Figure 4.

HBP1 is phosphorylated at Ser-372/Ser-380 by Pim-1. A, HBP1 is phosphorylated by Pim-1 in vivo. HEK293T cells were cotransfected HA-HBP1 with or without FLAG-Pim-1. An IP assay was carried out using anti-HA antibody followed by Western blotting with anti-HA, -phosphoserine, or -phosphothreonine antibodies. HeLa cells were stably transfected with pITA, pITA-FLAG-Pim-1, pLL3.7, or pLL3.7-shPim-1 through lentiviral infection, respectively. An IP assay was carried out using anti-HBP1 antibody followed by Western blotting with anti-HBP1 or anti-phosphoserine antibodies. B, HBP1 is phosphorylated by Pim-1 in vitro. Purified His-tagged HBP1 protein was incubated with or without His-Pim-1 in the presence of ATP. The reaction products were separated by SDS-PAGE and immunoblotted (IB) with the anti-His, -phosphoserine, or -phosphothreonine antibodies. C, schematic representation of wild-type HBP1 along with its single or double point mutants. D, HBP1 is phosphorylated at Ser-372 and Ser-380 by Pim-1 in vivo. HEK293T cells were cotransfected wild-type HBP1 or its single or double point mutants with or without HA-Pim-1. An IP assay was carried out using anti-FLAG, and the phosphorylation of wild-type and mutant HBP1 was assessed by Western blotting using antibodies against FLAG and phosphoserine (HBP1). E, HBP1 is phosphorylated at Ser-372 and Ser-380 by Pim-1 in vitro. Purified His-tagged wild-type HBP1 or its mutants were incubated with or without His-Pim-1 in the presence of ATP. The reaction products were separated by SDS-PAGE and immunoblotted with the anti-His or -phosphoserine (HBP1) antibodies. F, the increase of HBP1 protein levels induced by Pim-1 depend on Ser-372/Ser-380 phosphorylation. The protein levels of HBP1 and Pim-1 were measured by Western blotting in HEK293T cells transfected with FLAG-tagged HBP1, double point mutant S372A/S380A, or GFP with or without Pim-1 transfection. Levels of GFP protein are shown as equal transfection efficiency.