Reciprocal regulation of Abl and receptor tyrosine kinases.
ABSTRACT Previously, we showed that Abl kinases (c-Abl, Arg) are activated downstream of PDGF in a manner dependent on Src kinases and PLC-gamma1, and promote PDGF-mediated proliferation and migration of fibroblasts. We additionally demonstrated that Abl kinases bind directly to PDGFR-beta via their SH2 domains.In this study, we extend these findings by demonstrating that Abl kinases also are activated downstream of aPDGF autocrine growth loop in glioblastoma cells, indicating that the PDGFR-Abl signaling pathway also is likely to be important in glioblastoma development and/or progression.We recently showed that Abl kinases are highly active in many breast cancer cell lines, and the Her-2 receptor tyrosine kinase contributes to c-Abl and Arg kinase activation. In this study, we show that Abl kinase SH2 domains bind directly to Her-2, and like PDGFR-beta , Her-2 directly phosphorylates c-Abl. Previously, we demonstrated that PDGFR-beta directly phosphorylates Abl kinases in vitro, and Abl kinases reciprocally phosphorylate PDGFR-beta . Here, we show that PDGFR-beta-phosphorylation of Abl kinases has functional consequences as PDGFR-beta phosphorylates Abl kinases on Y245 and Y412, sites known to be required for activation of Abl kinases. Moreover, PDGFR-beta phosphorylates Arg on two additional unique sites whose function is unknown. Importantly, we also show that Abl-dependent phosphorylation of PDGFR-beta has functional and biological significances. c-Abl phosphorylates three tyrosine residues on PDGFR-beta (Y686, Y934, Y970), while Arg only phosphorylatesY686. Y686 and Y934 reside in PDGFR-beta catalytic domains, while Y970 is in the C-terminal tail. Using site-directed mutagenesis, we show that Abl-dependent phosphorylation of PDGFR-beta activates PDGFR-beta activity, in vitro, but serves to downregulate PDGFR-mediated chemotaxis. These data are exciting as they indicate that Abl kinases not only are activated by PDGFR and promote PDGFR-mediated proliferation and migration,but also act in an intricate negative feedback loop to turn-off PDGFR-mediated chemotaxis.
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ABSTRACT: Overexpression of the receptor tyrosine kinase (RTK) erbB2 (also designated neu or HER2) was implicated in causing a variety of human cancers, including mammary and ovarian carcinomas. Ligand-induced receptor dimerization is critical for stimulation of the intrinsic protein tyrosine kinase (PTK) of RTKs. It was therefore proposed that PTK activity is stimulated as a result of the reorientation of the cytoplasmic domains within receptor dimers, leading to transautophosphorylation and stimulation of enzymatic activity. Here, we propose a molecular mechanism for rotation-coupled activation of the erbB2 receptor. Using a computational exploration of conformation space of the transmembrane (TM) segments of an erbB2 homodimer, we found two stable conformations of the TM domain. We suggest that these conformations correspond to the active and inactive states of erbB2, and that the receptor molecules may switch from one conformation to the other without crossing exceedingly unfavorable states. This model provides an explanation for the biochemical and oncogenic properties of erbB2, such as the effects of erbB2 overexpression on kinase activity and cell transformation. Furthermore, the opposing effects of the neu* activating oncogenic point mutation and the Val-655-->Ile single-nucleotide polymorphism shown to be linked to reduced risk of breast cancer are explained in terms of shifts in the equilibrium between the active and inactive states of erbB2 in vivo.Proceedings of the National Academy of Sciences 12/2002; 99(25):15937-40. · 9.68 Impact Factor
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ABSTRACT: Tyrosine kinase oncogenes are formed as a result of mutations that induce constitutive kinase activity. Many of these tyrosine kinase oncogenes that are derived from genes, such as c-Abl, c-Fes, Flt3, c-Fms, c-Kit and PDGFRbeta, that are normally involved in the regulation of hematopoiesis or hematopoietic cell function. Despite differences in structure, normal function, and subcellular location, many of the tyrosine kinase oncogenes signal through the same pathways, and typically enhance proliferation and prolong viability. They represent excellent potential drug targets, and it is likely that additional mutations will be identified in other kinases, their immediate downstream targets, or in proteins regulating their function.Oncogene 06/2002; 21(21):3314-33. · 6.37 Impact Factor
Article: Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival.[show abstract] [hide abstract]
ABSTRACT: In spite of intensified efforts to understand cell signaling from endosomes, there is no direct evidence demonstrating that endosomal signaling is sufficient to activate signal transduction pathways and no evidence to demonstrate that endosomal signaling is able to produce a biological outcome. The lack of breakthrough is due in part to the lack of means to generate endosomal signals without plasma membrane signaling. In this paper, we report the establishment of a system to specifically activate epidermal growth factor (EGF) receptor (EGFR) when it endocytoses into endosomes. We treated cells with EGF in the presence of AG-1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks the recycling of EGFR. This treatment led to the internalization of nonactivated EGF-EGFR complexes into endosomes. The endosome-associated EGFR was then activated by removing AG-1478 and monensin. During this procedure we did not observe any surface EGFR phosphorylation. We also achieved specific activation of endosome-associated EGFR without using monensin. By using this system, we provided original evidence demonstrating that (i) the endosome can serve as a nucleation site for the formation of signaling complexes, (ii) endosomal EGFR signaling is sufficient to activate the major signaling pathways leading to cell proliferation and survival, and (iii) endosomal EGFR signaling is sufficient to suppress apoptosis induced by serum withdrawal.Molecular and Cellular Biology 11/2002; 22(20):7279-90. · 5.53 Impact Factor