A Mitochondrial Kinase Complex Is Essential to Mediate an ERK1/2-Dependent Phosphorylation of a Key Regulatory Protein in Steroid Biosynthesis (original) (raw)
Figure 4
PKA and ERK1/2 are strictly required to achieve maximal progesterone production by isolated mitochondria.
MA-10 cells were transiently transfected by electroporation with an empty vector or with a vector containing StAR cDNA (sense or antisense orientations). Mitochondria were incubated in the presence of cholesterol as substrate (a) in the presence of wild type ERK1-GST protein alone (b) or together with PKA catalytic subunit (d). The mutated inactive form of ERK1-GST (K71A) was also used (c and e). After the indicated incubations, mitochondria were pelleted and subjected to SDS-PAGE and Western blot (A). Specific antibodies that recognize StAR protein, the catalytic subunit of PKA, pMEK1/2, pERK1/2 and total ERK1/2 were used. The panel shows representative Western blots of three independently performed experiments. P4 production is shown in (B). Data are expressed as means±SD (n = 3). * p<0.05 bar b vs. bar a and § p<0.05 bar d vs. bar b. (C) MA-10 cells were treated with or without 0.5 mM of 8Br-cAMP for 3 hours; cytosolic and mitochondrial subcellular fractions were obtained and incubated in the presence or absence of human pERK1-GST bound to agarose beads. Input and pelleted proteins bound to pERK1-GST (complexes) were analyzed by SDS-PAGE and Western blot. The immunoblots show the bands corresponding to StAR and pERK1/2, as loading control. Data are representative of three independently performed experiments.