ARTICLE |
Correspondence to: Richard Blouin, Dépt. de Biologie, Faculté des Sciences, Université de Sherbrooke, 2500, Boulevard de l'Université, Sherbrooke, Québec, Canada J1K 2R1.
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Summary |
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DLK/MUK/ZPK is a serine/threonine kinase that belongs to the mixed-lineage (MLK) subfamily of protein kinases. As is the case for most members of this family, relatively little is known about the physiological role of DLK/MUK/ZPK in mammalian cells. Because analysis of subcellular distribution may provide important clues concerning the potential in vivo function of a protein, an antiserum was generated against the amino terminal region of murine DLK/MUK/ZPK and used for localization studies in wild-type NIH 3T3 cells. Light microscopic immunocytochemistry experiments performed with the antiserum revealed that DLK/MUK/ZPK was specifically localized in a juxtanuclear structure characteristic of the Golgi complex. In support of this, treatment of cells with brefeldin A, a drug known to disintegrate the Golgi apparatus, caused disruption of DLK/MUK/ZPK perinuclear staining. Ultrastructural observation of NIH 3T3 cells also confirmed this localization, showing that most of the immunoreactivity was detected on membranes of the stacked Golgi cisternae. Consistent with localization studies, biochemical analyses revealed that DLK/MUK/ZPK was predominantly associated with Golgi membranes on fractionation of cellular extracts and was entirely partitioned into the aqueous phase when membranes were subjected to Triton X-114 extraction. On the basis of these findings, we suggest that DLK/MUK/ZPK is a peripheral membrane protein tightly associated with the cytoplasmic face of the Golgi apparatus. (J Histochem Cytochem 47:12871296, 1999)
Key Words: DLK/MUK/ZPK, mixed-lineage kinase, Golgi apparatus, signal transduction
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Introduction |
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One of the most important signal transduction pathways in eukaryotes involves members of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases (, interleukin-1ß) and various forms of environmental stresses (e.g., heat shock, hyperosmolarity, UV and ionizing irradiation) that induce growth arrest, cellular repair, or apoptosis in most cell types (
Among the different MAPKKKs identified thus far and implicated in the regulation of the MAPK signaling pathways are members of the mixed-lineage kinase (MLK) family (
Despite their involvement in the JNK/SAPK cascade, relatively little is known about the mechanisms of regulation and the physiological roles of the different MLK family members in mammalian cells. A potential function for the DLK/MUK/ZPK gene in biological processes related to differentiation was recently postulated on the basis that its expression in embryonic and adult mouse tissues is restricted to highly specialized cell populations, such as neurons and epithelial cells (
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Materials and Methods |
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Production of DLK/MUK/ZPK-specific Antisera
A portion of the mouse DLK/MUK/ZPK cDNA (nucleotides 42711) (
Cell Culture
All cell lines used in this work were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml amphotericin B at 37C in a 5% CO2 atmosphere.
DLK/MUK/ZPK Expression in COS-1 Cells
The cDNA encoding mouse DLK/MUK/ZPK (
Preparation of Cell Lysates and Immunoblotting
Cells (control COS-1, DLK/MUK/ZPK-transfected COS-1, wild-type NIH 3T3 and Swiss 3T3, fibroblasts) were lysed for 30 min at 4C in PBS containing 1% Triton X-100, 10 mM NaPPi, 100 mM NaF, 0.2 mM Na3VO4, 0.2 mM PMSF, 2 µg/ml leupeptin, and 1 µg/ml aprotinin (PBS-T). Lysates were then centrifuged at 12,000 rpm for 15 min at 4C, and the concentration of total protein in the supernatant fraction was quantitated using the Bio-Rad protein assay (Bio-Rad Laboratories; Mississauga, Ontario, Canada). Proteins were separated on 10% SDS-PAGE under reducing conditions and transferred onto polyvinylidene difluoride (PVDF) membranes (Boehringer Mannheim; Laval, Quebec, Canada) using a semidry transfer apparatus (Amersham Pharmacia Biotech; Baie d'Urfé, Quebec, Canada). Membranes were blocked overnight at 4C in 20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20 (TBS-T) containing 5% skim milk powder. Blots were then incubated with the primary antibody for 1 hr at room temperature and the immunoreactivity was detected by enhanced chemiluminescence, using as secondary antibody a horseradish peroxidase-linked anti-rabbit antibody (ECL Western blotting kit; Amersham Pharmacia Biotech).
