Localization of the Mixed-lineage Kinase DLK/MUK/ZPK to the Golgi Apparatus in NIH 3T3 Cells

Douziech, Mélanie; Laberge, Gino; Grondin, Gilles; Daigle, Nathalie; Blouin, Richard

doi:10.1177/002215549904701008

Cited by 20 publications

(24 citation statements)

References 40 publications

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“…The data in this figure and all the other figures are representative of at least three independent experiments. 4°C with constant rotation using a polyclonal antibody against DLK (30) and protein A-Sepharose beads. After incubation, the immunocomplexes were washed 3 times with lysis buffer and 3 times with kinase buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl 2 , 0.5 mM dithiothreitol, 0.1 mM PMSF, 0.2 mM sodium orthovanadate , 1 g/ml leupeptin, and 1 g/ml aprotinin).…”

Section: Methodsmentioning

confidence: 99%

“…Lysates prepared from these cells were then subjected to immunoprecipitation analyses with an anti-DLK antibody (30), and the resulting immunocomplexes were probed with an anti-ubiquitin antibody. Because DLK kinase activity seems to be required for degradation, as suggested by our results with ectopically expressed DLK, cells were also exposed to the serine/threonine phosphatase inhibitor okadaic acid before being processed for immunoprecipitation to favor activation of endogenous DLK.…”

Section: Chip Is Required For Hsp70-mediated Degradation Of Dlk-mentioning

confidence: 99%

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

et al. 2006

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Chip Is Required For Hsp70-mediated Degradation Of Dlk-mentioning

confidence: 99%

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

et al. 2006

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“…The rabbit polyclonal antiserum to DLK was described previously. 41 Rabbit polyclonal or mouse monoclonal antibodies raised against cleaved caspase-9, cleaved caspase-3, poly(ADP-ribose)polymerase (PARP), cleaved PARP, and phospho-JNK were purchased from Cell Signaling Technology Inc. …”

Section: Chemicals and Antibodiesmentioning

confidence: 99%

Tissue transglutaminase triggers oligomerization and activation of dual leucine zipper-bearing kinase in calphostin C-treated cells to facilitate apoptosis

et al. 2004

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Although tissue transglutaminase (tTG) has been recognized as a mediator of apoptosis in various experimental models, little is currently known about the molecular mechanisms by which this protein modulates cell death. Recent work from our laboratory has shown that activation of tTG in cells exposed to the apoptotic inducer calphostin C triggers the crosslinking of dual leucine zipper-bearing kinase (DLK), a proapoptotic kinase acting as an essential component of the c-Jun aminoterminal kinase (JNK) signaling pathway. As a consequence of this observation, we have undertaken experiments to investigate the functional relevance of DLK oligomerization in tTG-mediated apoptosis. Our results indicate that, in cells undergoing calphostin C-induced apoptosis, tTG-dependent DLK oligomerization occurs early in the apoptotic response. Both immunocomplex kinase assays and immunoblotting with phosphospecific antibodies revealed that oligomer formation by tTG-mediated crosslinking reactions significantly enhanced the kinase activity of DLK and its ability to activate the JNK pathway. Moreover, functional studies demonstrate that tTG-mediated oligomerization of wild-type DLK sensitizes cells to calphostin C-induced apoptosis, while crosslinking of a kinase-inactive variant of DLK does not. Collectively, these data strongly suggest that tTG facilitates apoptosis, at least partly, by oligomerization and activation of the proapoptotic kinase DLK.

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“…MLK3 localizes with golgin-160 in the Golgi region DLK, a protein kinase related to MLK3, has been localized to the Golgi region (Douziech et al, 1999), and overexpressed MLK2 can interact with microtubules (Nagata et al, 1998). Moreover, MLK3 localization to the centrosomes and perinuclear regions in interphase and mitotic cells (Swenson et al, 2003) suggests that MLK3 and Golgi proteins reside in similar intracellular locations.…”

Section: N-terminal Region Of Golgin-160 Interacts With Mlk3mentioning

confidence: 99%

“…MLK proteins might also regulate physiological functions by being targeted to specific intracellular structures. For example, DLK has been shown to localize to the Golgi complex but its function there is unknown (Douziech et al, 1999). In addition, overexpressed MLK2 associates with microtubules and might regulate JNK activity and motor protein function (Nagata et al, 1998).…”

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confidence: 99%

Phosphorylation of golgin-160 by mixed lineage kinase 3

Cha

Smith

Gallo

et al. 2004

Journal of Cell Science

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Golgin-160 is a member of the coiled-coil family of golgin proteins, which are proposed to regulate the structure of the Golgi complex. The C-terminal two-thirds of golgin-160 is predicted to form a coiled-coil domain and the N-terminal head domain contains several putative binding domains, regulatory motifs and phosphorylation sites. Recently, it has been demonstrated that caspase-dependent cleavage of the golgin-160 head domain occurs rapidly after induction of apoptosis. The role of golgin-160 phosphorylation and the functional implications for Golgi structure have not been defined. In this study, we investigated the kinase(s) responsible for phosphorylation of golgin-160. Signaling through the small G-protein Rac and mixed-lineage-kinase-3 (MLK3) resulted in increased phosphorylation of golgin-160. The intracellular distribution of MLK3 overlapped with that of golgin-160 and the two proteins could be co-immunoprecipitated. In vitro kinase assays demonstrated that MLK3 directly phosphorylates golgin-160 in the N-terminal head region between residues 96 and 259. Overexpression of MLK3 caused an enhanced caspase-dependent cleavage of golgin-160 at Asp139. Golgin-160 is the first non-kinase substrate of MLK3 identified, and phosphorylation by MLK3 might modulate cleavage of golgin-160 during apoptosis.

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Localization of the Mixed-lineage Kinase DLK/MUK/ZPK to the Golgi Apparatus in NIH 3T3 Cells

Cited by 20 publications

References 40 publications

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

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

Tissue transglutaminase triggers oligomerization and activation of dual leucine zipper-bearing kinase in calphostin C-treated cells to facilitate apoptosis

Phosphorylation of golgin-160 by mixed lineage kinase 3

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