Tyrosine Kinase Adaptor Protein Research Articles

Abstract 1007: The Redox/Fyn/c-Cbl pathway and its interaction with Cool-1: A novel pathway that regulates chemosensitivity in glioblastoma

Abstract Glioblastomas (GBMs) are the most common primary brain tumor, accounting for 52 percent of all diagnosed brain tumors. They are also the most devastating of all brain tumors, with a five-year survival rate of 3.3 percent. One of the reasons for this poor prognosis is resistance to treatment with chemotherapy. We have discovered a novel means by which GBMs become chemoresistant, based on inhibition of the redox/Fyn/c-Cbl pathway by overexpression of Cool-1. The protein c-Cbl, a receptor tyrosine kinase adaptor protein and E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases critical for cell division and cell survival. c-Cbl is activated by Fyn kinase, which itself can be activated by pro-oxidants. In normal glial progenitor cells of the CNS, exposure to chemotherapy oxidizes cells, leading to sequential activation of Fyn and c-Cbl. With GBM cells, in contrast, exposure to BCNU or to chemical pro-oxidants does not lead to c-Cbl activation. Further studies have demonstrated that these agents do cause Fyn activation, suggesting that GBMs inhibit Fyn-mediated c-Cbl phosphorylation. This would lead to decreased degradation of EGFR, thus enabling continued cell proliferation and providing resistance to chemotherapy. We therefore tested the hypothesis that reversal of this inhibition would decrease cell division and enhance susceptibility to chemotherapy-induced cell death. In this work we discovered that oxidant-associated c-Cbl activation can be restored by suppressing expression of the protein Cool-1, which is over-expressed in many cancers including GBM. Our data further demonstrate that Cool-1 and c-Cbl interact, that genetic reduction in Cool-1 levels with small inhibitory RNAs restores normal c-Cbl-mediated degradation of RTKs (including EGFR) in response to exposure to pro-oxidants and BCNU, and that restoration of normal c-Cbl activation also increases sensitivity to this and other chemotherapeutic agents. The translation of these findings into an in vivo xenograft model shows that reduction in Cool-1 levels leads to decreased tumor take, decreased tumor growth, and increased survival. Specific pharmacological inhibitors of Cool-1 are not available and it was therefore necessary to explore other means of chemically inhibiting Cool-1 activation. We have data suggesting that pharmacological inhibition of Focal Adhesion Kinase leads to decreases in Cool-1 activation and a rescue of oxidant-induced c-Cbl activation and subsequent RTK degradation. Together these results support c-Cbl and Cool-1's roles in tumor development and increased resistance to chemotherapeutics. Rescuing the activation of c-Cbl mediated RTK degradation through Cool-1 inhibition is a novel method of treating GBMs that increases both sensitivity to chemotherapeutics and animal survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1007. doi:10.1158/1538-7445.AM2011-1007

SKAP2, a novel target of HSF4b, associates with NCK2/F-actin at membrane ruffles and regulates actin reorganization in lens cell

In addition to roles in stress response, heat shock factors (HSFs) play crucial roles in differentiation and development. Heat shock transcription factor 4 (HSF4) deficiency leads to defect in lens epithelial cell (LEC) differentiation and cataract formation. However, the mechanism remains obscure. Here, we identified Src kinase-associated phosphoprotein 2 (SKAP2) as a downstream target of HSF4b and it was highly expressed at the anterior tip of lens elongating fibre cells in vivo. The HSF4-deficient lenses showed reduced SKAP2 expression and defects in actin reorganization. The disassembly of stress fibres and formation of cortical actin fibres are critical for the initiation of LEC differentiation. SKAP2 localized at actin-rich ruffles in human LECs (SRA01/04 cells) and knockdown SKAP2 using RNA interference impaired the disassembly of cellular stress fibres in response to fibroblast growth factor (FGF)-b. Overexpression of SKAP2, but not the N-terminal deletion mutant of SKAP2, induced the actin remodelling. We further found that SKAP2 interacted with the SH2 domain of non-catalytic region of tyrosine kinase adaptor protein 2 (NCK2) via its N-terminus. The complex of SKAP2-NCK2-F-actin accumulated at the leading edge of the lamellipodium, where FGF receptors and focal adhesion were also recruited. These results revealed an essential role for HSF4-mediated SKAP2 expression in the regulation of actin reorganization during lens differentiation, likely through a mechanism that SKAP2 anchors the complex of NCK2/focal adhesion to FGF receptors at the lamellipodium in lens epithelial cells.

