Figure 2

HIRA is phosphorylated by Akt1 at serine 650. (a) An in vitro kinase assay was performed with purified GST or GST-HIRA(640–655; WT or S650A) and 80 ng of Akt1. Reaction mixtures were subjected to SDS-PAGE followed by autoradiography to detect P³²-labeled HIRA. Akt1 shows autophosphorylation. *GST and partial degradation products. (b) Full-length HIRA WT or S650A (3 μg each) purified from insect cells was phosphorylated in the presence and absence of 80 ng of Akt1 and cold ATP. Phosphorylation of HIRA was detected using an anti-phospho-HIRA antibody (p-HIRA). (c) Verification of anti-phospho-HIRA antibody. The specificity of the purified p-HIRA antibody was tested by dot blot assay. The indicated amounts of peptides with phosphorylated Ser 650 (RPRKD(pS)RLMPV) or their unmodified counterparts (RPRKDSRLMPV) were spotted on the membrane, and immunoblotting was performed using an anti p-HIRA antibody. The antibody specifically recognized the p-peptides phosphorylated at Ser650. (d) Whole-cell extracts from 293T cells were subjected to IP using an anti-p-HIRA antibody in the presence or absence of phospho- or unmodified peptides acting as competitors. Immunoprecipitates were probed with WC119 to detect HIRA. Rabbit IgG was used as the negative control. (e) 293T cells were treated with MK2206 (10 μM) for 24 h and whole-cell extracts were used for immunoblotting with the indicated antibodies. Tubulin was used as the loading control. IP, immunoprecipitation; WT, wild type.