Fig. 7

NUAK1 interacts with Akt and phosphorylates it at Ser-473. A IB of NUAK1 and Akt1 in vitro interaction. Recombinant His-tagged NUAK1 and immunoprecipitated HA-tagged Akt1 were used. B Proximity ligation assay (PLA) in MDA-MB-231 cells expressing FLAG-tagged NUAK1 or Empty vector (EV) (used as a negative control) were serum-starved overnight and stimulated with EGF by 10 min (n = 3). Red dots indicate proximity of FLAG-NUAK1 and Akt. DAPI was used as a nuclear counterstain. C Table of putative phosphoresidues phosphorylated by NUAK1 on Akt1. A consensus phosphorylation motif for NUAK1 obtained from GPS5.0 was used. D Molecular docking between NUAK1-Akt1. NUAK1 kinase domain (Red), Akt hydrophobic motif (Blue), and Ser-473 (Black) are represented. E Autoradiography of in vitro kinase assay using recombinant active GST-tagged NUAK1 and purified Akt1 HA-tagged. F IB of in vitro kinase assay using recombinant active His-tagged NUAK1 and purified Akt1 HA-tagged. Akt Ser-473 and Thr-308 phosphorylation were determined using specific antibodies. G Quantification of Akt S473 phosphorylation from F. Each bar represents the mean ± SD, n = 3. Data from 3 independent experiments were analyzed by Student t test. H IB of in vitro kinase assays using recombinant His-tagged NUAK1 and recombinant GST-tagged Akt1 (left) or recombinant active GST-tagged NUAK1 and recombinant GST-tagged Akt1 (right), respectively. I IB of in vitro kinase assays using purified FLAG-NUAK1 WT or FLAG-NUAK1 K84A (kinase-dead) and purified Akt1 HA-tagged. J IB of in vitro kinase assay using recombinant active GST-tagged Akt1 and recombinant His-tagged NUAK1. NUAK1 Ser-600 phosphorylation was determined using a specific antibody. All kinase assays were performed at least three times, except the kinase assay in J, that was performed two times. K IB of Akt signaling and NUAK1 phosphorylation at Ser-600 after insulin stimulation. GAPDH was used as loading control