Abstract A159: The Redox/ Fyn/c-Cbl pathway and its interaction with Cool-1: A novel pathway that regulates chemosensitivity in glioblastoma

Abstract GBMs are the most common primary brain tumor, accounting for 52 percent of all diagnosed brain tumors. It is also the most devastating of all brain tumors, with a five-year survival rate of 3.3 percent. One of the reasons for this poor prognosis is resistance of tumor cells to treatment with chemotherapy. We have discovered a novel means by which GBMs become chemoresistant, based on inhibition of the redox/Fyn/c-Cbl pathway by overexpression of Cool-1. The protein c-Cbl, a receptor tyrosine kinase adaptor protein, is responsible for the ubiquitination and degradation of epidermal growth factor receptor (EGFR) and other receptor protein kinases critical in cell division and cell survival. c-Cbl is activated by Fyn kinase, which itself can be activated by agents that make cells more oxidized. In normal glia and glial progenitor cells of the CNS, exposure to chemotherapy makes cells more oxidized, leading to sequential activation of Fyn and c-Cbl. In GBMs, in contrast, exposure of GBM cells to BCNU or to chemical pro-oxidants does not lead to c-Cbl activation. Further studies demonstrated that these agents did cause Fyn activation, suggesting that GBMs inhibit Fyn-mediated c-Cbl activation. This would lead to decreased degradation of EGFR, thus enabling continued cell division and providing resistance to chemotherapy. We therefore set out to test the hypothesis that reversal of this inhibition would decrease cell division and enhance susceptibility to chemotherapy-induced cell death. In this work we discovered that oxidant-associated c-Cbl activation can be restored by suppressing expression of the protein Cool-1, which is over-expressed in many cancers including GBM. Our data further demonstrates that Cool-1 and Cbl interact, that genetic reduction in Cool-1 levels by expression of small inhibitory RNAs restores normal c-Cbl-mediated degradation of RTKs (including EGFR) in response to exposure to pro-oxidants and BCNU, and that restoration of normal c-Cbl activation also increases sensitivity to this and other chemotherapeutic agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A159.

Phospholipase Cγ2 Is Critical for Dectin-1-mediated Ca2+ Flux and Cytokine Production in Dendritic Cells

Dectin-1 is a C-type lectin that recognizes beta-glucan in the cell walls of fungi and plays an important role in anti-fungal immunity. It signals via tyrosine kinase Syk and adaptor protein Card9 to activate NF-kappaB leading to proinflammatory cytokine production in dendritic cells (DCs). Other than this, not much else is known of the mechanism of Dectin-1 signaling. We demonstrate here that stimulation of DCs with zymosan triggers an intracellular Ca2+ flux that can be attenuated by a blocking anti-Dectin-1 antibody or by pre-treatment of cells with the phospholipase C (PLC) gamma-inhibitor U73122, suggesting that Dectin-1 signals via a PLCgamma pathway to induce Ca2+ flux in DCs. Interestingly, treatment of DCs with particulate curdlan, which specifically engages Dectin-1, results in the phosphorylation of both PLCgamma1 and PLCgamma2. However, we show that PLCgamma2 is the critical enzyme for Dectin-1 signaling in DCs. PLCgamma2-deficient DCs have drastic impairment of Ca2+ signaling and are defective in their secretion of interleukin 2 (IL-2), IL-6, IL-10, IL-12, IL-23, and tumor necrosis factor alpha. PLCgamma2-deficient DCs also exhibit impaired activation of ERK and JNK MAPKs and AP-1 and NFAT transcription factors in response to Dectin-1 stimulation. In addition, PLCgamma2-deficient DCs are also impaired in their activation of NF-kappaB upon Dectin-1 engagement due to defective assembly of the Card9-Bcl10-Malt1 complex and impaired IKKalpha/beta activation and IkappaBalpha degradation. Thus, our data indicate that pattern recognition receptors such as Dectin-1 could elicit Ca2+ signaling and that PLCgamma2 is a critical player in the Dectin-1 signal transduction pathway.

