Commentary to: Mutations of the PI3KCA gene in ovarian and breast cancer

Objective: to conduct a mutational analysis of the PIK3CA gene in ovarian and breast tumors and correlate the molecular results with histological types

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

The erythroid differentiation of K562 cells depends on the nuclear translocation of Akt

Previous findings have demonstrated that Erythropoietin (EPO) treatment during erythroid differentiation of human erythroleukaemia cells activates PI3K (1,2) and that an active PI3K translocates into the nucleus (3). When PI3K activity is inhibited, cellular differentiation is blocked, showing the requirement of this kinase during cell differentiation (3). It is known that in the PI3K signalling pathway, the Ser/Thr kinase Akt is a downstream enzyme involved in many cellular processes including differentiation (4). Therefore we wanted to analyze the role of nuclear Akt during the erythroid differentiation process in K562 erythroleukaemia cells, following EPO treatment. Materials and methods: K562 cells coltures, EPO treatment, preparation of whole cell homogenates, isolation of nuclei, immunoblotting, Akt kinase assay and immunocytochemistry were accomplished as previously reported (3). Results: In whole cells homogenates after 10 min of EPO exposure we observed the highest level of Ser473 phosphorylated Akt. This kinase increases rapidly and transiently in response to EPO treatment its intranuclear amount, with a delay when compared with the cell homogenates. Infact we observed that into the nucleus Ser473 phosphorylated Akt was observable at highest levels after 15 min. In situ analysis by means of immunocytochemistry showed the nuclear translocation of Akt and of its phosphorylated form on Ser473 that peaked at 15 min and returned to control conditions after 30 min. Enzyme translocation and erythroid differentiation were blocked by the specific Akt pharmacological inhibitor acting on its PH activation domain, 1L-6-Hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (from Alexis). Almost all Akt kinase observed into the nucleus was phosphorylated on Serine 473 of the hydrophobic regulative activation domain, as demonstrated by using double round of immunoprecipitation (IP) experiments in which the first round of IP was performed with anti phospho- Akt (Ser473) antibody and the second round with total anti Akt antibody. Using kinase assay we observed a peak of Akt activity after 10 min of EPO treatment in whole cell homogenates and an intranuclear peak of activity after 15 min of EPO exposure, that were both blocked by pre-treatment of cells by Akt inhibitor. Conclusions: EPO induces the nuclear translocation of Akt in a time dependent and ordered manner. Furthermore, when cells were treated with an Akt inhibitor, the nuclear translocation of the enzyme was blocked as well as the differentiation process. These findings strongly suggest the requirement of active Akt translocation into the nucleus as an important step in the PI3K/Akt signaling pathway to enter into EPO-mediated erythroid differentiation

AKT1 (v-akt murine thymoma viral oncogene homolog 1). Atlas Genet Cytogenet Oncol Haematol. May2009. http://AtlasGeneticsOncology.org/Genes/AKT1ID355ch14q32.html (This paper should be referenced as such)

none5Atlas Genet Cytogenet Oncol Haematol. May 2009 . URL : http://AtlasGeneticsOncology.org/Genes/AKT1ID355ch14q32.htmlopenEtro D; Missiroli S; Buontempo F; Neri LM; Capitani S.Etro, Daniela; Missiroli, Silvia; Buontempo, Francesca; Neri, Luca Maria; Capitani, Silvan