Threonine 308 phosphorylated form of Akt translocates to the nucleus of PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin.
We have examined the issue of whether or not in PC12 cells it may be observed a nerve growth factor (NGF) nuclear translocation of an active (phosphorylated) Akt. Western blot analysis with antibodies to either total or phosphorylated Akt showed a maximal nuclear translocation after 15 min of NGF stimulation. NGF increased rapidly and transiently the enzymatic activity of immunoprecipitable nuclear Akt and after 45 min the values returned to a level close to the basal one. Enzyme translocation was blocked by the selective phosphoinositide 3-kinase inhibitor, LY294002. Confocal microscopy of samples stained with antibody to Akt showed an evident increase in immunostaining intensity in the nuclear interior after NGF treatment. Treatment of cells with inhibitors of protein phosphatase PP2A, calyculin A, or okadaic acid, maintained the phosphorylation levels of nuclear Akt. Immunoprecipitation experiments revealed an association between Akt and PP2A that was maximal when nuclear Akt activity was decreased. Both total and active Akt associated with the nuclear matrix and, in particular, with the protein nucleolin, with which Akt co-immunoprecipitated. These findings strongly suggest that the intranuclear translocation of active Akt is an important step in the signaling pathways elicited by the neurotrophin NGF and that the intranuclear control of Akt is achieved through the action of PP2A.
Borgatti
Paola
P
Dipartimento di Morfologia ed Embriologia, Sezione di Anatomia Umana, Università di Ferrara, Ferrara, Italy.
Martelli
Alberto M
AM
Tabellini
Giovanna
G
Bellacosa
Alfonso
A
Capitani
Silvano
S
Neri
Luca M
LM
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology*; D015530:Nuclear Matrix / Q000187:drug effects / Q000378:metabolism*; D016716:PC12 Cells; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D000097796:Nucleolin
D020932:Nerve Growth Factor; D010750:Phosphoproteins; D010769:Phosphothreonine; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C494920:Akt1 protein, rat