C-terminus of heat shock protein 70-interacting protein facilitates degradation of apoptosis signal-regulating kinase 1 and inhibits apoptosis signal-regulating kinase 1-dependent apoptosis

Abstract

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is regulated under conditions of cellular stress. ASK1 phosphorylates c-Jun N-terminal kinase (JNK) and elicits an apoptotic response. ASK1 activity is regulated at multiple levels, 1 of which is through inhibition by cytosolic chaperones of the heat shock protein (Hsp) 70 family. Among the proteins that determine Hsp70 function, CHIP (C-terminus of Hsp70-interacting protein) is a cochaperone and ubiquitin ligase that interacts with Hsp70 through an amino-terminal tetratricopeptide repeat (TPR) domain. Prominent among the cellular functions mediated by CHIP is protection against physiologic stress. Because ASK1 is known to contain a TPR-acceptor site, we examined the role of CHIP in regulating ASK1 function. CHIP interacted with ASK1 in a TPR-dependent fashion and induced ubiquitylation and proteasome-dependent degradation of ASK1. Targeting of ASK1 by CHIP inhibited JNK activation in response to oxidative challenge and reduced ASK1-dependent apoptosis, whereas short interfering RNA (siRNA)-dependent depletion of CHIP enhanced JNK activation. Consistent with its ability to reduce cytoplasmic ASK1 levels, CHIP triggered the translocation of ASK1 partner protein death-associated protein (Daxx) into the nucleus, where it is known to activate an antiapoptotic response. These results indicate that CHIP regulates ASK1 activity by inducing its ubiquitylation and degradation, which, together with its effects on Daxx localization, provides a mechanism for the antiapoptotic effects of CHIP observed in the face of cellular and physiologic stress.

Fig 1.

ASK1 expression is reduced in the presence of overexpressed CHIP. COS-7 cells were transiently transfected with HA-ASK1 with or without CHIP (wild type and H260Q). Twenty-four hours after transfection, cells were lysed in radioimmune precipitation buffer, and the cell lysates were blotted with anti-HA, anti-CHIP, and anti-β-actin, as indicated. IB, immunoblotting

Fig 2.

CHIP stimulates ubiquitylation of ASK1. (A) COS-7 cells were transiently transfected with HA-ASK1 with or without Myc-CHIP and treated with 25 μM MG132 or vehicle for 8 hours before harvesting. Cell lysates were immunoblotted for HA-ASK1, Myc-CHIP, and β-actin to normalize protein amounts. (B) COS-7 cells were transfected as in panel A and immunoprecipitated with anti-HA antibody. Immunoprecipitated proteins were immunoblotted with anti-ubiquitin antibody. (C) In vitro ubiquitylation assay was performed using HA-ASK1 as a substrate with the indicated components of the ubiquitylation reaction. Ub-ASK1, ubiquitylated ASK1; WCL, whole cell lysates; IP, immunoprecipitation

Fig 3.

CHIP interacts with ASK1 via its TPR domain. COS-7 cells were transiently transfected with HA-ASK1 and either vector, wild-type Myc-CHIP (Myc-CHIP WT) or Myc-CHIP lacking the TPR domain (Myc-CHIP ΔTPR). Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Myc. Whole cell lysates and the immunoprecipitated proteins were immunoblotted with anti-HA, anti-Myc, and anti–β-actin, as indicated

Fig 4.

Ubiquitylation and degradation of ASK1 mediated by CHIP reduces JNK1 activity under stress. (A) COS-7 cells stably transfected with Myc-CHIP (COS-7/Myc-CHIP). COS-7/Myc-CHIP as well as COS-7 cells were transiently transfected with either HA-ASK1 or vector. Twenty-four hours after transfection, cells were treated with or without 2 mM H₂O₂ for 20 minutes before harvesting. Cell lysates were blotted with anti-HA, anti-Myc, and anti-β-actin. Quantitative densitometric analysis of JNK1 activity (n = 3) is shown in the bar graph. (B) The same cell lysates as in panel A were immunoprecipitated with anti-JNK1 antibody and immunoprecipitated proteins were subjected to an in vitro JNK1 activity assay using c-Jun (79) as a substrate as described in Materials and Methods. ³²P-radiolabeled c-Jun was observed after separation on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and exposure to film (upper panel). The same cell lysates were also used for immunoblotting JNK1 expression level (lower panel) with anti-JNK1 antibody

Fig 5.

Regulation of JNK1 activity by endogenous CHIP levels. COS-7 cells were transiently transfected with CHIP and control siRNA expression vectors. Cells were treated with or without 2 mM H₂O₂ for 20 minutes before harvesting, and JNK1 activity was measured. Quantitative densitometric analysis of JNK1 activity (n = 3) is shown in the bar graph

Fig 6.

CHIP triggers translocation of Daxx into nucleus. COS-7 cells were transiently transfected with Flag-Daxx with wild-type CHIP, CHIP H260Q mutant, or the empty vector. Twenty-four hours after transfection, cells were fractionated to nucleus and cytoplasm, and localization of Flag-Daxx and CHIP were determined by immunoblotting using antibodies against Flag and CHIP, respectively. Anti-Oct-1 and anti-GAPDH were used for quantitating nuclear and cytoplasmic proteins, respectively. Nu, nucleus; Cy, cytoplasm; Vec, empty vector (pcDNA3); WT, wild-type CHIP; H260Q, CHIP H260Q mutant