that EGFR in endosomal DRMs are more resistant to tyrosine dephosphorylation. In accordance with the higher level of Tyr phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc to endosomal DRMs compared with whole endosomes. Furthermore, a proteomic analysis identified a selective increase of many alpha-subunits of heterotrimeric G proteins in endosomal DRMs in response to EGF. These observations suggest that a distinctive pool of endocytic EGFR, potentially competent for signaling, is actively trafficking through intracellular compartments with the characteristic of lipid rafts.     

Nucleobindin-2 is a positive modulator of EGF-dependent signals leading to enhancement of cell growth and suppression of adipocyte differentiation

Nucleobindin-2 is a 420-amino-acid EF-hand calcium-binding protein that undergoes proteolytic processing to generate an 82-amino-acid amino-terminal peptide termed nesfatin-1. To determine whether nucleobindin-2 has any biological function, nucleobindin-2 was either overexpressed or knocked down by short hairpin RNA in cultured CHO cells expressing the human insulin and epidermal growth factor (EGF) receptors (CHO/IE) and in 3T3-L1 cells. Reduction in nucleobindin-2 expression inhibited EGF-stimulated MAPK kinase (S217/S221) and Erk phosphorylation (T202/Y204). In contrast, there was no significant effect on EGF-stimulated EGF receptor phosphorylation, EGF receptor internalization, or 52-kDa Shc and c-Raf phosphorylation. Although kinase suppressor of Ras-1 and protein phosphatase 2A expression was not changed, intracellular calcium concentrations and PP2A activity was significantly increased in nucleobindin-2 knocked-down cells. Concomitant with these alterations in EGF-stimulated signaling, cell proliferation was significantly reduced in nucleobindin-2 knocked-down cells. Moreover, reduced nucleobindin-2 expression in 3T3-L1 preadipocytes resulted in a greater extent of 3T3-L1 cell adipocyte differentiation. Taken together, these data indicate that nucleobindin-2 regulates EGF-stimulated MAPK kinase/Erk signaling, cell proliferation, and adipocyte differentiation.     

Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor-induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration

Stem cell factor (SCF) has crucial roles in proliferation, survival, and differentiation of hematopoietic stem cells and mast cells through binding to c-Kit receptor (KIT). Chemotaxis is another unique function of SCF. However, little is known about the intracellular signaling pathway of SCF/KIT-mediated cell migration. To investigate the signaling cascade, we made a series of 22 KIT mutants, in which tyrosine (Y) residue was substituted for phenylalanine (F) in the cytoplasmic domain, and introduced into BAF3 cells or 293T cells. On stimulation with SCF, BAF3 expressing KIT(WT)(WT) showed cell migration and Ca(2+) mobilization. Among 22 YF mutants, Y567F, Y569F, and Y719F showed significantly reduced cell migration and Ca(2+) mobilization compared to WT. In Y567F, Lyn activation on SCF stimulation decreased and C-terminal Src kinase (Csk) suppressed KIT-mediated Ca(2+) influx and cell migration, suggesting that Y567-mediated Src family kinase (SFK) activation leads to Ca(2+) influx and migration. Furthermore, we found that p38 mitogen-activated protein kinase (p38 MAPK) and Erk1/2 were also regulated by Y567/SFK and involved in cell migration, and that p38 MAPK induced Ca(2+) influx, thereby leading to Erk1/2 activation. In Y719F, the binding of phosphatidylinositol 3'-kinase (PI3K) to KIT was lost and KIT-mediated cell migration and Ca(2+) mobilization were suppressed by PI3K chemical inhibitors or dominant-negative PI3K, suggesting the involvement of Y719-mediated PI3K pathway in cell migration. Combination of Csk and the PI3K inhibitor synergistically reduced cell migration, suggesting the cooperation of SFK and PI3K. Taken together, these results indicate that 2 major KIT signaling pathways lead to cell migration, one is Y567-SFK-p38 MAPK-Ca(2+) influx-Erk and the other is Y719-PI3K-Ca(2+) influx.     

Human somatic PTPN11 mutations induce hematopoietic-cell hypersensitivity to granulocyte-macrophage colony-stimulating factor

Juvenile myelomonocytic leukemia (JMML) is a lethal disease of young children characterized by hypersensitivity of hematopoietic progenitors to granulocyte-macrophage colony-stimulating factor (GM-CSF). Mutations in PTPN11, which encodes the protein tyrosine phosphatase Shp-2, are common in JMML. We hypothesized that PTPN11 mutations induce hypersensitivity of hematopoietic progenitors to GM-CSF and confer increased GM-CSF-stimulated phospho-extracellular signal-regulated kinase (Erk) levels. To test this hypothesis, the wild-type (WT) and 3 mutant Ptpn11 cDNAs (E76K, D61V, and D61Y) were transduced into murine bone marrow cells to examine GM-CSF-stimulated granulocyte-macrophage colony-forming unit (CFU-GM) growth, macrophage progenitor proliferation, and activation of the Ras signaling pathway. Expression of the Shp-2 mutants induced progenitor cell hypersensitivity to GM-CSF compared with cells transduced with vector alone or WT Shp-2. Macrophage progenitors expressing the Shp-2 mutants displayed both basal and GM-CSF-stimulated hyperproliferation compared with cells transduced with vector alone or WT Shp-2. Consistently, macrophage progenitors transduced with the Shp-2 mutants demonstrated constitutively elevated phospho-Erk levels and sustained activation of phospho-Erk following GM-CSF stimulation compared with vector alone or WT Shp-2. These data support the hypothesis that PTPN11 mutations induce hematopoietic progenitor hypersensitivity to GM-CSF due to hyperactivation of the Ras signaling axis and provide a basis for the GM-CSF signaling pathway as a target for rational drug design in JMML.