Global Phosphoproteomics Reveals Crosstalk Between Bcr-Abl and Negative Feedback Mechanisms Controlling Src Signaling

Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Science Signaling (Impact Factor: 6.28). 07/2011; 4(166):ra18. DOI: 10.1126/scisignal.2001314
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In subtypes and late stages of leukemias driven by the tyrosine kinase fusion protein Bcr-Abl, signaling by the Src family kinases (SFKs) critically contributes to the leukemic phenotype. We performed global tyrosine phosphoprofiling by quantitative mass spectrometry of Bcr-Abl-transformed cells in which the activities of the SFKs were perturbed to build a detailed context-dependent network of cancer signaling. Perturbation of the SFKs Lyn and Hck with genetics or inhibitors revealed Bcr-Abl downstream phosphorylation events either mediated by or independent of SFKs. We identified multiple negative feedback mechanisms within the network of signaling events affected by Bcr-Abl and SFKs and found that Bcr-Abl attenuated these inhibitory mechanisms. The C-terminal Src kinase (Csk)-binding protein Pag1 (also known as Cbp) and the tyrosine phosphatase Ptpn18 both mediated negative feedback to SFKs. We observed Bcr-Abl-mediated phosphorylation of the phosphatase Shp2 (Ptpn11), and this may contribute to the suppression of these negative feedback mechanisms to promote Bcr-Abl-activated SFK signaling. Csk and a kinase-deficient Csk mutant both produced similar globally repressive signaling consequences, suggesting a critical role for the adaptor protein function of Csk in its inhibition of Bcr-Abl and SFK signaling. The identified Bcr-Abl-activated SFK regulatory mechanisms are candidates for dysregulation during leukemia progression and acquisition of SFK-mediated drug resistance.

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Available from: Matteo Pellegrini, Jan 12, 2014
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    • "PTPN18 is an interesting candidate for several reasons. First, it is involved in the negative feedback mechanisms controlling the Bcr-Abl fusion tyrosine kinase-signalling network by inhibiting the phosphorylation of Src family kinase (Rubbi et al, 2011). Second, PTPN18 can induce actin cytoskeleton reorganisation (Shiota et al, 2003), suggesting a plausible explanation of the morphological changes observed in the imatinib-resistant GIST cells (Mahadevan et al, 2007). "
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    ABSTRACT: Background: Gastrointestinal stromal tumour (GIST) is mainly initialised by receptor tyrosine kinase gene mutations. Although the tyrosine kinase inhibitor imatinib mesylate considerably improved the outcome of patients, imatinib resistance still remains a major therapeutic challenge in GIST therapy. Herein we evaluated the clinical impact of microRNAs in imatinib-treated GISTs. Methods: The expression levels of microRNAs were quantified using microarray and RT–qPCR in GIST specimens from patients treated with neoadjuvant imatinib. The functional roles of miR-125a-5p and PTPN18 were evaluated in GIST cells. PTPN18 expression was quantified by western blotting in GIST samples. Results: We showed that overexpression levels of miR-125a-5p and miR-107 were associated with imatinib resistance in GIST specimens. Functionally, miR-125a-5p expression modulated imatinib sensitivity in GIST882 cells with a homozygous KIT mutation but not in GIST48 cells with double KIT mutations. Overexpression of miR-125a-5p suppressed PTPN18 expression, and silencing of PTPN18 expression increased cell viability in GIST882 cells upon imatinib treatment. PTPN18 protein levels were significantly lower in the imatinib-resistant GISTs and inversely correlated with miR-125a-5p. Furthermore, several microRNAs were significantly associated with metastasis, KIT mutational status and survival. Conclusions: Our findings highlight a novel functional role of miR-125a-5p on imatinib response through PTPN18 regulation in GIST.
