Proteomics exploration reveals that actin is a signaling target of the kinase Akt.
The serine/threonine kinase Akt is a key mediator of cell survival and cell growth that is activated by most growth factors, but its downstream signaling largely remains to be elucidated. To identify signaling partners of Akt, we analyzed proteins co-immunoprecipitated with Akt in MCF-7 breast cancer cells. Mass spectrometry analysis (MALDI-TOF and MS-MS) of SDS-PAGE-separated Akt co-immunoprecipitates allowed the identification of 10 proteins: alpha -actinin, valosin-containing protein, inhibitor kappaB kinase, mortalin, tubulin beta, cytokeratin 8, actin, 14-3-3sigma, proliferating cell nuclear antigen, and heat shock protein HSP27. The identification of these putative Akt binding partners were validated with specific antibodies. Interestingly, the major protein band observed in Akt co-immunoprecipitates was found to be the cytoskeleton protein actin for which a 14-fold increase was observed in Akt-activated compared with non-activated conditions. The interaction between Akt and actin was further confirmed by reverse immunoprecipitation, and confocal microscopy demonstrated a co-localization specifically induced under growth factor stimulation. The use of wortmannin indicated a dependence on the phosphatidylinositol 3-kinase pathway. Using a phospho-Akt substrate antibody, the phosphorylation of actin on an Akt consensus site was detected upon growth factor stimulation, both in cellulo and in vitro, suggesting that actin is a substrate of Akt kinase activity. Interestingly, cortical remodeling of actin associated with cell migration was reversed by small interfering RNA directed against Akt, indicating the involvement of Akt in the dynamic reorganization of actin cytoskeleton germane to breast cancer cell migration. Together these data identify actin as a new functional target of Akt signaling.
Vandermoere
Franck
F
ERI-8 INSERM (JE-2488), University of Sciences and Technologies Lille, Villeneuve d'Ascq, France.
El Yazidi-Belkoura
Ikram
I
Demont
Yohann
Y
Slomianny
Christian
C
Antol
Johann
J
Lemoine
Jérôme
J
Hondermarck
Hubert
H
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity / Q000187:drug effects; D014407:Tumor Cells, Cultured
D000199:Actins; D009363:Neoplasm Proteins; D016222:Fibroblast Growth Factor 2; D051057:Proto-Oncogene Proteins c-akt