Abstract
Current evidence suggests that the mixed lineage kinase family member dual leucine zipper-bearing kinase (DLK) might play a significant role in the regulation of cell growth and differentiation, particularly during the process of tissue remodeling. To further explore this working model, we have investigated the regulation of host and recombinant DLK in NIH3T3 and COS-1 cells undergoing apoptosis. Using calphostin C, a potent and selective inhibitor of protein kinase C and a recognized apoptosis inducer for various cell types, we demonstrate, by immunoblot analysis, that DLK protein levels are rapidly and dramatically down-regulated during the early phases of apoptosis. Down-regulation in calphostin C-treated cells was also accompanied by the appearance of SDS- and mercaptoethanol-resistant high molecular weight DLK immunoreactive oligomers. Experiments aimed at elucidating the mechanism(s) underlying DLK oligomerization revealed that the tissue transglutaminase (tTG) inhibitor monodansylcadaverine antagonized the effects of calphostin C almost completely, thereby suggesting the involvement of a tTG-catalyzed reaction as the root cause of DLK down-regulation and accumulation as high molecular weight species. In support of this notion, we also show that DLK can serve as a substrate for tTG-dependent cross-linking in vitro and that this covalent post-translational modification leads to the functional inactivation of DLK. Taken together, these observations suggest that transglutamination and oligomerization may constitute a relevant physiological mechanism for the regulation of DLK activity.
Highlights
A variety of extracellular “stress” stimuli responsible for the induction of growth arrest and apoptosis in multicellular organisms are transduced from the cell membrane to the nucleus via a phosphorylation cascade involving members of the c-Jun NH2-terminal kinase (JNK)1 subgroup of mitogen-activated
Advances over the past few years have contributed to our general understanding of how the mixed lineage kinase dual leucine zipper-bearing kinase (DLK) regulates JNK signaling, little is currently known about the mechanisms affecting its own regulation, about the proteins it is most likely to interact with within the cell, and about its physiological function(s)
We provide evidence that calphostin C induces a rapid and dramatic decrease in the levels of the 150-kDa membrane/Golgi-associated form of DLK in NIH 3T3, Neuro-2a, and AtT-20 cells as well as in COS-1 cells transiently transfected with a functional DLK expression vector
Summary
Chemicals and Antibodies—Calphostin C, bisindolylmaleimide I, staurosporine, MG-132, EST, calpeptin, DEVD-CHO, and Z-VAD-FMK, were purchased from Calbiochem-Novabiochem Co. (San Diego, CA). Plasmids and Transfection—A BamHI fragment of the mouse DLK cDNA (amino acids 30 – 662) [35] was inserted in frame with a hexahistidine (His) tag sequence in the bacterial expression vector pET-30b (Novagen Inc., Madison, WI). A COOH-terminal truncated construct encoding amino acid residues 1–719 of DLK was generated by PCR using Pwo polymerase and subcloned into the mammalian expression vector pLXSN (CLONTECH Laboratories Inc., Palo Alto, CA). A NH2- and COOH-terminal truncated DLK construct (amino acid 160 –511) tagged with the FLAG epitope sequence (DYKDDDDK) was generated by PCR and inserted into pcDNA3 (Invitrogen Corp.). Thereafter, cells were transfected with 5 g of the various DLK expression vectors using Superfect (Qiagen Inc., Mississauga, Ontario, Canada) according to the manufacturer’s protocol. Membranes were “blocked” overnight at 4 °C in 20 mM Tris, pH 7.5, 150 mM
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