Epidermal growth factor treatment enhances the kinase activity of kinase suppressor of Ras Journal Article
| Authors: | Xing, H. R.; Lozano, J.; Kolesnick, R. |
| Article Title: | Epidermal growth factor treatment enhances the kinase activity of kinase suppressor of Ras |
| Abstract: | In Drosophila melanogaster and Caenorhabditis elegans, kinase suppressor of Ras (KSR) functions as a positive modulator of Ras-dependent signaling either upstream of or parallel to Raf. Attempts to characterize the biochemical and biological properties of mammalian KSR, however, have had limited success. Although some studies demonstrated a requirement of KSR kinase activity for its action, others indicated the kinase function of KSR is dispensable and suggested that KSR acts primarily as a scaffold protein. Interpretations of KSR function are further hampered by the lack of a standardized assay for its kinase activity in vitro. To address this issue, we established a two-stage in vitro kinase assay in which KSR never comes in contact with any recombinant kinases other than c-Raf-1. Using this assay, we show that KSR immunoprecipitated from quiescent COS-7 cells overexpressing Flag-tagged KSR was inactive, but its activity was rapidly and markedly induced upon epidermal growth factor treatment. Moreover, KSR-reconstituted mitogen-activated protein kinase activation was detected in KSR immunoprecipitates depleted of all contaminating kinases (c-Raf-1, MEK1, ERK2) by multiple high salt washes. Only full-length kinase-active KSR was capable of signaling c-Raf-1-dependent activity as kinase inactive and C- and N-terminal deletion mutants were without effect. Furthermore, endogenous KSR isolated from A431 cells, which contain high levels of activated EGF receptor, displays constitutively enhanced kinase activity. Hence, KSR kinase activity is not an artifact of overexpression but a property intrinsic to this protein. The recognition of EGF as a potent activator of KSR kinase activity and the availability of a well defined in vitro kinase assay should facilitate the definition of the function of KSR as a Ras-effector molecule. |
| Keywords: | epidermal growth factor; mitogen activated protein kinase; carcinoma, squamous cell; nonhuman; animal cell; animals; gene overexpression; protein kinases; enzyme activation; tumor cells, cultured; enzyme activity; transfection; cos cells; bioassay; recombinant proteins; mitogen-activated protein kinases; phosphotransferase; proto-oncogene proteins c-raf; inhibitor protein; precipitation; humans; priority journal; article |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 275 |
| Issue: | 23 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2000-06-09 |
| Start Page: | 17276 |
| End Page: | 17280 |
| Language: | English |
| DOI: | 10.1074/jbc.C900989199 |
| PUBMED: | 10764733 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 18 November 2015 -- Source: Scopus |
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