Faculty Bibliography

Interleukin-6 (IL-6) activates the Jak/STAT pathway as well as the mitogen-activated protein kinase cascade. Tyrosine 759 of the IL-6 signal-transducing receptor subunit gp130 has been identified as being involved in negative regulation of IL-6-induced gene induction and activation of the Jak/STAT pathway. Because this site is known to be a recruitment motif for the protein-tyrosine phosphatase SHP2, it has been suggested that SHP2 is the mediator of tyrosine 759-dependent signal attenuation. We recently observed that the suppressor of cytokine-signaling SOCS3 also acts through the tyrosine motif 759 of gp130. However, the relative contributions of SHP2 and SOCS3 to the repression of IL-6 signaling are not understood. Therefore, we designed experiments allowing the independent recruitment of each of these proteins to the IL-6-receptor complex. We show that receptor- and membrane-targeted SHP2 counteracts IL-6 signaling independent of SOCS3 binding to gp130. On the other hand, SOCS3 inhibits signaling in cells expressing a truncated SHP2 protein, which is not recruited to gp130. These data suggest, that there are two, largely distinct modes of negative regulation of gp130 activity, despite the fact that both SOCS3 and SHP2 are recruited to the same site within gp130.

Increased expression of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. Although the biology of this receptor has been the subject of intense investigation, surprisingly little is known about how increased expression of the wild-type EGFR affects downstream signal transduction in cells. We show that increasing the expression of the receptor results in dramatic shifts in signaling with attenuation of EGF-induced Ras, extracellular signal-related kinases (ERKs), and Akt activation, as well as amplification of STAT1 and STAT3 signaling. In this study, we focus on the mechanism of attenuated ERK signaling and present evidence suggesting that the mechanism of attenuated ERK signaling in EGFR-overexpressing cells is a sequestration of ERKs at the cell membrane in EGFR-containing complexes. Increased expression of the EGFR results in an aberrant localization of ERKs to the cell membrane. Furthermore, ERKs become associated with the EGFR in a physical complex in EGFR-overexpressing cells but not in control cells. The EGFR-ERK association is detected in unstimulated cells or on exposure to a low concentration of EGF; under these conditions, ERK activation is minimal. Exposure of these cells to saturating concentrations of EGF results in a decreased membrane localization of ERKs, a concomitant dissociation of ERKs from the EGFR, and restores ERK activation. A similar association can be detected between the EGFR and MEK1 in receptor-overexpressing cells, suggesting that multiple components of the ERK signaling pathway may become trapped in complexes with the EGFR. These findings can be demonstrated in cells transfected to express high levels of the EGFR as well as in cancer cells which naturally overexpress the EGFR and, thus, may be representative of altered EGFR signaling in human cancer.

We previously reported that RSV-transformed quail neuroretina cells (QNR-ts68) were highly resistant to apoptosis provoked by serum withdrawal, and that this property was due to v-Src kinase activity. The present study investigates the cytotoxic effect and the functional mechanism of carbazolequinone-mediated cell death in this system. QNR-ts68 cells were subjected to carbazolequinone treatment and both growth inhibition and cell death induction were examined using formazan assays. Cell death mechanism (both apoptosis and necrosis) was confirmed through phosphatidyl serine exposure and propidium iodide incorporation. Furthermore, the effect of active carbazolequinone was inhibited by a pan caspase inhibitor. Cytofluorimetric and immunofluorescence data demonstrated the activation of caspase-3 and the involvement of mitochondria. Therefore, this study clearly indicates that carbazolequinones could induce cell death in transformed cells displaying high levels of antiapoptotic tyrosine kinase activity. Further investigations would be necessary to elucidate the mechanisms by which these carbazolequinones act as antitumor agents.

The PTEN tumor suppressor gene encodes a phosphatidylinositol 3'-phosphatase that is inactivated in a high percentage of human tumors, particularly glioblastoma, melanoma, and prostate and endometrial carcinoma. Previous studies showed that PTEN is a seryl phosphoprotein and a substrate of protein kinase CK2 (CK2). However, the sites in PTEN that are phosphorylated in vivo have not been identified directly, nor has the effect of phosphorylation on PTEN catalytic activity been reported. We used mass spectrometric methods to identify Ser(370) and Ser(385) as in vivo phosphorylation sites of PTEN. These sites also are phosphorylated by CK2 in vitro, and phosphorylation inhibits PTEN activity towards its substrate, PIP3. We also identify a novel in vivo phosphorylation site, Thr(366). Following transient over-expression, a fraction of CK2 and PTEN co-immunoprecipitate. Moreover, pharmacological inhibition of CK2 activity leads to decreased Akt activation in PTEN+/+ but not PTEN-/- fibroblasts. Our results contrast with previous assignments of PTEN phosphorylation sites based solely on mutagenesis approaches, suggest that CK2 is a physiologically relevant PTEN kinase, and raise the possibility that CK2-mediated inhibition of PTEN plays a role in oncogenesis.

