Figure 7.

NRBF2 is required for CCZ1-MON1A-associated PIK3C3 kinase activity and complex formation. (A) Endogenous CCZ1 interacts with endogenous PIK3C3 in the mouse brain as reflected by immunoprecipitation. (B) Endogenous CCZ1 interacts with endogenous PIK3C3 in N2a cells as reflected by immunoprecipitation. (C-E) CCZ1 interacts with PIK3C3, and nrbf2 KO attenuates the interaction of CCZ1 with PIK3C3 in mouse brains. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the relative control. (F and G) nrbf2 KO attenuates the colocalization of CCZ1-MON1A with GFP-2xFYVE, a chimeric protein that specifically binds PtdIns3P. After N2a cells were transiently transfected with GFP-2xFYVE plus FLAG-CCZ1 or FLAG-MON1A plasmids, the colocalization was visualized and representative images were shown. Scale bar: 2.5 μm. (H) Quantification of CCZ1-IPed PIK3C3 kinase activity in WT and nrbf2^-/- mice brain. (I and J) Quantification of CCZ1-IPed PIK3C3 kinase activity in WT and nrbf2^-/- N2a cells, and the rescue of impaired CCZ1-associated PIK3C3 kinase activity by overexpressing Flag-NRBF. (K) Rescue of impaired interaction between CCZ1 and PIK3C3, RAB7 in nrbf2^-/- cells by overexpressing Flag-NRBF2