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First published online 20 January 2004
doi: 10.1242/jcs.00897

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Phosphorylation of golgin-160 by mixed lineage kinase 3

¹ Department of Pharmaceutical Sciences, University of Maryland-School of Pharmacy, Baltimore, MD 21201, USA
² Department of Cell Biology, Johns Hopkins University-School of Medicine, Baltimore, MD 21205, USA
³ Department of Physiology, Michigan State University, East Lansing, MI 48824, USA

View larger version (26K): [in a new window]   Fig. 2. The MLK inhibitor CEP-11004 blocks MLK3-induced phosphorylation of golgin-160. (A) HeLa cells were transiently transfected with Myc-tagged golgin-160 in the absence or presence of MLK3 and then treated in the absence or presence of CEP-11004 (0.5 µM) during the last 5 hours of the experiment. Protein lysates were separated by SDS-PAGE for 20 hours at 20 mA to achieve separation of phosphorylated forms of golgin-160 (top) and MLK3 (bottom). The gel shift of MLK3 is due to autophosphorylation. (B) CEP-11004 blocks MLK3 but not active DLK. Cells were transfected with HA-tagged JNK1 alone or in the presence of DLK or MLK3 followed by treatment with or without 0.5 µM CEP-11004. Lysates were immunoblotted for phosphorylated JNK1 (pJNK), HA-JNK1, DLK (Flag tag) or MLK3 expression. The activation level of JNK shown in the lower graph was determined by densitometry as described in Materials and Methods. (C) CEP-11004 does not inhibit MKK1. Cells transfected with wild-type or inactive (KR) MLK3 or active MKK1 were treated in the absence or presence of CEP-11004. Lysates were immunoblotted for active ERK (ppERK, top) or -tubulin (bottom) as a protein loading control. The lower graph shows densitometry analysis of the relative amounts of ppERK in each condition. The activation level of ERK was determined by densitometry, as described in Materials and Methods. Data represent two separate experiments.

View larger version (34K): [in a new window]   Fig. 1. Phosphorylation of golgin-160 by MLK3- and Rac-dependent pathways. (A) Phosphorylation of endogenous golgin-160 is enhanced in doxorubicin-treated cells. HeLa cells were metabolically labeled with 35S-methionine and 35S-cysteine for 2 hours and chased in normal growth medium in the absence or presence of doxorubicin (120 µM) for 4 hours. Immunoprecipitated golgin-160 was mock-treated or treated with protein phosphatase and visualized by fluorigraphy after SDS-PAGE. Elimination of the slower migrating forms in the presence of phosphatase indicates that golgin-160 is a phosphoprotein. Enhanced phosphorylation was observed in doxorubicin-treated cells. (B) Phosphorylation was examined by analysing slower-migrating forms of expressed golgin-160 in CHO cells by immunoblotting, using the anti-Myc (9E10) antibody, for Myc-tagged golgin-160 in cells transfected with wild-type MLK3 (WT) or catalytically inactive MLK3 (KR). The gel was run for 20 hours at 20 mA to enhance the separation of the phosphorylated forms of golgin-160. (C) Myc immunoblot of expressed golgin-160 in cells transfected with cDNA for wild-type (WT) or inactive (KR) MLK3 and inactive (N-) or active (V-) Rac. (D) Myc immunoblot of expressed golgin-160 in cells transfected with cDNA for v-Rac, dual leucine zipper-bearing kinase (DLK), wild-type MKK1 (WT) or a constitutively active MKK1 mutant. The upper and lower arrows in (B-D) indicate the position of the hyperphosphorylated (p) and hypophosphorylated (g160) forms of golgin-160, respectively. Data represent at least four experiments.

View larger version (71K): [in a new window]   Fig. 3. MLK3 interacts with the N-terminal region of golgin-160. (A) EGFP-tagged golgin-160 deletion mutants containing N-terminal regions spanning amino acids 1-59 (lane 1), 1-139 (lane 2) or 96-393 (lane 3) were transiently expressed with wild-type MLK3 in CHO cells. Following GFP immunoprecipitation, protein complexes were resolved by SDS-PAGE and immunoblotted for MLK3 (top) and GFP (bottom). (Left) The MLK3 and EGFP-tagged golgin-160 proteins in the immunoprecipitations. (Right) The expression of MLK3 and EGFP-tagged golgin-160 proteins in the total cell lysates (10% of the input for the immunoprecipitations). Data represent three separate experiments. (B) MLK3 interacts with GST-tagged golgin-160(1-393). MLK3 or Akt-1 were transcribed and translated in vitro in the presence of 35S-methionine. GST or GST/golgin-160(1-393) pre-bound to glutathione-agarose beads were incubated with the labeled proteins and bound material was resolved by SDS-PAGE and autoradiography. The input is 10% of the starting material added to each pull-down. These data are representative of three independent experiments.