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.

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and a CML-like disease, as well as lymphoid leukemia, in mice. p210 BCR/ABL is an activated tyrosine kinase that phosphorylates itself and several cellular signaling proteins. The autophosphorylation site tyrosine 177 binds the adaptor Grb2 and helps determine the lineage and severity of BCR/ABL disease: Tyr177 mutation (BCR/ABL-Y177F) dramatically impairs myeloid leukemogenesis, while diminishing lymphoid leukemogenesis. The critical signal(s) from Tyr177 has remained unclear. We report that Tyr177 recruits the scaffolding adaptor Gab2 via a Grb2/Gab2 complex. Compared to BCR/ABL-expressing Ba/F3 cells, BCR/ABL-Y177F cells exhibit markedly reduced Gab2 tyrosine phosphorylation and association of phosphatidylinositol-3 kinase (PI3K) and Shp2 with Gab2 and BCR/ABL, and decreased PI3K/Akt and Ras/Erk activation, cell proliferation, and spontaneous migration. Remarkably, bone marrow myeloid progenitors from Gab2 (-/-) mice are resistant to transformation by BCR/ABL, whereas lymphoid transformation is diminished as a consequence of markedly increased apoptosis. BCR/ABL-evoked PI3K/Akt and Ras/Erk activation also are impaired in Gab2 (-/-) primary myeloid and lymphoid cells. Our results identify Gab2 and its associated proteins as key determinants of the lineage and severity of BCR/ABL transformation.

Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. We have found that the protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3'-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1 compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. Our results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner.

CD22, a negative regulator of B cell antigen receptor signaling, binds glycoconjugates terminating in alpha2, 6 sialic acid. The physiological ligand(s) for CD22 remain unknown. We asked whether the sialic acid binding domains are necessary for CD22 to function as a negative regulator. We generated two mutants that lack sialic acid binding activity and expressed them in a novel CD22(-/-) murine B cell line. Anti-IgM activated B cells expressing either CD22 mutant had greater Ca(2+) responses than cells expressing wild-type CD22. Each variant also had reduced CD22 tyrosine phosphorylation and Src homology 2 domain-containing protein tyrosine phosphatase-1 association. These data suggest that the alpha2, 6 sialic acid ligand binding activity of CD22 is critical for its negative regulatory functions.

We have developed a mathematical theory that describes the regulation of signaling pathways as a function of a limited number of key parameters. Our analysis includes linear kinase-phosphatase cascades, as well as systems containing feedback interactions, crosstalk with other signaling pathways, and/or scaffolding and G proteins. We find that phosphatases have a more pronounced effect than kinases on the rate and duration of signaling, whereas signal amplitude is controlled primarily by kinases. The simplest model pathways allow amplified signaling only at the expense of slow signal propagation. More complex and realistic pathways can combine high amplification and signaling rates with maintenance of a stable off-state. Our models also explain how different agonists can evoke transient or sustained signaling of the same pathway and provide a rationale for signaling pathway design.

Mice lacking the protein-tyrosine phosphatase PTP1B are hypersensitive to insulin and resistant to obesity. However, the molecular basis for resistance to obesity has been unclear. Here we show that PTP1B regulates leptin signaling. In transfection studies, PTP1B dephosphorylates the leptin receptor-associated kinase, Jak2. PTP1B is expressed in hypothalamic regions harboring leptin-responsive neurons. Compared to wild-type littermates, PTP1B(-/-) mice have decreased leptin/body fat ratios, leptin hypersensitivity, and enhanced leptin-induced hypothalamic Stat3 tyrosyl phosphorylation. Gold thioglucose treatment, which ablates leptin-responsive hypothalamic neurons, partially overcomes resistance to obesity in PTP1B(-/-) mice. Our data indicate that PTP1B regulates leptin signaling in vivo, likely by targeting Jak2. PTP1B may be a novel target to treat leptin resistance in obesity.