Down-regulation of the Mixed-lineage Dual Leucine Zipper-bearing Kinase by Heat Shock Protein 70 and Its Co-chaperone CHIP

Alex Daviau,Marco Di Fruscio,Roxanne Proulx,Jacques Landry,Cam Patterson,Yves Durocher,Richard Blouin,Robert M Tanguay,Karine Robitaille

doi:10.1074/jbc.m607612200

Abstract

Dual leucine zipper-bearing kinase (DLK) is a mixed-lineage kinase family member that acts as an upstream activator of the c-Jun N-terminal kinases. As opposed to other components of this pathway, very little is currently known regarding the mechanisms by which DLK is regulated in mammalian cells. Here we identify the stress-inducible heat shock protein 70 (Hsp70) as a negative regulator of DLK expression and activity. Support for this notion derives from data showing that Hsp70 induces the proteasomal degradation of DLK when both proteins are co-expressed in COS-7 cells. Hsp70-mediated degradation occurs with expression of wild-type DLK, which functions as a constitutively activated protein in these cells but not kinase-defective DLK. Interestingly, the Hsp70 co-chaperone CHIP, an E3 ubiquitin ligase, seems to be indispensable for this process since Hsp70 failed to induce DLK degradation in COS-7 cells expressing a CHIP mutant unable to catalyze ubiquitination or in immortalized fibroblasts derived from CHIP knock-out mice. Consistent with these data, we have found that endogenous DLK becomes sensitive to CHIP-dependent proteasomal degradation when it is activated by okadaic acid and that down-regulation of Hsp70 levels with an Hsp70 antisense attenuates this sensitivity. Therefore, our studies suggest that Hsp70 contributes to the regulation of activated DLK by promoting its CHIP-dependent proteasomal degradation.

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

EXPERIMENTAL PROCEDURES

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)

After nonradioactive translation

RESULTS

DISCUSSION