PKA Phosphorylation Site Influences The Function Of Mitofusin 2 Gene

Partâ… Effect of mitofusin 2 with protein kinase A phosphorylation site deletion on the proliferation and apoptosis of vascular smooth muscle cells1. ObjectiveTo investigate the effect of mitofusin 2 (Mfn2) gene with the protein kinase A (PKA) phosphorylation site deletion [Mfn2-PKA(â–³)] on inhibiting the proliferation and inducing the apoptosis of vascular smooth muscle cells (VSMCs) and related signaling pathway.2. MethodsVSMCs of rats were infected by recombinational adenovirus carrying green fluorescent protein (GFP), Mfn2-PKA (â–³) or Mfn2 gene (Adv-GFP, Adv-Mfn2-PKA (â–³), Adv-Mfn2). The abundance of Mfn2-PKA(â–³) protein and Mfn2 protein were determined by Western blot analysis using Mfn2 antibody. Laser confocal microscopy (LCMS) was used to observe the locations of the proteins. The growth curve of the VSMCs was explored by MTT. The effect of Adv-Mfn2-PKA(â–³) on the apoptosis of VSMCs was explored by ELISA. Western blot were used to detect the expression of phosphorylation of ERK1/2 (p-ERK1/2) and protein kinase B (p-Akt).3. ResultsThe Mfn2 and Mfn2-PKA (â–³) both expressed protein-specific bands in VSMCs. Two kinds of gene expression products were mainly located at the out membrane of mitochondria. Compared with the control group and Adv-GFP group, the absorbance values at 3,4,5,6 days were significantly reduced in Adv-Mfn2 group (P< 0.01), and no obvious changes were observed in Adv-Mfn2-PKA (â–³) group. Mfn2-PKA(â–³) had no effect on promoting the apoptosis of VSMCs compared with Mfn2 (P<0.01). Overexpression of Mfn2-PKA(â–³) gene could not down-regulate the expression of p-ERK1/2 and p-Akt (P< 0.01).4. ConclusionsMfn2-PKA (â–³), located at the out membrane of mitochondria, has no effect on suppressing the proliferation and on inducing apoptosis of VSMCs. Mfn2-PKA (â–³) has no inhibition effect on ERK1/2 and Akt signaling pathway. PKA phosphorylation site plays an important role in regulating the function of Mfn2 gene.Partâ…¡Mutation of the protein kinase A phosphorylation site influences the anti-proliferative activity of mitofusin 21. ObjectiveMitofusin 2 (Mfn2) is an important suppressor of vascular smooth muscle cells (VSMCs) proliferation. It contains a protein kinase A (PKA) phosphorylation site at serine 442 (S442) and can be phosphorylated by PKA. This study examined the role of phosphorylating specific sites on the regulation of Mfn2 protein activity in vitro and in vivo.2. MethodsTo investigate the functional significance of Mfn2 phosphorylation, we introduced two separate mutations at the codon for serine 442 in the rat Mfn2 gene. Replacing serine with alanine provided a nonphosphorylatable residue with minimal structural changes to the protein, while replacing the serine residue with aspartic acid mimicked constitutive phosphorylation at this site. In this study, we evaluated changes in Mfn2 protein abundance, mitochondrial morphology and membrane fusion in adenovirus infected rat VSMCs. Since Mfn2 has profound suppressive effects on Ras in VSMCs and might therefore inhibit cell proliferation by modulating the activity of the Ras-Raf-ERK1/2 signaling pathway and the cell cycle, we chose to test the effects of these mutations on proliferation of VSMCs in vitro and in vivo.3. ResultsOur results indicated that, in VSMCs, Mfn2 expression and mitochondrial morphology are affected by adenoviral-mediated overexpression of the two Mfn2 mutant proteins in the same way as the wild-type Mfn2 protein. Specifically, overexpression of the protein harboring the phospho-deficient mutation Mfn2-S442A (serine replaced by alanine at residue 442) increased the inhibitory effects of Mfn2 on proliferation of VSMCs, and stimulation effects on apoptosis of VSMCs in culture. On the other hand, the phospho-mimetic mutation Mfn2-S442D (serine replaced by aspartic acid at residue 442) led to loss of growth suppressor activity.4. ConclusionsThese results suggest that this specific PKA phosphorylation site plays a key role in Mfn2-mediated suppression of VSMC growth, which is independent of its effects on modulation of mitochondrial morphology.Partâ…¢Mutation of the protein kinase A phosphorylation site influences the pro-apoptosis activity of mitofusin 21. ObjectiveTo study the role of Mfn2 gene with protein kinase A phosphorylation site mutations in the apoptosis of VSMCs and related signaling pathways. 2. MethodsTwo novel mutations were constructed, Adv-Mfn2-alaPKA and Adv-Mfn2-asnPKA, then VSMCs were infected with them. The effect of mutations on the apoptosis of VSMCs was explored by flow cytometry analysis. The cell mitochondrial membrane potential was detected by using JC-1 staining method. Western blot were used to detect the expression of Mfn2,p-Akt and cleaved caspase-9.3. ResultsThe expression of Mfn2 protein has no significant difference in Adv-Mfn2, Adv-Mfn2-S442A and Adv-Mfn2-S442D groups. Flow cytometry analysis showed both of Mfn2-S442A and Mfn2 had stronger effect in promoting the apoptosis of VSMCs (P<0.01). The mitochondrial membrane potential in these two groups remarkably decreased (P<0.01). The results of Western blot indicated that the protein expression of p-Akt remarkably decreased, whereas cleaved capased-9 protein highly expressed in Adv-Mfn2-S442A and Adv-Mfn2 group (P<0.01). Mfn2-S442A was superior to Mfn2 in promoting the apoptosis of VSMCs (P<0.01). The effect of Mfn2-S442D was similar to the control.4. ConclusionsMfn2-S442A has more stronger inducing effect on the apoptosis of VSMCs than Mfn2, while Mfn2-S442D has no effect. We can conclude that PKA phosphorylation site plays an important role in regulating the function of Mfn2 gene.