APS-mediated Ubiquitination of the Insulin Receptor Enhances its Internalization, but does not Induce its Degradation

APS, a tyrosine kinase adaptor protein with pleckstrin homology and Src homology 2 domains, is rapidly and strongly tyrosine-phosphorylated by insulin receptor kinase upon insulin stimulation. We have previously shown that APS knockout mice have increased insulin sensitivity, and that this enhancement is possibly due to increased insulin-response on adipose tissues. However, the function of APS in insulin signaling has so far been controversial. Here, we report that APS enhanced ligand-dependent multi-ubiquitination of the insulin receptor (IR) in CHO cells overexpressing the IR. APS-mediated ubiquitination of the IR induced enhancement of the IR internalization, but did not affect the IR degradation. This finding shows one of the pleiotropic functions of APS in insulin signaling.

Rapid WAVE dynamics in dendritic spines of cultured hippocampal neurons is mediated by actin polymerization

The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE) complex has been proposed to link Rho GTPase activity with actin polymerization but its role in neuronal plasticity has never been documented. We now examined the presence, distribution and dynamics of WAVE3 in cultured hippocampal neurons. WAVE3 was localized to dendritic spines via its N-terminal domain. Green fluorescent protein (GFP)-tagged WAVE3 clusters demonstrate an F-actin-dependent high rate of local motility. Constitutive Rac activation translocates WAVE3 (via the N-terminus), to the leading edge of lamellipodia. Also, spinogenesis is associated with actin-based motility of the WAVE3 protein. Brain specific WAVE1 showed similar localization and effects on spine density. Cytoplasmic fragile X mental retardation protein interacting protein (CYFIP) and non-catalytic region of tyrosine kinase adaptor protein 1 (NCK-1), proteins that are assumed to complex with WAVE, have a somewhat similar cellular distribution and motility. We propose that the WAVE complex is a downstream effector of the Rac signaling cascade, localized to sites of novel synaptic contacts by means of its N-terminal domain, to guide local actin polymerization needed for morphological plasticity of neurons.

Increased insulin sensitivity and hypoinsulinemia in APS knockout mice.

A tyrosine kinase adaptor protein containing pleckstrin homology and SH2 domains (APS) is rapidly and strongly tyrosine phosphorylated by insulin receptor kinase upon insulin stimulation. The function of APS in insulin signaling has heretofore remained unknown. APS-deficient (APS(-/-)) mice were used to investigate its function in vivo. The blood glucose-lowering effect of insulin, as assessed by the intraperitoneal insulin tolerance test, was increased in APS(-/-) mice. Plasma insulin levels during fasting and in the intraperitoneal glucose tolerance test were lower in APS(-/-) mice. APS(-/-) mice showed an increase in the whole-body glucose infusion rate as assessed by the hyperinsulinemic-euglycemic clamp test. These findings indicated that APS(-/-) mice exhibited increased sensitivity to insulin. However, overexpression of wild-type or dominant-negative APS in 3T3L1 adipocytes did not affect insulin receptor numbers, phosphorylations of insulin receptor, insulin receptor substrate-1, or Akt and mitogen-activated protein kinase. The glucose uptake and GLUT4 translocation were not affected by insulin stimulation in these cells. Nevertheless, the insulin-stimulated glucose transport in isolated adipocytes of APS(-/-) mice was increased over that of APS(+/+) mice. APS(-/-) mice also showed increased serum levels of leptin and adiponectin, which might explain the increased insulin sensitivity of adipocytes.