1. GAK phosphorylates Atp1a3 at novel site T705 in HEK 293Ts.
GAK phosphorylates Atp1a3 at novel site T705 in HEK 293Ts. (A) Atp1a3 is immunoprecipitated by GAK from HEK 293T cells co-transfected with myc-tagged FL GAK WT and HA-tagged Atp1a3 WT (left panel). Atp1a3 is also able to be immunoprecipitated by myc when cells are co-transfected with myc-tagged GAK1–400 and HA-tagged Atp1a3 WT (right panel). HA-tagged and myc-tagged pRK5 vectors have been used as IP controls. (B) Atp1a3 pT705 is present in HEK 293T samples co-transfected with Atp1a3 WT and either FL GAK WT or GAK1–400 (left). Atp1a3 T705A mutants show reduced pT705, as do GAK KD mutants (centre). GAK1–400 pT705 levels are similar compared to FL GAK (right). Graph data represent means ± SEM, n = 3 independent experiments, GAK FL (WT/TA P = 0.02, KD/WT P = 0.001, KD/TA P = 0.005), GAK1–400 (WT/TA P = 0.04, KD/WT P = 0.05, KD/TA P = 0.009), *P < 0.05, **P < 0.01, ***P < 0.001, t test. (C) HEK 293T cells co-transfected with HA-tagged FL GAK WT and HA-Atp1a3 WT and pretreated in serum-free media were incubated with either DMSO or 5 μM of GAK inhibitor 12g, 12i, or 12g+i for 1 h n = 3 independent in duplo experiments. Graph data represent means ± SEM, 12g P = 0.009, 12i P = 0.009, 12g+i P = 0.03; *P < 0.05, **P < 0.01, t test. (D) Co-transfections of HEK 293T cells with HA-tagged Atp1a3 WT and either HA-tagged GAK or AAK1 demonstrate that GAK is the primary kinase phosphorylating Atp1a3 at T705. n = 5 independent experiments.
Amy W Lin et al. LSA 2018;1:e
© 2018 Lin et al.