Figure 2.
NPHP-disease mutants of Nek8 retain full kinase activity. (A) Schematic diagram of Nek8 showing the N-terminal kinase and C-terminal RCC1-like domains. The start and end positions as well as the amino acids mutated in this study are indicated. (B). Kinase assays were performed and analysed as described in Figure 1 on immunoprecipitates prepared from HEK 293 cells transfected for 24 h with the constructs indicated and using histone H1 as substrate. (C) The kinase activity of the different Nek8 proteins relative to the wild-type is indicated. (D) Kinase assays were performed and analysed as described in Figure 1 on immunoprecipitates prepared from HEK 293 cells that were either mock-transfected or transfected for 24 h with wild-type Nek8, the kinase domain fragment (Kin) or the RCC1 domain fragment (RCC1). (E) The kinase activity of the different Nek8 proteins relative to the wild-type is indicated. (F) HEK 293 cells were transiently transfected with GFP alone or GFP-RCC1 for 24 h before cells were lysed and immunoprecipitates prepared with anti-GFP antibodies. Immunoprecipitates were then subject to western blot with anti-GFP antibodies (IP-Blot) or used in kinase assays with the commercial Nek8 protein. Kinase assays were then subject to SDS–PAGE and autoradiography (32P). Data in (C) and (E) represent means (±SD) of three separate experiments. Molecular weights (kDa) are indicated on the left of gels in (D) and (F).