Lamin A Ser404 Is a Nuclear Target of Akt Phosphorylation in C2C12 Cells

• Published in: Journal of proteome research 2008-11, Vol.7 (11), p.4727-4735
• Main Authors: Cenni, Vittoria, Bertacchini, Jessika, Beretti, Francesca, Lattanzi, Giovanna, Bavelloni, Alberto, Riccio, Massimo, Ruzzene, Maria, Marin, Oriano, Arrigoni, Giorgio, Parnaik, Veena, Wehnert, Manfred, Maraldi, Nadir M, de Pol, Anto, Cocco, Lucio, Marmiroli, Sandra
• Format: Article
• Language: English
• Subjects: Animals; Cell Line; Cell Nucleus - genetics; Cell Nucleus - metabolism; Humans; Kidney - cytology; Lamin Type A - genetics; Lamin Type A - metabolism; Mice; Myoblasts - metabolism; Phosphorylation; Proteomics - methods; Proto-Oncogene Proteins c-akt - genetics; Proto-Oncogene Proteins c-akt - metabolism; Recombinant Proteins - metabolism; Serine - metabolism; Transfection
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr800262g

Akt/PKB is a central activator of multiple signaling pathways coupled with a large number of stimuli. Although both localization and activity of Akt in the nuclear compartment are well-documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. A proteomic-based search for nuclear substrates of Akt was undertaken, exploiting 2D-electrophoresis/MS in combination with an anti-Akt phosphosubstrate antibody. This analysis indicated lamin A/C as a putative substrate of Akt in C2C12 cells. In vitro phosphorylation of endogenous lamin A/C by recombinant Akt further validated this result. Moreover, by phosphopeptide analysis and point mutation, we established that lamin A/C is phosphorylated by Akt at Ser404, in an evolutionary conserved Akt motif. To delve deeper into this, we raised an antibody against the lamin A Ser404 phosphopeptide which allowed us to determine that phosphorylation of lamin A Ser404 is triggered by the well-known Akt activator insulin, and is therefore to be regarded as a physiological response. Remarkably, expression of S404A lamin A in primary cells from healthy tissue caused the nuclear abnormalities that are a hallmark of Emery-Dreifuss muscular dystrophy (EDMD) cells. Indeed, it is known that mutations at several sites in lamin A/C cause autosomal dominant EDMD. Very importantly, we show here that Akt failed to phosphorylate lamin A/C in primary cells from an EDMD-2 patient with lamin A/C mutated in the Akt consensus motif. Together, our data demonstrate that lamin A/C is a novel signaling target of Akt, and implicate Akt phosphorylation of lamin A/C in the correct function of the nuclear lamina.