Article

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: 7.65). 07/2011; 4(166):ra18. DOI: 10.1126/scisignal.2001314
Source: PubMed

ABSTRACT 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.

Download full-text

Full-text

Available from: Matteo Pellegrini, Jan 12, 2014
0 Followers
 · 
107 Views
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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 · 4.82 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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 · 2.06 Impact Factor
  • Source
    • "In particular, tyrosine kinases account for 0.3% of the genome yet contribute to a disproportionately large percentage (30%) of the known 100 dominant oncogenes [14]. In particular, the tyrosine kinase Bcr-Abl is deregulated in leukemia, HER-2 is amplified in breast cancer, and EGFR, MET, and PDGFR are over-expression and mutated across a variety of cancers [15] [16] [17]. Thus, despite the relative low levels of tyrosine phosphorylation , it is clear that tyrosine kinases are essential in mediating cellular communication and are critically important in the regulation of cell growth and oncogenic transformation [1] [2] [4] [18]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tyrosine phosphorylation is a dynamic reversible post-translational modification that regulates many aspects of cell biology. To understand how this modification controls biological function, it is necessary to not only identify the specific sites of phosphorylation, but also to quantify how phosphorylation levels on these sites may be altered under specific physiological conditions. Due to its sensitivity and accuracy, mass spectrometry (MS) has widely been applied to the identification and characterization of phosphotyrosine signaling across biological systems. In this review we highlight the advances in both MS and phosphotyrosine enrichment methods that have been developed to enable the identification of low level tyrosine phosphorylation events. Computational and manual approaches to ensure confident identification of phosphopeptide sequence and determination of phosphorylation site localization are discussed along with methods that have been applied to the relative quantification of large numbers of phosphorylation sites. Finally, we provide an overview of the challenges ahead as we extend these technologies to the characterization of tyrosine phosphorylation signaling in vivo. With these latest developments in analytical and computational techniques, it is now possible to derive biological insight from quantitative MS-based analysis of signaling networks in vitro and in vivo. Application of these approaches to a wide variety of biological systems will define how signal transduction regulates cellular physiology in health and disease.
    Seminars in Cell and Developmental Biology 06/2012; 23(8). DOI:10.1016/j.semcdb.2012.05.008 · 5.97 Impact Factor
Show more