Figure 4.

AKAP121 increases src-dependent phosphorylation and activity of components of the mitochondrial respiratory chain. (A) HEK293 cells were transiently transfected with vectors encoding AKAP121, PTPD1, and kinase-inactive form of Src (src K^–). Where indicated, cells were treated with the src inhibitor PP2, 30 min before harvesting. Mitochondrial fractions were immunoblotted with anti-phosphotyrosine antibody. A representative set of autoradiograms is shown. (B) HEK293 cells were transiently transfected with control (CMV), AKAP121, or AKAP84_Δ1–30 vectors and harvested 48 h after transfection. Where indicated, PP2 was added to the medium 30 min before harvesting. COX activity was assayed with purified mitochondria. Inset, immunoblot analysis of cell lysates with anti-cytochrome c oxidase II subunit (1, CMV; 2, AKAP121; 3, AKAP84_Δ1–30; AKAP121 +PP2). The data represent the mean ± SEM of five independent experiments. (C) Semiquantitative PCR of total genomic DNA extracted from HEK293 cells stably or transiently transfected with the AKAP121 expression vector. Mitochondrial (cytochrome B, Nadhd) and nuclear (β globin) genomic DNAs were amplified as described in Materials and Methods. The data are shown as fold increase of mitochondrial versus nuclear genomic DNA. * p < 0.05 vs. CMV-transfected cells; ** p < 0.01 vs. CMV-transfected cells. (D) Southern blot analysis of total genomic DNA extracted from stably transfected HEK293 cells. Mitochondrial (mito) and nuclear (β globin) cDNAs were used as probes. Indicated are the fold increases of mitochondrial versus β globin DNA. Values from control, CMV cells (0-time point) were set as 1.