Immunocytochemistry
Light and electron microscopic immunocytochemistry experiments were carried out as described previously (
Subcellular Fractionation
Subcellular fractionation of NIH 3T3 cells was performed on self-forming Percoll gradient as described previously (-mannosidase II (Man II) antiserum (1:2000 dilution; supplied by Dr. K.W. Moremen, University of Georgia). A rabbit antiserum directed against G-protein ß-subunits (1:1000 dilution; Chemicon International, Temecula, CA) was also used in these experiments as a marker for the plasma membrane.
Extraction and Trypsin Treatment of Membranes
NIH 3T3 cells were homogenized in 250 mM sucrose, 10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM PMSF, 2 µg/ml leupeptin, and 1 µg/ml aprotinin with a Dounce homogenizer, followed by centrifugation at 600 x g for 3 min. The supernatant from this centrifugation was then spun at 100,000 x g for 1 hr at 4C in a Beckman SW50.1 rotor, and the resulting pellet (crude membrane fraction) was resuspended in the homogenization buffer containing either 1 M NaCl, 1 M KCl, or 0.1 M Na2CO3, pH 11.5. After incubation on ice for 30 min, the samples were centrifuged again at 100,000 x g for 30 min to obtain supernatants and membranes pellets. Extraction of crude membrane fraction with Triton X-114 was carried out as described by
Crude membrane fractions prepared as described above were also resuspended in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, with or without 1% Triton X-100. After incubation on ice for 5 min, trypsin (0.1 µg/ml) was added and the digestion was allowed to proceed at 4C for 20 min. Then protease inhibitors (2 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml aprotinin) were added to neutralize the trypsin and the membranes were diluted with an equal volume of digestion buffer containing either 1% or 2% Triton X-100. After a 30-min incubation at 4C, the samples were centrifuged at 12,000 rpm in a microfuge for 15 min. The supernatants of these incubations were then immunoprecipitated for 3 hr at 4C with constant rotation, using either DLK/MUK/ZPK antiserum (1:250 dilution) or Man II antiserum (1:500 dilution) and protein AAgarose beads. At the end of the incubation period, the immunocomplexes were collected by centrifugation at 12,000 rpm for 30 sec and washed twice with digestion buffer containing 1% Triton X-100. The final pellet was resuspended in 2 x Laemmli sample buffer, heated to 95C for 5 min, and fractionated by SDS-PAGE. DLK/MUK/ZPK and Man II were detected by immunoblotting.
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Results |
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Characterization of Anti-DLK/MUK/ZPK Antisera
To investigate the biological function of the mixed-lineage kinase DLK/MUK/ZPK, we raised antisera in rabbits against a recombinant E. coli-produced fragment of the mouse DLK/MUK/ZPK protein. The specificity of one of these antisera was assayed by immunoblotting of cell lysates prepared from control and DLK/MUK/ZPK-transfected COS-1 cells and from wild- type NIH 3T3 and Swiss 3T3 fibroblasts. In immunoblot analysis carried out with lysates of COS-1 cells, the antiserum recognized a unique protein band of ~130 kD only in DLK/MUK/ZPK transfectants (Figure 1A). The size of this protein corresponds to what has been previously reported for transfected mouse and rat DLK/MUK/ZPK (
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Localization of DLK/MUK/ZPK in Wild-type NIH 3T3 Cells
Subcellular localization of DLK/MUK/ZPK was initially examined in NIH 3T3 cells by immunohistochemistry with DLK/MUK/ZPK antiserum and alkaline phosphatase-conjugated secondary antibody. All interphase cells showed a reactivity with juxtanuclear structures characteristic of the Golgi apparatus (Figure 2A2C). Staining of the same cell structures was also observed in murine Swiss 3T3, P19, and AtT-20 cell lines (not shown). By contrast, no immunoreactivity was seen in any of the cell types incubated with the preimmune serum (not shown). Interestingly, the perinuclear distribution of DLK/MUK/ZPK was disrupted in mitotic cells in a manner consistent with a Golgi localization. During metaphase, when cells are rounded, DLK/MUK/ZPK was localized to vesicle-like structures uniformly distributed throughout the cytoplasm, whereas in early telophase an intense signal was detected in the centrosomal region of the mitotic spindle (Figure 2A and Figure 2D2F).
