Nucleolin: a nuclear Akt substrate

Nucleolin is one of the most representative proteins in nucleoli of actively dividing cells, potentially involved in a large number of nuclear processes like transcription, chromatin organization, rRNA maturation and ribosome assembly (1-2). We observed the presence in nucleolin of the Akt phosphorylation motif, which is highly conserved in several species, raising the hypothesis that nucleolin could be a specific substrate of Akt. However, modulation of nucleolin functions by the PI3K/Akt signaling pathway has not been still investigated. With this study we aimed to demonstrate the functional interaction between Akt kinase and nucleolin. Since Akt translocates into the nucleus after stimulation of several cell lines (3), our goal was to explore Akt and nucleolin binding and Akt enzymatic activity toward this potential nuclear substrate after treatment in different cell models. Materials and methods: we used HL60, Jurkat and PC12 cell in coltures, stimulated with different growth factors. Preparation of whole cell homogenates, immunoprecipitation, immunoblotting and Akt kinase assay were accomplished as previously reported (4-6). Results: nucleolin was recognized in immunoblotting after immunoprecipitation with anti Akt antibody; conversely, Akt was detected in immunoblotting after nucleolin immunoprecipitation. Interestingly, bands displayed various intensity in different cell models, being more intense in cell lines with the PI3K/Akt signaling pathway iper-activated (7-8), such as HL60-AR (apoptosis resistant) and Jurkat cells. In vitro Akt kinase assay on nucleolin, performed using the immunoprecipitated proteins, showed the functional interaction between the two molecules, since nucleolin was recognized by the antibody against the phosphorylated Akt sequence (PAS). Moreover, second round of immunoprecipitation using antibody against nucleolin after a first round using anti-PAS antibody, showed a faint amount of unphosphorylated nucleolin, thus showing elevated levels of phospho-nucleolin in Jurkat cells. Nucleolin phosphorylation was modulated by cell treatment, as demonstrated in HL60 human acute promyelocytic leukaemia cells stimulated with IGF-I and in PC12 murine pheochromocytoma cell line treated with NGF. Nevertheless, HL60-AR and Jurkat cell lines, which have the PI3K/Akt signaling pathway iper-activated, showed high basal level of nucleolin phosphorylation. Conclusions: We showed that nucleolin is a direct target for Akt kinase activity. Nucleolin and Akt differently interact in several cell lines, as shown by their higher binding in models where the PI3K/Akt signaling pathway is activated. Our findings strongly document the Akt and nucleolin functional interaction that seems to have a role in relaying signals from cell surface to the nucleus in several cell lines.