    British Journal of Cancer 10/2014; 111(11). DOI:10.1038/bjc.2014.548 · 4.84 Impact Factor
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    • "Metformin increases amyloid-␤ production and secretion by AMPK activation and increased BACE1 level, suggesting that it is potentially accelerating Alzheimer's manifestation in patients with T2D [12]. Phosphoproteomic analysis is particularly useful in recognizing the alteration of signaling pathways by a drug [13] [14]. Herein, we determined the alteration of phosphoproteome in the brain of metformin-administrated mice to understand the action of metformin in the CNS. "
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    ABSTRACT: Metformin, a potent antihyperglycemic agent is recommended as the first-line oral therapy for type 2 diabetes (T2D). Recently, metformin has been reported to be beneficial to neurodegenerative disease models. However, the putative mechanisms underlying the neuroprotective effects of metformin in disease models are unknown. Thus, we applied LC-MS/MS-based pattern analysis and two-dimensional electrophoresis (2DE)-based proteomic approach to understand the global phosphoproteomic alteration in the brain of metformin-administrated mice. Collectively, LC-MS/MS-based pattern analysis reveals that 41 phosphoproteins were upregulated and 22 phosphoproteins were downregulated in the brain of metformin-administrated mice. In addition, 5 differentially expressed phosphoproteins were identified upon metformin administration by 2DE coupled with mass spectrometry. The phosphorylation status of metabolic enzymes was decreased while that of mitochondrial proteins was increased by metformin. Interestingly, phosphorylated α-synuclein was significantly decreased by metformin administration. Taken together, our results might provide potential pathways to understand the pharmacological effect of metformin on neuroprotection.
    Neuroscience Letters 07/2014; 579. DOI:10.1016/j.neulet.2014.07.029 · 2.03 Impact Factor
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    • "The protein Cbp/PAG1 [15,17,18] is a scaffold molecule involved in recruiting both the inhibitory kinase Csk, which inturn also recruits the inhibitory phosphatase PTPN18 [93], as well as E3 ubiquitin ligase SOCS1 to active Lyn, via facilitating the enzymatic inactivation of Lyn (through Csk phosphorylation of the C-terminal tyrosine of Lyn, and PTPN18 dephosphorylation of the activation loop motif) as well as degradation of Lyn via the proteasome through its poly-ubiquitination mediated by SOCS1. In these CML cells the Bcr-Abl kinase overpowers the negative feedback loops initiated by its activation of Lyn through activation of the phosphatase Shp2 that is able to dephosphorylate Cbp/PAG1 thus mitigating its ability to turn off the Lyn signals [92]. It is also very interesting to note that the second generation (T315I non-effective, e.g. "
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    ABSTRACT: Src family kinases such as Lyn are important signaling intermediaries, relaying and modulating different inputs to regulate various outputs, such as proliferation, differentiation, apoptosis, migration and metabolism. Intriguingly, Lyn can mediate both positive and negative signaling processes within the same or different cellular contexts. This duality is exemplified by the B-cell defect in Lyn−/− mice in which Lyn is essential for negative regulation of the B-cell receptor; conversely, B-cells expressing a dominant active mutant of Lyn (Lynup/up) have elevated activities of positive regulators of the B-cell receptor due to this hyperactive kinase. Lyn has well-established functions in most haematopoietic cells, viz. progenitors via influencing c-kit signaling, through to mature cell receptor/integrin signaling, e.g. erythrocytes, platelets, mast cells and macrophages. Consequently, there is an important role for this kinase in regulating hematopoietic abnormalities. Lyn is an important regulator of autoimmune diseases such as asthma and psoriasis, due to its profound ability to influence immune cell signaling. Lyn has also been found to be important for maintaining the leukemic phenotype of many different liquid cancers including acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML) and B-cell lymphocytic leukaemia (BCLL). Lyn is also expressed in some solid tumors and here too it is establishing itself as a potential therapeutic target for prostate, glioblastoma, colon and more aggressive subtypes of breast cancer. Lay Abstract To relay information, a cell uses enzymes that put molecular markers on specific proteins so they interact with other proteins or move to specific parts of the cell to have particular functions. A protein called Lyn is one of these enzymes that regulate information transfer within cells to modulate cell growth, survival and movement. Depending on which type of cell and the source of the information input, Lyn can positively or negatively regulate the information output. This ability of Lyn to be able to both turn on and turn off the relay of information inside cells makes it difficult to fully understand its precise function in each specific circumstance. Lyn has important functions for cells involved in blood development, including different while blood cells as well as red blood cells, and in particular for the immune cells that produce antibodies (B-cells), as exemplified by the major B-cell abnormalities that mice with mutations in the Lyn gene display. Certain types of leukaemia and lymphoma appear to have too much Lyn activity that in part causes the characteristics of these diseases, suggesting it may be a good target to develop new anti-leukaemia drugs. Furthermore, some specific types, and even specific subtypes, of solid cancers, e.g. prostate, brain and breast cancer can also have abnormal regulation of Lyn. Consequently, targeting this protein in these cancers could also prove to be beneficial.
    Cell Communication and Signaling 07/2012; 10(1):21. DOI:10.1186/1478-811X-10-21 · 3.38 Impact Factor
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