Kinase suppressor of Ras transphosphorylates c-Raf-1 Journal Article
| Authors: | Zafrullah, M.; Yin, X.; Haimovitz-Friedman, A.; Fuks, Z.; Kolesnick, R. |
| Article Title: | Kinase suppressor of Ras transphosphorylates c-Raf-1 |
| Abstract: | Whether kinase suppressor of Ras1 (KSR1) is an active kinase that phosphorylates c-Raf-1 or a scaffold that coordinates signaling along the Ras/ERK1 signaling module is actively debated. In this study, we generated a monoclonal antibody against a c-Raf-1 peptide containing phosphorylated Thr<sup>269</sup>, the putative target for KSR1 kinase activity. We show that this antibody detects Thr<sup>269</sup>-phosphorylated c-Raf-1 in A431 cells upon epidermal growth factor (EGF) stimulation, preceding MEK1 activation. Furthermore, this antibody detects in vitro phosphorylation of FLAG-c-Raf-1 and kinase-dead FLAG-c-Raf-1(K375M) by immunopurified KSR1, but fails to detect phosphorylation of FLAG-c-Raf-1(K375M/T269V), engineered with a Thr<sup>269</sup> to valine substitution. To provide unequivocal evidence that KSR1 is a legitimate kinase, we purified KSR1 to homogeneity, confirmed by mass spectrometry, renatured it in-gel, and demonstrated that it phosphorylates BSA-conjugated c-Raf-1 peptide at Thr<sup>269</sup>. These studies add to emerging data validating KSR1 as a kinase that phosphorylates c-Raf-1. © 2009 Elsevier Inc. |
| Keywords: | epidermal growth factor; controlled study; unclassified drug; human cell; raf protein; mass spectrometry; animals; mice; amino acid substitution; protein kinases; protein; enzyme activity; cercopithecus aethiops; cos cells; phosphorylation; monoclonal antibody; antibodies, monoclonal; enzyme phosphorylation; enzyme analysis; threonine; mapk; proto-oncogene proteins c-raf; c-raf-1; egf; ksr; scaffold; protein ksr1; antibodies, phospho-specific |
| Journal Title: | Biochemical and Biophysical Research Communications |
| Volume: | 390 |
| Issue: | 3 |
| ISSN: | 0006-291X |
| Publisher: | Elsevier Science, Inc. |
| Date Published: | 2009-12-18 |
| Start Page: | 434 |
| End Page: | 440 |
| Language: | English |
| DOI: | 10.1016/j.bbrc.2009.09.048 |
| PUBMED: | 19766101 |
| PROVIDER: | scopus |
| PMCID: | PMC2800940 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 1" - "Export Date: 30 November 2010" - "CODEN: BBRCA" - "Source: Scopus" |
Altmetric
Citation Impact
BMJ Impact Analytics
MSK Authors
Related MSK Work