Lamin A Ser404 is a nuclear target of Akt phosphorylation in C2C12 cells.
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.
Cenni
Vittoria
V
Department of Anatomy and Histology and CIPro Proteomics Centre, University of Modena and Reggio Emilia, Via Del Pozzo 71, I-41100 Modena, Italy.
Bertacchini
Jessika
J
Beretti
Francesca
F
Lattanzi
Giovanna
G
Bavelloni
Alberto
A
Riccio
Massimo
M
Ruzzene
Maria
M
Marin
Oriano
O
Arrigoni
Giorgio
G
Parnaik
Veena
V
Wehnert
Manfred
M
Maraldi
Nadir M
NM
de Pol
Anto
A
Cocco
Lucio
L
Marmiroli
Sandra
S
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D034904:Lamin Type A / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D032446:Myoblasts / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D014162:Transfection
D034904:Lamin Type A; D011994:Recombinant Proteins; D012694:Serine; D051057:Proto-Oncogene Proteins c-akt