A role of Gab2 association in Flt3 ITD mediated Stat5 phosphorylation and cell survival

Experimental Clinical Chemistry, Department of Laboratory Medicine, Malmö University Hospital, Lund University, Malmö SE-20502, Sweden.
British Journal of Haematology (Impact Factor: 4.71). 06/2009; 146(2):193-202. DOI: 10.1111/j.1365-2141.2009.07725.x
Source: PubMed


The haematopoietic growth factor receptor Flt3 has been implicated as major cause of transformation in acute myeloid leukaemia. Intracellular signals mediated by wild-type Flt3 are involved in cell differentiation and survival whereas signalling via the mutant Flt3 ITD (internal tandem duplication) promotes enhanced cell growth. In this study, we identified tyrosines 768, 955 and 969 of Flt3 as phosphorylation sites and mediators of growth factor receptor binding protein 2 (Grb2) interaction, leading to the association of Grb2 associated binder 2 (Gab2) and contributing to proliferation and survival. Ba/F3 cells were transfected with either the wild-type Flt3 or the ITD, with or without a triple mutation of the Grb2 binding sites, and characterised in terms of proliferation and viability. Interestingly, the Flt3 ITD promoted increased survival but after introducing the triple mutation, this phenotype was lost. When looking into different downstream pathways, this effect was mainly caused by decreased phosphoinositide 3-kinase and Stat5 signalling, and the Flt3 ITD carrying the Grb2 binding mutations showed less Akt and Stat5 activation compared to the regular Flt3 ITD receptor. These findings not only reveal novel phosphorylation sites in Flt3 but contribute to the understanding of the molecular mechanism by which Flt3 ITD functions in pathological conditions.

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    • "Detailed molecular classification of phosphoproteins (representing the phosphopeptide probes) interacting with GRB2 revealed that the majority of these are associated with receptors (GF, T cell, B cell and cytokine signaling), adaptor proteins including tyrosine phosphatases, cytoskeletal regulators, nuclear transporters and RNA binding proteins (Supplementary Table S5). Almost all of these categories had phophoprotein-GBR2 interactions leading to a ras-Erk cascade that are reported to confer serum independent growth phenotype [23], ERK activation mediated proliferation and survival [24], [25]. "
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    ABSTRACT: The architecture of cellular proteins connected to form signaling pathways in response to internal and external cues is much more complex than a group of simple protein-protein interactions. Post translational modifications on proteins (e.g., phosphorylation of serine, threonine and tyrosine residues on proteins) initiate many downstream signaling events leading to protein-protein interactions and subsequent activation of signaling cascades leading to cell proliferation, cell differentiation and cell death. As evidenced by a rapidly expanding mass spectrometry database demonstrating protein phosphorylation at specific motifs, there is currently a large gap in understanding the functional significance of phosphoproteins with respect to their specific protein connections in the signaling cascades. A comprehensive map that interconnects phospho-motifs in pathways will enable identification of nodal protein interactions that are sensitive signatures indicating a disease phenotype from the physiological hemostasis and provide clues into control of disease. Using a novel phosphopeptide microarray technology, we have mapped endogenous tyrosine-phosphoproteome interaction networks in breast cancer cells mediated by signaling adaptor protein GRB2, which transduces cellular responses downstream of several RTKs through the Ras-ERK signaling cascade. We have identified several previously reported motif specific interactions and novel interactions. The peptide microarray data indicate that various phospho-motifs on a single protein are differentially regulated in various cell types and shows global downregulation of phosphoprotein interactions specifically in cells with metastatic potential. The study has revealed novel phosphoprotein mediated signaling networks, which warrants further detailed analysis of the nodes of protein-protein interaction to uncover their biomarker or therapeutic potential.
    PLoS ONE 09/2013; 8(6):e67634. DOI:10.1371/journal.pone.0067634 · 3.23 Impact Factor
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    • "Particularly, MAPK pathway is activated by FLT3 through multiple ways: (i) interaction of GRB2/SOS to tyrosines 768, 955, and 969 induces ERK phosphorylation; (ii) the interaction of GRB2 and GAB2 to these tyrosine residues recruits SHP2 and results in ERK phosphorylation; (iii) mutation of SRC binding sites of FLT3 results in reduced FLT3-dependent ERK activation [28]. "
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    ABSTRACT: The development of the genetic studies on acute myeloid leukemias (AMLs) has led to the identification of some recurrent genetic abnormalities. Their discovery was of fundamental importance not only for a better understanding of the molecular pathogenesis of AMLs, but also for the identification of new therapeutic targets. In this context, it is essential to identify AML-associated "driver" mutations, which have a causative role in leukemogenesis. Evidences accumulated during the last years indicate that activating internal tandem duplication mutations in FLT3 (FLT3-ITD), detected in about 20% of AMLs, represents driver mutations and valid therapeutic targets in AMLs. Furthermore, the screening of FLT3-ITD mutations has also considerably helped to improve the identification of more accurate prognostic criteria and of the therapeutic selection of patients.
    07/2013; 2013:275760. DOI:10.1155/2013/275760
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    • "This further illustrates that interactions between Gab2 and partners like SHP2 is critical for development of MPD, in vivo. However, the relative contribution of the Gab2/SHP2 interaction for Erk activation versus the reciprocal inactivation of STAT5 is highly complex and difficult to discern [21, 90, 91, 100]. Both Erk and STAT5 can drive myeloproliferation, and the degree to which they cooperate in normal and leukemic hematopoiesis is not well defined. "
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    ABSTRACT: The Grb-2 associated binder (Gab) family of scaffolding/adaptor/docking proteins is a group of three molecules with significant roles in cytokine receptor signaling. Gabs possess structural motifs for phosphorylation-dependent receptor recruitment, Grb2 binding, and activation of downstream signaling pathways through p85 and SHP-2. In addition, Gabs participate in hematopoiesis and regulation of immune response which can be aberrantly activated in cancer and inflammation. The multifunctionality of Gab adapters might suggest that they would be too difficult to consider as candidates for "targeted" therapy. However, the one drug/one target approach is giving way to the concept of one drug/multiple target approach since few cancers are addicted to a single signaling molecule for survival and combination drug therapies can be problematic. In this paper, we cover recent findings on Gab multi-functionality, binding partners, and their role in hematological malignancy and examine the concept of Gab-targeted therapy.
    Advances in Hematology 01/2012; 2012:380635. DOI:10.1155/2012/380635
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