A Ligand-inducible Epidermal Growth Factor Receptor/Anaplastic Lymphoma Kinase Chimera Promotes Mitogenesis and Transforming Properties in 3T3 Cells (original) (raw)
Related papers
Experimental Cell Research, 2001
Two residues have been shown to be critical for the kinase activity of the receptor for epidermal growth factor (EGF): lysine-721, which functions in the binding of ATP by correctly positioning the ␥-phosphate for phosphoryl transfer, and aspartate-813, which functions as the catalytic base of the kinase. Mutation of either of these two residues has been shown to disrupt kinase activity of the receptor. However, studies performed in different laboratories had suggested that while EGF receptors mutated at lysine-721 are unable to stimulate significant increases of [ 3 H]thymidine incorporation into DNA in response to EGF treatment, cells expressing EGF receptors mutated at aspartate-813 do stimulate significant incorporation of [ 3 H]thymidine into DNA in response to EGF. In the present study, EGF receptors mutated at lysine-721 or aspartate-813 (K721R and D813A, respectively), as well as wild-type EGF receptors, were expressed in the same cellular background, Chinese hamster ovary cells, and side-by-side experiments were performed to investigate possible signaling-related differences. Our results indicate that while there are measurable differences in the abilities of the two mutant receptors to stimulate [ 3 H]thymidine incorporation between 20 and 24 h after addition of EGF, these differences cannot be correlated with significant differences in EGFstimulated tyrosine phosphorylation of mutant EGF receptor and endogenous ErbB2, the extent of receptor internalization, EGF-stimulated ion uptake, stimulation of SHC activity, or receptor association with Grb2. Flow cytometric data suggest that populations of cells expressing either kinase-impaired mutant EGF receptor progress similarly into S phase in response to addition of EGF. These observations suggest that D813A and K721R retain similar ability to stimulate mitogenic signaling events through transactivation of ErbB2 with only subtle temporal differences, and they emphasize the importance of expressing mutant receptors in an identical cellular context to make valid comparisons of functions.
A ligand-inducible anaplastic lymphoma kinase chimera is endocytosis impaired
Oncogene, 2004
Ligand-induced membrane trafficking of the anaplastic lymphoma kinase (ALK) was studied using a chimeric receptor in which the extracellular and transmembrane domain of ALK was substituted for the corresponding regions of epidermal growth factor receptor (EGFR). Wild-type EGFR, EGFR/ALK and an EGFR/ALK kinase negative mutant were independently expressed in mouse NR6 fibroblasts. The capacity of EGFR/ALK to mediate [ 125 I]-EGF internalization, receptor degradation and downregulation, which has never been previously described, was assayed. The rate of [ 125 I]-EGF-induced internalization mediated by the cytoplasmic domain of ALK was reduced several fold compared with the wildtype EGFR. The low rate of EGF internalization promoted by EGFR/ALK correlated with an impaired degradation and downregulation of the receptor and indicate that ALK is not subject to traditional mechanisms used to regulate receptor tyrosine kinase function. Accordingly, ALK-activated intracellular domain does not associate in vivo with c-cbl and does not undergo ligand-mediated ubiquitination. The current study provides new insight into the function and regulation of ALK suggesting that the relative long membrane residence of activated ALK might confers a more potent and prolonged signaling activity. Indeed NR6-EGFR/ALK cells exhibited a B3-fold increase in a maximal mitogenic response than NR6-EGFR.
Journal of Cellular Physiology, 1992
Ohio 44 106 A population of stable NIH 3T3 transfectants with two molecular weight classes of membrane-bound EGF receptors encoded by a human CCF receptor cDNA has been identified and characterized. In addition to intact EGF receptors, these cells also express a molecule with an extensive cytosolic deletion. This deletion includes the ligand-activated intrinsic protein tyrosine kinase catalytic domain. Treatment with EGF caused dimerization of intact and truncated receptors, allowing us to assess protein tyrosine kinase activity in the heterodimer isolated from living cells. In contrast to honiodimeric complexes with intact EGF receptor only, heterodimers were deficient in protein tyrosine kinase activity. Moreover, physical association between intact and truncated molecules suppressed receptor autophosphorylation by EGF receptor protein tyrosine kinase activated by antibody binding in vitro. Evidence presented here !,upports the idea that protein tyro3ine kinase activation is facilitated by interaction between adjacent receptor molecules with intact catalytic domains. Furthermore, molecules with cytoplasmic deletions that are physically associated with kinase-active ECF receptors appear to behave as dominant negative mutations. The HerC cI cells used in this study were selected with methotrexate to amplify the EGF receptor cDNA, and in that sense may resemble certain tumor-derived cells characterized by overexpressed and rearranged EGF receptor genes.
Proceedings of The National Academy of Sciences, 2000
Overexpression of ErbB-2͞Neu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)͞Neu ؉ MMTV͞transforming growth factor ␣ bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogenactivated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27 Kip1 . In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly in vitro by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1͞2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 and MAPK precipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant in vitro. Cdk2 and MAPK precipitates from AG-1478treated tumors were unable to phosphorylate p27 in vitro. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression.