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Cell adhesion differentially regulates the nucleocytoplasmic distribution of active MAP kinases

Andrew E. Aplin1,2,*, Brian P. Hogan1, Jeannie Tomeu1 and R. L. Juliano1

1 Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
2 Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA



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  		Fig. 1. Anisomycin treatment activates JNK regardless of the state of anchorage. Serum-starved detached NIH 3T3 cells were either maintained in suspension (Sus) or plated onto fibronectin (Fn)-coated dishes for 2 hours in DMEM/BSA. (A) Cells at the 2 hour time point were stimulated appropriately with increasing doses of anisomycin (0, 5, 20, 50 and 100 ng/ml) for either 15 minutes or 30 minutes, as indicated. (B) Cells were stimulated with 50 ng/ml anisomycin for 0, 5, 15, 30, 45 and 60 minutes, as indicated. Under each condition, cells were lysed in modified RIPA buffer and endogenous JNK activity determined by immunoprecipitation with C-17 and in vitro kinase assay using GST-c-Jun as the substrate. Incorporation of 32P into GST-c-Jun was determined by autoradiography.

 


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  		Fig. 2. Activated JNK phosphorylates the nuclear transcription factor c-Jun regardless of adhesion. Serum-starved detached NIH 3T3 cells were either plated onto fibronectin (Fn)-coated coverslips or maintained in suspension (Sus) for 2 hours in DMEM/BSA. At this time, cells were stimulated appropriately with anisomycin (50 ng/ml) for 30 minutes. Suspended cells were plated on poly-lysine-coated coverslips for the final 15 minutes. Cells were washed briefly in PBS before fixation. Cells were processed by immunofluorescence for phospho-JNK (A), phosphoserine-63 c-Jun (B), and total c-Jun (B,C). Primary antibody staining was detected with the appropriate fluorophore-conjugated anti-rabbit and anti-mouse IgG. In A, inserts show the overlay between phospho-JNK and nuclear staining with Hoechst 33342 reagent. Bars, 20 µM (A); 50 µM (B,C).

 


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  		Fig. 3. Phosphorylation of c-Jun by activated JNK is adhesion independent over a range of anisomycin concentrations. Serum-starved detached NIH 3T3 cells were either plated onto fibronectin (Fn)-coated dishes or maintained in suspension (Sus) for 2 hours in DMEM/BSA. At this time point, cells were stimulated (A) with increasing doses of anisomycin (0, 5, 20, 50 and 100 ng/ml) for 30 minutes or (B) with 50 ng/ml anisomycin for the times indicated. Under each condition, cells were lysed in modified RIPA buffer and lysates analyzed by western blotting for phosphoserine-63 c-Jun (Phospho63-cJun), phosphoserine-73 c-Jun (Phospho 73-cJun) and total c-Jun. The results shown are representative of three independent experiments.

 


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  		Fig. 4. Phosphorylation of Elk-1 is anchorage-dependent upon activation of the ERK pathway, but anchorage-independent upon activation of the JNK pathway. (A) NIH 3T3 cells were transfected with either {Delta}MEKK1 or 22W Raf. Cells were replated onto fibronectin-coated coverslips. The localization of MEKK1 and Raf were determined by immunofluorescence using either TRITC or FITC-conjugated anti-rabbit secondary antibodies. Bar, 25 µM. (B) Cells expressing FLAG-Elk-1 were plated onto Fn-coated dishes or maintained in suspension (Sus) and analyzed by immunofluorescence with anti-Elk-1 antibodies. Insets show the overlay of Elk-1 and nuclear staining. (C) Cells were transfected with FLAG-Elk-1 and either vector 22W Raf or {Delta}MEKK1. After 24 hours, transfected cells were serum-starved before being replated in DMEM/BSA onto Fn-coated dishes or maintained in suspension (Sus) for a further 3 hours. Ectopically expressed Elk-1 was immunoprecipitated from cell lysates from each condition with an M2 FLAG epitope antibody. FLAG-Elk-1 immunoprecipitates were analyzed by western blotting for levels of Ser383-phosphorylated and total Elk-1. The results shown are representative of at least three independent experiments with equivalent results.

 


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  		Fig. 5. Anisomycin treatment activates p38 in an anchorage-independent manner. Serum-starved detached NIH 3T3 cells were either maintained in suspension (Sus) or plated onto fibronectin (Fn)-coated dishes for 2 hours in DMEM/BSA. (A) At the 2 hour time point, cells were stimulated appropriately with increasing doses of anisomycin (0, 25, 50 and 200 ng/ml) for the 30 minutes. (B) Cells were treated with 50 ng/ml anisomycin for the times indicated. Under each condition, cells were lysed in modified RIPA buffer and cell lysates probed with antibodies to phosphorylated and total p38.

 


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  		Fig. 6. Phosphorylated p38 accumulates in the nucleus in both adherent and non-adherent cells. Serum-starved detached NIH 3T3 cells were either plated onto fibronectin (Fn)-coated dishes or maintained in suspension (Sus) for 2 hours in DMEM/BSA before being stimulated with 50 ng/ml anisomycin for 30 minutes. Suspended cells were allowed to adhere to poly-lysine-coated coverslips for the final 15 minutes. Fixed cells were analyzed by immunofluorescence for phospho-p38. Bars, 20 µM. Inserts show the overlay between phospho-p38 and nuclear staining with Hoechst 33342 reagent.

 


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  		Fig. 7. Expression of ERK2-MEK1-LA phosphorylates Elk-1 efficiently in suspended cells. NIH 3T3 cells were transfected with either pCMV5 (Vec), 22W Raf, or pCMV5-ERK2-MEK1-LA alone (A) or in combination with pCMV5-FLAG-Elk-1 (B). Serum-starved cells were either replated on fibronection (Fn) or maintained in suspension (Sus) for 2 hours in DMEM/BSA. (A) ERK activity was determined by immunoprecipitation with anti-myc antibody followed by western blotting with anti-active ERK antibodies and by in vitro kinase assay using recombinant GST-Elk-1 as the substrate. (B) Elk-1 phosphorylation was determined by immunoprecipition from cell lysates with an anti-FLAG antibody and western blotting for levels of Ser383 phosphorylated and total Elk-1. The results shown are representative of three separate experiments.

 

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