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To provide additional evidence for the localization of DLK/MUK/ZPK to the Golgi region, we carried out immunohistochemical studies with DLK/MUK/ZPK antiserum on NIH 3T3 cells that were exposed to brefeldin A (BFA). BFA is a lipophilic fungal toxin that induces segregation of Golgi stacks into the endoplasmic reticulum and collapses of the trans-Golgi network on the microtubule-organizing center (
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Location of DLK/MUK/ZPK in NIH 3T3 cells was also examined at the ultrastructural level by immunoelectron microscopy. Because initial attempts to localize DLK/MUK/ZPK by immunogold labeling did not give satisfactory results, we used as an alternative approach a pre-embedding immunoperoxidase technique for electron microscopic localization of DLK/MUK/ZPK (Figure 4). As expected from the light microscopic immunocytochemistry, specific labeling for DLK/MUK/ ZPK was found in the area of the Golgi complex (Figure 4A and Figure 4C). Apparently, this labeling was associated with membranes of the stacked Golgi cisternae and associated vesicles. No staining was observed in the lumen of Golgi vesicles (Figure 4D). Negative control with preimmune serum gave no signal (Figure 4B).
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Co-purification of DLK/MUK/ZPK with the Golgi Apparatus in Fractionated Cell Extracts
To confirm at the biochemical level the predicted Golgi association of DLK/MUK/ZPK, NIH 3T3 cells were fractionated using a self-forming Percoll gradient protocol that allows good resolution of plasma membranes, Golgi apparatus, endoplasmic reticulum, and lysosomes (-mannosidase II (Man II) and G-protein ß-subunits, two marker proteins representative of the Golgi apparatus and plasma membrane, respectively. As shown in Figure 5, Western blot analysis revealed that DLK/MUK/ZPK and the Golgi marker Man II had overlapping fractionation profiles. Both proteins were particularly enriched in the heaviest fractions of the Percoll gradient and a considerable proportion of them co-purified in fractions 9 and 10. A mutually exclusive enrichment of DLK/MUK/ZPK and Man II was seen in fractions 8 and 11 respectively, but the significance of this observation is not known. The plasma membrane, as demonstrated by G-protein ß-subunit distribution, was predominantly found in fractions 24, in which no DLK/MUK/ZPK was detected (Figure 5). These results, together with the localization studies, strongly support the association of DLK/MUK/ZPK with the Golgi complex in NIH 3T3 cells.
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Membrane Association and Topology of DLK/MUK/ZPK
The nature of the association of DLK/MUK/ZPK with Golgi membranes was investigated using different extraction procedures, followed by Western blot analysis. As shown in Figure 6A, all DLK/MUK/ZPK immunoreactivity was associated with the membrane fraction prepared from NIH 3T3 cells (Figure 6, Lanes 1 and 2). This immunoreactivity could not be removed from particulate fractions even after treatment with 1 M NaCl, 1 M KCl, or 0.1 M Na2CO3, pH 11.5 (Figure 6, Lanes 38). When total cell membranes were subjected to Triton X-114 extraction, which allows discrimination between membrane-associated and integral membrane proteins, DLK/MUK/ZPK entirely partitioned into the aqueous phase (Figure 6, Lanes 9 and 10). These results therefore indicate that DLK/MUK/ZPK is a peripheral protein strongly associated with Golgi membranes.