View larger version (49K): [in a new window]   Fig. 4. MLK3 is localized in the juxtanuclear Golgi region. HeLa cells were left untreated or transiently transfected with HA-tagged wild-type MLK3. The localization of endogenous (A) and expressed MLK3 (B) was examined by immunofluorescence in HeLa cells that were co-stained with GM130 as a marker of the Golgi complex. The merged image (lower right) of MLK3 (Texas Red) and GM130 (FITC) demonstrates overlap between the two proteins in the juxtanuclear Golgi region. The nuclear DNA staining with DAPI is shown for reference. Scale bar, 10 µm.

View larger version (41K): [in a new window]   Fig. 5. MLK3 phosphorylates the N-terminal head region of golgin-160. HeLa cells were transfected with GFP golgin-160 mutants encompassing specific amino acid regions of the protein. The golgin-160 proteins were immunoprecipitated and incubated with expressed MLK3 isolated from CHO cells and phosphate incorporation was determined in a kinase assay. (A) (Top) GFP immunoblot of golgin-160 proteins in the kinase reaction mixture after incubation for 60 minutes in the absence or presence of MLK3. (Bottom) Relative phosphate incorporation into golgin-160 wild-type and mutant proteins in the absence (open bars) or presence (closed bars) of MLK3. The phosphorylation of the golgin-160 mutants was normalized to the phosphorylation of golgin-160 wild type in the absence of MLK3, which was set at a value of 1. (B) (Top) Immunoblot of GFP/golgin-160(1-393) and GFP/golgin-160(393-1498) in the kinase reaction mixture after incubation in the absence or presence of MLK3 for 20 minutes or 40 minutes. (Bottom) Phosphate incorporation in the golgin-160(1-393) mutant incubated in the absence (open bars) or presence (closed bars) of MLK3 for 20 or 40 minutes. No phosphate incorporation was observed in the golgin-160(393-1498) fragment, even though the expression levels for both mutant proteins were the same (top). MLK3 increased phosphorylation of golgin-160 wild type and the 1-393 N-terminal mutant by 2.5-6.2 times in three or four separate experiments.

View larger version (56K): [in a new window]   Fig. 6. In vitro phosphorylation of the N-terminal region of golgin-160 by MLK3. Bacterially expressed, purified GST or GST-tagged golgin-160 mutants (1-393) or (96-259) were incubated with MLK3 immunoprecipitated from HeLa cells for 20 minutes or 60 minutes in an in vitro kinase assay. (A) (Left) The incorporation of phosphate into GST or GST/golgin-160(1-393) after a 60 minute incubation in the absence or presence of MLK3. (Right) The immunoblot for GST. (B) (Top) The incorporation of phosphate into GST/golgin-160(96-259) after a 20 minute or 60 minute incubation in the absence or presence of MLK3. (Middle, bottom) The immunoblots for GST (middle) and MLK3 proteins (bottom). (C) Purified GST/golgin-160(1-393) was incubated with or without immunoprecipitated MLK3 in the absence or presence of CEP11004 (0.5 µM). Golgin-160 phosphorylation and MLK3 expression are shown in the top two panels. Total GST/golgin-160(1-393) expression by Coomassie staining (CS) or immunoblotting (IB) are shown in the lower two panels.

View larger version (56K): [in a new window]   Fig. 7. MLK3 enhances golgin-160 cleavage at aspartate at residue 139. HeLa cells stably expressing EGFP-tagged golgin-160 were transfected with wild-type (WT) or catalytically inactive (KR) MLK3. Cell lysates were fractionated into soluble cytosolic and membrane protein fractions, and 10% of each fraction was analysed by immunoblotting. (A) (Top) An immunoblot for GFP. The arrows indicate the position of full-length EGFP-tagged golgin-160 and EGFP/golgin-160(1-139). The presence of the caspase inhibitor Boc-D-FMK (10 µM) in wild-type MLK3 transfected cells (WT/CI) during the last 24 hours of incubation inhibited production of the golgin-160 cleavage product in the cytosolic protein fraction. Protein loading controls show the expression of p97 golgin and -tubulin. MLK3 was found primarily in association with the membrane fraction. (B) (Top) A GFP immunoblot of the cytosolic fraction from stable cells transfected with wild-type or inactive MLK3 and compared with an EGFP-tagged golgin-160 mutant spanning residues 1-139. (Middle) Immunoblot of golgin-160 using an antibody specific for the N-terminus. (Bottom) Immunoblot for -tubulin as a protein loading control. (C) Immunoprecipitation of EGFP-tagged proteins from soluble cytosolic fraction isolated from stable cells transfected with wild-type or inactive MLK3 using a monoclonal GFP antibody. EGFP-tagged proteins were visualized following immunoblotting with a polyclonal GFP antibody. Data represent at least three separate experiments.