The Scaffolding Adapter Gab2, via Shp-2, Regulates Kit-evoked Mast Cell Proliferation by Activating the Rac/JNK Pathway

Yu, Min; Luo, Jincai; Yang, Wentian; Wang, Yongping; Mizuki, Masao; Kanakura, Yuzuru; Besmer, Peter; Neel, Benjamin G.; Gu, Haihua

doi:10.1074/jbc.m603742200

Cited by 82 publications

(96 citation statements)

References 57 publications

(74 reference statements)

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“…In NIH3T3 cells (which have low levels of GAB2), both isoforms activate PI3K/Akt signalling but the GNNK À variant also strongly activates MAPK signalling in a Src-dependent manner (Caruana et al, 1999;Voytyuk et al, 2003;Young et al, 2007). However, in haematopoietic cells, PI3K activity is at least partly dependent on Src-family kinase mediated phosphorylation of GAB2 and this can lead to isoform-specific c-Kit activation of PI3K signalling (Yu et al, 2006;Sun et al, 2008). In NIH3T3 cells both isoforms promote resistance to anoikis, but GNNK À also strongly promotes loss of contact inhibition and increases tumourigenicity (Caruana et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

et al. 2011

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BRCA1 mutation-associated breast cancer originates in oestrogen receptor-alpha-negative (ER À ) progenitors in the mammary luminal epithelium. These cells also express high levels of the Kit gene and a recent study demonstrated a correlation between Brca1 loss and Kit over-expression in the mammary epithelium. However, the functional significance of c-Kit expression in the mammary gland is unknown. To address this, c-Kit À and c-Kit þ mammary epithelial subsets were isolated by flow cytometry, characterised for expression of lineage-specific cell markers and functionally analysed by in vitro colony forming and in vivo transplantation assays. The results confirm that the majority of luminal ER À progenitors are c-Kit þ , but also that most stem cells and the differentiated cell populations are c-Kit À . A subset of c-Kit þ cells with high proliferative potential was found in the luminal ER þ population, however, suggesting the existence of a distinct luminal ER þ progenitor cell type. Analysis of mouse Brca1 mammary tumours demonstrated that they expressed Kit and its downstream effector Lyn at levels comparable to the most strongly c-Kit þ luminal ER À progenitors. Consistent with c-Kit being a progenitor cell marker, in vitro threedimensional differentiation of c-Kit þ cells resulted in a loss of c-Kit expression, whereas c-Kit over-expression prevented normal differentiation in vivo. Furthermore, c-Kit was a functional marker of proliferative potential, as c-Kit inhibition by short hairpin knockdown prevented normal epithelial growth and caused cells to undergo apoptosis. Therefore, c-Kit defines distinct progenitor populations in the mammary epithelium and is critical for mammary progenitor survival and proliferation. Importantly, c-Kit is only the second mammary epithelial stem/progenitor marker to be shown to have a functional role in the mammary epithelium and the first marker to be shown to be required for progenitor cell function. The c-Kit signalling network has potential as a target for therapy and/or prevention in BRCA1-associated breast cancer.

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Section: Discussionmentioning

confidence: 99%

c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

et al. 2011

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“…GAB2 is also known to be tyrosine phosphorylated by oncoproteins including SRC and BCR-ABL (44). In contrast, SHP2 has been shown to regulate PI3K and c-Kit activity (39,45) and is essential for maintaining ERK signaling (46).…”

Section: Analysis Of Activating Signaling Pathways Using a Multifacetedmentioning

confidence: 99%

Detection of a rare BCR–ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS)

Breitkopf

Yuan

Pihán

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.targeted therapies | personalized medicine | protein-protein interactions P roteomics studies over the last decade have provided significant advancements in our understanding of the functional landscape of proteins, their posttranslational modifications and the protein-protein interactions (PPI) (1-3). This is especially important for diseases such as cancer because proteins and their transient posttranslational modifications (PTMs) reflect the changes cells undergo during pathogenic development. Modern high-resolution mass spectrometers are well suited for these analyses and have led the efforts in this field of proteomics, allowing researchers to analyze dynamic changes in cell lines or tissue and to distinguish between normal and unhealthy states (4, 5). PPI networks and phosphoproteomics are important for understanding the function and regulation of pathways leading to cell growth and proliferation in diseases such as cancer. The advantage of analyzing purified protein complexes is the ability to identify specific interacting proteins and posttranslational modifications that may otherwise go undetected in large-scale global analyses. This can be achieved by performing an immunoprecipitation (IP) with a bait protein and analyzing the prey proteins using microcapillary-tandem mass spectrometry (LC-MS/MS) (6-1...

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“…Previous analyses have identified signaling molecules that can bind to Gab2 on receptor activation, including the tyrosine phosphatase Ptpn11/Shp2, leading to the activation of Erk and Jnk (Yu et al, 2006), the p85 subunit of PI3K, leading to Akt activation (Bouscary et al, 2001), and Src family kinases (Kong et al, 2003). Therefore, to dissect the signaling pathways downstream of Gab2 that could mediate anchorage-independent growth, we examined the effects on cell vitality of a panel of small molecule inhibitors targeting the abovementioned signaling kinases ( Figure 4a).…”

Section: Gab2-driven Anchorage Independence Requires Src and Involvesmentioning

confidence: 99%

The GAB2 signaling scaffold promotes anchorage independence and drives a transcriptional response associated with metastatic progression of breast cancer

et al. 2009

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Acquisition of independence from anchorage to the extracellular matrix is a critical event for onset and progression of solid cancers. To identify and characterize new genes conferring anchorage independence, we transduced MCF10A human normal breast cells with a retroviral cDNA expression library and selected them by growth in suspension. Microarray analysis targeted on library-derived transcripts revealed robust and reproducible enrichment, after selection, of cDNAs encoding the scaffolding adaptor Gab2. Gab2 was confirmed to strongly promote anchorageindependent growth when overexpressed. Interestingly, downregulation by RNA interference of endogenous Gab2 in neoplastic cells did not affect their adherent growth, but abrogated their growth in soft agar. Gab2-driven anchorage independence was found to specifically involve activation of the Src-Stat3 signaling axis. A transcriptional 'signature' of 205 genes was obtained from GAB2-transduced, anchorageindependent MCF10A cells, and found to contain two main functional modules, controlling proliferation and cell adhesion/migration/invasion, respectively. Extensive validation on breast cancer data sets showed that the GAB2 signature provides a robust prognostic classifier for breast cancer metastatic relapse, largely independent from existing clinical and genomic indicators and from estrogen receptor status. This work highlights a pivotal role for GAB2 and its transcriptional targets in anchorage-independent growth and breast cancer metastatic progression.

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The Scaffolding Adapter Gab2, via Shp-2, Regulates Kit-evoked Mast Cell Proliferation by Activating the Rac/JNK Pathway

Cited by 82 publications

References 57 publications

c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

Detection of a rare BCR–ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS)

The GAB2 signaling scaffold promotes anchorage independence and drives a transcriptional response associated with metastatic progression of breast cancer

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