Fig. 2

Interaction of Akt and VRK2 in mammalian cells. a, b HT-1080 cells (ATCC) were cultured in the presence of 25 μM chloroquine before rapamycin treatment (10 μM) or HBSS treatment for 4 h to induce autophagy. Endogenous Akt interacted with VRK2 in HT-1080 cells (lanes 2, 4, and 6 in the top panel: non-, HBSS-, or rapamycin-treated groups, respectively) in coimmunoprecipitation assays [76, 98, 99]. Expression of endogenous VRK2 under the three treatment conditions (top panel): total Akt (CST, #9272, second panel) is shown (right-hand panels). Note that the interaction of Akt with VRK2 appears to be enhanced after HBSS treatment, compared with the untreated cells (a, second panel, compare lane 2 with 4, and non- vs. HBSS-treated cells, respectively). c FLAG-VRK2, HA-Akt2, and EGFP-Phafin2 were cotransfected into 293T cells, and coimmunoprecipitation assays were performed [76]. Kinase-dead (KD) Akt (T308A or T308A/S473A) failed to interact with VRK2. Please note that S473A Akt only weakly interacts with VRK2, compared to T308A/S473A double mutant Akt. Expression and schematic structure of each construct are shown (right-hand panels). d KD VRK2 (K61A/K169E) interacted weakly with Akt as compared to the WT VRK2 in a coimmunoprecipitation assay using 293T cells. The schematic structure and expression of each construct are shown (right-hand panels). e FLAG-VRK2, HA-Akt2, and EGFP-Phafin2 were cotransfected into 293T cells, and coimmunoprecipitation assays were performed with anti-FLAG and anti-GFP antibodies and immunoblotting with an anti-GFP antibody (top panel), anti-HA antibody (second panel), and anti-FLAG antibody (third panel). Akt interacted with Phafin2 and VRK2 (lanes 2 and 4, middle panel), but no interaction between Phafin2 and VRK2 was detected (third panel)