Abstract
Down-regulation of the Mixed-lineage Dual Leucine Zipper-bearing Kinase by Heat Shock Protein 70 and Its Co-chaperone CHIP
Highlights
Dual leucine zipper-bearing kinase (DLK)2 is a serine/threonine kinase that belongs to a family of mitogen-activated protein kinase kinase kinases, known as mixed-lineage kinases (MLKs) [1]
Down-regulation of DLK by heat shock protein 70 (Hsp70)—During the course of a study recently conducted in our laboratory, we noticed that overexpression of wild-type DLK in MCF-7 human breast cancer cells resulted in the accumulation of a protein with a molecular weight (Mr) of ϳ70,000 (p70), as revealed by Coomassie Blue staining of cell extracts fractionated on reducing SDSPAGE
These results confirm that the ectopic expression of DLK in COS-7 cells is sufficient to up-regulate in a specific manner Hsp70 protein levels and suggest that DLK kinase activity is required for Hsp70 induction
Summary
Reagents, and Antibodies—The proteasome inhibitor lactacystin and the rabbit polyclonal antibody against actin were purchased from Sigma-Aldrich. Medium was removed 48 h after transfection, and cells were treated with lactacystin (10 M) and/or okadaic acid (400 nM) in 3 ml of DMEM supplemented with 10% (v/v) heat-inactivated FBS, 100 units/ml penicillin, 100 g/ml streptomycin, and 25 g/ml amphotericin B/60 mm at 37 °C. Preparation of Cell Lysates and Immunoblotting—Cells were lysed for 30 min at 4 °C in 15 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 150 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 g/ml leupeptin, and 1 g/ml aprotinin. In Vitro Interaction Assay—The full-length coding sequence of wildtype DLK was inserted in-frame with a His-tag sequence in the pTriEx-4 expression vector (Novagen) for production of recombinant proteins using the TNT T7 Coupled Reticulocyte Lysate system (Promega Corp., Madison, WI)
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.
