Fig. 4

MAPK14 phosphorylates CRN5 at serine 423. a GFP-CRN5 was immunoprecipitated from lysates of U373 cells using mAb K77-578-1. The GFP-CRN5 band at 83 kDa was cut from the Coomassie stained gel, subjected to mass spectrometry, and a phosphorylation was identified at serine 423 (highlighted in black). Underlined, MAPK14 phosphorylation consensus target motif. Amino acid sequences highlighted in grey indicate the sequence coverage by mass spectrometry. b GST-MAPK14 expressed in E. coli BL21 and HEK293 cells. MAPK14 derived from the latter is phosphorylated at Thr180 and Tyr182 as determined by phospho-MAPK14 pAb D3F9, and is visible at a slightly higher molecular weight than the non-phosphorylated protein derived from the bacteria. c Co-immunoprecipitation studies were carried out using the CRN5 mAb K77-578-1 coupled to Protein G-coated magnetic microbeads (Miltenyi), which were first incubated with GST-CRN5 purified from E. coli BL21 and afterwards incubated with either GST-MAPK14 purified from HEK293 cells or GST purified from E. coli BL21 for control. Analysis was done by GST rabbit pAb [21] immunoblotting. d A pull-down assay was performed using GST-CRN5 or GST coated glutathione-sepharose beads together with non-tagged MAPK14. All three recombinant proteins were purified from E. coli BL21 as GST-fusion proteins; from MAPK14 the tag was cleaved by TEV-protease. Analysis was performed using CRN5 mAb K77-578-1, MAPK14 rabbit pAb, and the GST pAb. c, d For illustration purposes the order of lanes from the original immunoblots were digitally re-arranged to omit dispensable lanes. e Immunofluorescence images show an enrichment and co-localization of CRN5 and MAPK14 at the front of lamellipodia (arrows). U373 cells stably over-expressing GFP-CRN5 were methanol fixed; detection of MAPK14 was done with pAb #9212 and secondary antibody goat anti-mouse AlexaFluor 568. f In vitro kinase assay employing full-length GFP-CRN5 and GST-tagged MAPK14 purified from HEK293 cells. Time course (10, 20, 60, 90 min) of phosphate incorporation from [γ-³²P]ATP into GFP-CRN5. As controls the selective, ATP-competitive MAPK14 kinase inhibitor SB202190 was used, and samples lacking GST-MAPK14 or GFP-CRN5. Left panel, autoradiograph (³²P); right panel, corresponding Coomassie brilliant blue stained gel. Note, that the part of the autoradiograph showing the time-dependent phosphorylation of GFP-CRN5 is presented with increased contrast settings (dotted rectangle), because of the high background signals from MAPK14 autophosphorylation