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  • Refametinib br Materials and methods br Results br Discussio

    2024-02-23


    Materials and methods
    Results
    Discussion In the present study, we identified HBP1 as a novel substrate of AKT. This was demonstrated by in vitro phosphorylation assays and western blotting using phosphosite-specific antibodies. Three sites were identified by mass spectrometry and mutagenesis: Ser380, Thr484 and Ser509. The two serine residues match the reported AKT consensus perfectly [42] and were the most important phosphorylation sites in vitro. Phosphosite-specific Refametinib also demonstrated the phosphorylation of these two sites in cells in an AKT-dependent manner (Fig. 1, Fig. 5 and Suppl. Fig. 1). Thr484 lacks the classical arginine at position −5 and contributed to a lesser extent to HBP1 phosphorylation by recombinant AKT in vitro. In transfected cells, Thr484 mutation tended to decrease the binding of the AKT substrate antibody to HBP1. Nevertheless, anti-phospho-Thr484 antibodies were not available to fully demonstrate endogenous HBP1 phosphorylation on that site by AKT. HBP1 recruitment by AKT is also supported by co-immunoprecipitation experiments. Our results suggest that AKT interacts with the N-terminal and C-terminal parts of HBP1, whereas the three phosphorylation sites are located in the C-terminal part. AKT binding to HBP1 was not affected by mutating the phosphorylation sites. This is in agreement with findings on other AKT substrates, such as FOXO3, in which the docking point is distinct from phosphorylation motifs [43]. HBP1 phosphorylation was induced by growth factors, such as EGF and IGF-1, which activate AKT. Conversely, HBP1 phosphorylation was significantly decreased by AKT pharmacological inhibition using MK-2206 and by AKT1/2 down-regulation using siRNA. Nevertheless, significant residual phosphorylation was observed in the absence of active AKT, suggesting that others kinases may phosphorylate HBP1 on the same sites as AKT. Accordingly, it was reported recently that the Pim-1 kinase phosphorylates HBP1 on Ser380 (and Ser372) under oxidative stress [44]. AKT and Pim-1 are known to share many substrates [[45], [46], [47]]. Furthermore, we also noticed that the sequence surrounding the Ser380 corresponds to a PKA consensus site and we observed that recombinant PKA kinase was able to phosphorylate HBP1 in vitro on Ser380 and Thr484 (data not shown). An overlap between AKT and PKA substrates has also been reported [48,49]. Using multiple model systems, our experiments suggest that HBP1 phosphorylation blocks its transcriptional activity and its ability to inhibit the cell cycle, in line with the oncogenic properties of AKT. First, mutations that mimic phosphorylation (HBP1-EEE) abolished HBP1 activity, as measured by luciferase reporter assays, gene expression studies and proliferation tests. Single and double mutants had an intermediate effect compared to the triple mutant. Second, mutation of the three phosphorylation sites to alanines (HBP1-AAA) enhanced the regulation of cell cycle genes and the anti-proliferative activity of HBP1. This effect was not seen in luciferase reported assays, possibly because a maximal effect is already reached by transfection of wild type HBP1. Nevertheless, HBP1-AAA was resistant to inhibition by constitutively-activated AKT, compared with wild-type HBP1. Altogether, our data suggest that HBP1 phosphorylation by AKT negatively regulates its function. The three phosphorylation sites are located in the repression domain (Ser380) or the DNA-binding domain (Thr484 and Ser509), suggesting that HBP1 phosphorylation affects DNA-binding or the recruitment of cofactors to the repression domain. In this respect, the regulation of several HBP1 target genes depends on RB-binding [3,12,35,36]. However, we demonstrated that the interaction with RB was not affected by mutation of HBP1 phosphorylation sites. The exact mechanism of HBP1 regulation by phosphorylation remains to be identified. HBP1 deficiency was shown to reduce the duration of the cell cycle in neural stem cells and progenitors in vivo [11]. Downregulation of HBP1 by miR-96 and miR-155 was also reported to promote glioma cell proliferation [17,18]. Accordingly, we showed that HBP1 knockdown by shRNA increased glioblastoma cell proliferation. Conversely, HBP1 overexpression using retroviral particles decreased proliferation, survival and foci formation by these cells. This effect was enhanced with HBP1-AAA, while it was lost with the HBP1-EEE mutant, suggesting that HBP1 phosphorylation, most likely by AKT, controls its impact on the cell cycle. This conclusion is also supported by the well-established role of AKT in tumor cell growth, and in particular in glioblastoma [[37], [38], [39]].