Abstract 389: Mitochondrial p27 Is Absolutely Required for Endothelial Cell Migration and ATP Production
The cell cycle inhibitor p27 has been shown to exert non-nuclear functions. Moreover, migration of fibroblasts depends on p27 expression. However, the underlying mechanisms of this phenomenon are largely unknown. Therefore, we first investigated the role of p27 in endothelial cell (EC) migration and showed that its downregulation abrogated migration. Since we previously demonstrated that migration of EC requires intact mitochondria, we analyzed whether it depends on the localization of p27 within the cell. We generated p27 variants, which are exclusively localized either in the nucleus (nucp27) or in the mitochondria (mitop27). Overexpression of mitop27 enhanced EC migration, whereas nucp27 decreased it. Along the same line, mitop27 induced ATP production, which is required for migratory processes. As a potential mechanism for the increased ATP production, we found that mitochondrial p27 interacts with prohibitin, which forms multimeric complexes thereby enhancing respiration and ATP production. Next, we analyzed, which domains in p27 are involved in these processes. Therefore, we created p27 deletion mutants and found that both the N- and the C-terminus are necessary for cell migration and ATP-production. These regions contain several phosphorylation sites, two of which (Ser 10 and Thr 187) are phosphorylated by pro-migratory stimuli. Thus, we cloned corresponding nonphosphorylatable (p27 S10A/T187A) and phosphomimetic (p27 S10D/T187D) mutants of mitop27. Overexpression of the nonphosphorylatable mutant decreased EC migration, while the phosphomimetic mutant increased it. Finally, we investigated the mitochondrial import mechanism of p27. Since p27 lacks a typical mitochondrial targeting sequence, we determined whether it interacts with cytosolic heat shock protein 60 (HSP60), which is known to import proteins into the mitochondria. Indeed, cytosolic p27 is associated with HSP60 and this association is increased under promigratory stimuli.
Taken together, these data demonstrate that mitochondrial p27 and its phosphorylation at the N- and C-termini are required for migration and ATP production and that p27 is imported into the mitochondria in a HSP60 dependent manner.
- © 2012 by American Heart Association, Inc.