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

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 coexpressed 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 CHIPdependent proteasomal degradation.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). Members of this family, which also include MLK1, MLK2, MLK3, MLK4, leucine zipper-bearing kinase, and leucine zipper and sterile ␣-motif kinase (1), are characterized at the structural level by the presence of a catalytic domain bearing amino acid motifs found in serine/threonine and tyrosine kinases and one or two leucine zipper motifs, which regulate their activity by mediating protein dimerization or oligomerization (2-5). A number of other interesting motifs that are likely important for protein binding have also been identified in specific members of the MLK family. For instance, MLK2 and MLK3 contain a Src homology 3 (SH3) domain in their N-terminal region that binds, respectively, the GTPase dynamin and the Ste20-related protein kinase HPK1 (6, 7). Both MLK proteins also possess a functional Cdc42/Rac interactive binding (CRIB) motif that mediates association with Cdc42 and Rac1 in a GTP-dependent manner (8, 9).The importance of the MLKs as signaling molecules is highlighted by the fact that these proteins act as key regulators of the c-Jun N-terminal kinase (JNK) subgroup of mitogen-activated protein kinases (1). Specifically, all MLK family members regulate the JNK pathway by phosphorylating and activating the JNK direct upstream activators . In addition to their role in catalyzing JNK activation, MLKs are also known to contribute to apoptosis in neuronal cells. Indeed, when dominant negative forms of MLKs...

CHIP Overexpression Reduces Mutant Androgen Receptor Protein and Ameliorates Phenotypes of the Spinal and Bulbar Muscular Atrophy Transgenic Mouse Model

“…A wide range of different proteins have been identified as CHIP substrates, including members of the steroid hormone receptor family (Connell et al, 2001;Tateishi et al, 2004;Wang and DeFranco, 2005), the cystic-fibrosis transmembraneconductance regulator (Meacham et al, 2001;Younger et al, 2006), E2A transcription factors (Huang et al, 2004), raf-1 protein kinase (Demand et al, 2001), ErbB2 (Zhou et al, 2003), nucleophosminanaplastic lymphoma kinase (Bonvini et al, 2004), dual leucine zipper-bearing kinase (Daviau et al, 2006), caytaxin (Grelle et al, 2006), ␣B-crystallin (Chavez Zobel et al, 2003), tau (Hatakeyama et al, 2004;Petrucelli et al, 2004;Sahara et al, 2005;Dickey et al, 2006), ␣-synuclein (Shin et al, 2005), the p53 tumor suppressor (Esser et al, 2005), apoptosis signal-regulating kinase 1 (Hwang et al, 2005), and polyQ-disease causative proteins (Jana et al, 2005;Miller et al, 2005;Al-Ramahi et al, 2006). CHIP can directly interact with and degrade the wildtype AR in a phosphorylation-dependent or -independent manner (Cardozo et al, 2003;Rees et al, 2006) and can repress AR transcriptional activity, suggesting that CHIP may play a role in regulating AR function in the cell (He et al, 2004).…”

Molecular Mechanism of the Negative Regulation of Smad1/5 Protein by Carboxyl Terminus of Hsc70-interacting Protein (CHIP)

“…For instance, CHIP can facilitate the chaperone-dependent degradation of a number of signature proteins involved in neurodegenerative diseases (22). Accumulated evidence has also linked CHIP to tumor development, as several oncogenic proteins are its substrates (23)(24)(25)(26). A recent study observed that expression of CHIP suppressed tumor cell growth and metastasis in breast cancer, suggesting that CHIP may as well have a role in controlling tumor progression (27).…”

The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed-Lineage Kinase 3

“…CHIP/Hsp70 complexes promote ubiquitination and proteasome-dependent degradation of protein substrates (27,37,38). Thus, the effect of CHIP and Hsp70 on endogenous MLK3 ubiquitination and protein levels was analyzed.…”

“…CHIP knockdown cells had impaired heat shock-or osmotic shock-induced MLK3 protein degradation, and JNK activation was enhanced. Possibly, CHIP knockdown resulted in the upregulation of MAP3Ks such as MLK3, ASK1, or DLK and/or inhibited MAPK phosphatases, all of which could have contributed to the enhanced JNK activation (38,45).…”