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Next, to determine the topology of DLK/MUK/ZPK in Golgi membranes, particulate fractions prepared from NIH 3T3 cells were subjected to trypsin digestion in the presence or absence of a detergent. Figure 6B shows that DLK/MUK/ZPK was completely digested by trypsin even in the absence of a detergent (upper panel). Under the same conditions, Man II, an integral membrane protein facing the Golgi lumen, was resistant to digestion (Figure 6B, lower panel). Man II immunoreactivity was lost only when digestion was carried out in the presence of a detergent (Figure 6B, lower panel). Taken together, these results suggest that DLK/MUK/ZPK is a peripheral protein anchored to the cytoplasmic face of the Golgi complex.
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Discussion |
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An antiserum against DLK/MUK/ZPK was produced to examine, for the first time, its endogenous subcellular distribution in cultured cells. The specificity of this antiserum was demonstrated on cell extracts prepared from transfected COS-1 cells and from wild-type NIH 3T3 and Swiss 3T3 fibroblasts. On the basis of these observations, which provide strong evidence for the suitability of this antiserum, the localization of DLK/MUK/ZPK was then investigated in NIH 3T3 cells by a combination of microscopic and biochemical experimental approaches. The results of these studies demonstrate that DLK/MUK/ZPK is specifically localized to the Golgi apparatus. We further found that DLK/MUK/ZPK has two of the properties expected for a peripheral and cytoplasmically disposed Golgi membrane protein. It partitioned solely into the aqueous phase of Triton X-114 extraction and was completely digested with trypsin under conditions in which the integrity of Man II was retained. In view of these findings, we propose that DLK/MUK/ZPK is a peripheral protein bound to the cytoplasmic face of Golgi membranes in NIH 3T3 cells. Support for this idea is provided by the observation that the amino acid sequence of DLK/MUK/ZPK has neither a putative signal peptide nor a hydrophobic membrane-spanning domain (
Because DLK/MUK/ZPK behaves like a hydrophilic protein, as suggested by Triton X-114 extraction, its association with Golgi membranes may be mediated via binding to the cytoplasmic tail of an integral Golgi membrane protein. Like other members of the MLK family, DLK/MUK/ZPK contains domains that are beleived to be involved in proteinprotein interactions, including several putative proline-rich SH3 binding motifs and a leucine/isoleucine zipper domain. These motifs may contribute to the interaction or association of DLK/MUK/ZPK with a specific integral membrane protein. Recently, it was demonstrated that MLK2/MST and MLK3/SPRK/PTK1 interacts in vitro with the small GTP binding proteins Rac and Cdc42, which are known to activate the JNK/SAPK and p38 MAPK pathways (
The finding that DLK/MUK/ZPK is localized to the Golgi complex in NIH 3T3 fibroblasts raises the intriguing possibility that this protein kinase plays a regulatory role in vesicle biogenesis and/or transport. Intracellular trafficking through the eukaryotic secretory pathway is a complex process controlled by the action of multiple mechanisms, including phosphorylation events (
Indirect support for the idea that other MLK family members may somehow contribute to Golgi function is provided by the findings of
In summary, we have demonstrated that endogenous DLK/MUK/ZPK is specifically localized to the Golgi apparatus in NIH 3T3 cells, in which it behaves like a peripheral membrane protein facing the cytosol. These observations raise the possibility that this protein kinase is involved in the processes of intracellular lipid and protein transport, although there is no experimental evidence at present. Studies addressing the identification of the protein partners and physiological targets of DLK/MUK/ZPK should help us to determine whether this kinase could contribute to secretion.
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Acknowledgments |
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Supported by a grant from the Natural Sciences and Engineering Research Council of Canada. M.D. is the recipient of a studentship from the Natural Sciences and Engineering Research Council of Canada.
We thank Dr Adrien R. Beaudoin and Ms Sheila MacLean for critical reading of the manuscript.
Received for publication February 3, 1999; accepted April 29, 1999.
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