Abstract
Kinase Suppressor of Ras1 (KSR1) functions as a positive modulator of Ras-dependent signaling either upstream of or parallel to Raf-1, and pharmacologic inactivation of KSR1 may serve as a treatment for Rasdriven malignancies such as pancreatic cancer (Xing, H. R., Cordon-Cardo, C., Deng, X., Tong, W., Campodonico, L., Fuks, Z., and Kolesnick, R. (2003) Nat. Med. 9, 1266-1268). Although some studies demonstrated a requirement for KSR1 kinase activity for its action, others suggested KSR1 acts primarily as a scaffold facilitating assembly of the c-Raf-1/MEK module. We recently established a two-stage in vitro reconstitution assay to measure KSR1 kinase activity (Xing, H. R., Lozano, J., and Kolesnick, R. (2000) J. Biol. Chem. 275, 17276-17280). In this assay, KSR1, immunopurified to apparent homogeneity, never comes in contact with recombinant kinases other than c-Raf-1. In the first assay stage, activated KSR1 is incubated with recombinant c-Raf-1 and ATP. In the second stage, activated c-Raf-1 is separated from KSR1, and incubated with unactivated MEK1, unactivated MAPK, Elk-1, and ATP. Elk-1 phosphorylation serves as a specific readout for MAPK activation. However, because KSR1 constitutively associates with MEK1 and this interaction appears critical for KSR1 scaffolding function, it has been argued that the kinase activity detected is an artifact of KSR1-bound MEK1. To address these concerns, we depleted as much as 90% of KSR1-bound MEK1 by high salt washing without altering KSR1 kinase activity. Further, a complete inactivation of KSR1-bound MEK1 by pretreating with the MEK inhibitor PD 98059 prior to the first assay stage did not alter KSR1 kinase activity. In addition, the omission of exogenous recombinant GST-MEK1 from the reaction mixture during the second assay stage abolished Elk-1 phosphorylation confirming KSR1-bound MEK1 does not support MAPK activation in our in vitro assay. Moreover, a kinase-inactive mutant, FLAG-Ki-KSR1(D683A/D700A), which efficiently interacts with endogenous MEK1, lacks kinase activity. These results collectively support our contention that the kinase activity of KSR1 is an intrinsic property of this protein independent of KSR1-bound endogenous MEK.
Highlights
Kinase Suppressor of Ras1 (KSR1) was originally identified in Drosophila melanogaster and Caenorhabditis elegans to function as a positive modulator of Ras-mitogen-activated protein kinase (MAPK)1 signaling either upstream of or parallel to c-Raf-1 [1,2,3]
Because MEK1 and KSR1 association is constitutive and such interaction is critical for the scaffolding function of KSR1 in MAPK signaling, others suggest that KSR1-bound MEK1 may be responsible for the kinase activity of KSR1 detected in our two-stage assay [15]
Multiple High Salt Washes Remove MEK1 from KSR1 Immune Complexes without Altering KSR1-specific Activity—The inability to detect the kinase activity of KSR1 arose in part from the lack of a standardized assay
Summary
Expression of KSR1 in COS-7 Cells and EGF Treatment—COS-7 cells (American Type Culture Collection) were maintained in high glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen), penicillin, and streptomycin at 37 °C in 5% CO2. FLAGKSR1 or FLAG-Ki-KSR1, immunoprecipitated from 500 g of total cell lysates with 60 l of agarose-conjugated anti-FLAG M2 antibody (Sigma), were washed with low salt- (0.15 M NaCl ϫ 5) or high salt- (1.0 M NaCl ϫ 5) containing Nonidet P-40 buffer. PD 98059 or vehicle was incubated with immunopurified KSR1 or 0.5 unit (200 ng) of human active GST-MEK1 (Upstate Biotechnology catalog number 14-206) for 20 min at room temperature before initiating the kinase assay. KSR1 immune complexes were collected by centrifugation and washed five times with Nonidet P-40 lysis buffer containing either 0.15 M NaCl or 1.0 M NaCl. 40 g of total COS-7 cell lysates were used to estimate the percentage of cellular MEK1 associated with FLAG-KSR1 and FLAG-Ki-KSR1 by densitometry. FLAG-KSR1, FLAG-Ki-KSR1, and their associated MEK1 were resolved by 7.5% SDS-PAGE and visualized by Western blot analysis using mouse anti-FLAG M2-peroxidase conjugated antibody (Sigma catalog number A-8592) and rabbit anti-MEK1 antibody (Cell Signaling, catalog number 9122), respectively
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
