Objective: Mitofusin 2 (Mfn2) is an important suppressor of vascular smooth muscle cell (VSMC) 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. Methods and results: We introduced two mutations at S442 in rat Mfn2, and investigated their effects using cultured rat VSMCs and the balloon injury model. Our 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 in culture, and neointimal hyperplasia and restenosis in the rat carotid artery balloon injury model at days 14 after injury. 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. Conclusions: These 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. (C) 2010 Elsevier Ireland Ltd. All rights reserved.