Phospho-mTOR ( Ser 2481 ) colocalizes with condensed chromosomes during metaphase

V. R. Kosach, I. O. Tykhonkova, O. V. Cherednyk © 2016 V. R. Kosach et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.112.7 + 576.353 + 576.316 + 576.31


Introduction
The mammalian target of rapamycin (mTOR) is a protein kinase involved in the regulation of cell growth and aging affecting numerous biosynthetic processes and cell metabolism [1].Deregulation of mTOR is observed in many pathologies, including cancer, metabolic and neurodegenerative disorders [2,3].
Phosphorylation is one of the main posttranslational modifications that regulate kinase activity [4].Usually, gradual modification of several phosphorylation sites is needed for full activation of protein kinases.Phospho ry lation could also play an important role in the regulation of kinase subcellular localization [5,6].
mTOR kinase has four characterized phosphorylation sites: Ser1261 localized at the HEAT domain and three other sites Thr2446, Ser2448, Ser2481 -at regulatory domain, which is important for the mTOR kinase activity [7,8].Phosphorylation of serine 2481 is detected only in vertebrates and is known to be [an] autophosphorylation site.[9].
Previous studies showed that phospho-mTOR (Ser2481) is located at the midzone and cleavage furrow of the dividing cells.It was proposed that phospho-mTOR (Ser2481) could be a mitotic kinase [10].However, the role of Ser2481 phosphorylation and targets of phospho-mTOR (Ser2481) during mitosis are unknown.
In this study we have shown for the first time that phospho-mTOR (Ser2481) localized as discrete foci at metaphase plate during mitosis in human cell lines MCF-7 and HepG2.Our data support the hypothesis proposed earlier that phospho-mTOR (Ser 2481) could act as a CPP (chromosomal passenger protein)like kinase during mitosis [11].

Immunofluorescent analysis
Cultured MCF-7 or HepG2 cells were fixed with methanol for 10 min at room temperature.Thereafter, the cells were permeabilized with 0.2 % Triton X-100 in PBS for 10 min and incubated for 30 min at room temperature in 10 mM cupric sulphate and 50 mM ammonium acetate, pH 5.0 to eliminate autofluorescence.Non-specific binding was blocked after incubation with 10 % FCS in PBS for 30 min at 37°C.Between incubations the cells were washed with PBS three times for 3 min.Double immunofluorescent analysis was performed by addition of the primary antibody mix: rabbit ā-phospho-mTOR (Ser2481) + mouse ā-Ki-67, ā -mTOR antibodies for 1 h at 37 °C in humidifying chamber.
The secondary FITC conjugated anti-mouse antibodies, Texas Red conjugated anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, Pennsylva nia, USA) were applied as a mix in dilution 1:400 for 45 min at 37°C in humidifying chamber.In some cases DNA was counterstained with Hoechst 33342.Samples were washed and embedded into Mowiol medium (Sigma-Aldrich, St. Louis, USA) containing 2.5 % DABCO (Sigma-Aldrich, St. Louis, USA).

Results
Double immunofluorescent analysis was performed on cell lines MCF-7 and HepG2 to study the peculiarities of the phospho-mTOR (Ser2481) subcellular localization.Antibody to the proliferating cell antigen Ki-67 was applied to visualize and distinct the different stages of cell cycle.
Interphase cells showed very low level of phospho-mTOR (Ser2481) fluorescence signal.In contrast, mitotic cells demonstrated bright staining, which had dramatically different subcellular localization of phospho-mTOR (Ser2481) during mitosis progression.
Phospho-mTOR (Ser2481) was detected at the midbody and the cleavage furrow in the anaphase and telophase (Fig. 1 A, small arrow) as it was reported earlier [11].However, we observed phospho-mTOR (Ser2481) as bright dots among the arms of condensed chromosomes in metaphase for the first time (Fig. 1 A, long arrows).Double immunofluorescent analysis revealed phospho-mTOR (Ser2481) at the metaphase plates of HepG2 (Fig. 1 B) and MCF-7 (Fig. 1 C) cells.
At the same time the antibodies against different parts of non-phosphorylated mTOR molecule showed diffuse staining in the cytoplasm of mitotic cells (Fig. 2).Namely the antibodies against N-terminal, C-terminal regions and central part of mTOR molecule were applied.No convincing reaction among the arms of condensed chromosomes was revealed.Only after chromosomes separation in anaphase, a bright positive reaction on mTOR was detected (data not shown).Phosphorylation of mTOR on Ser2481 could change the conformation of molecule that will make phospho-mTOR (Ser2481) inaccessible for the antibodies against non-phosphorylated mTOR.

Discussion
Serine 2481 is one of four characterized phosphosites of mTOR kinase.It is known to be autophosphorylated by mTOR.Serine 2481 localizes at the regulatory domain of the kinase and could be involved in the modulation of its activity [8,9].In mammalian cells mTOR exists as a core enzyme of two different complexes: mTORC1 and mTORC2 [13,14,15].Earlier studies showed that specific mTOR inhibitor rapamycin treatment did not affect Serine 2481 phosphorilation.So, it was hypothesized that phospho-mTOR (Ser2481) is a unique feature of mTORC2 [16].However, further investigations demonstrated that the growth factors such as insulin stimulate the autophosphorylation of mTOR associated with both mTORC1 and mTORC2.Also, it was revealed that phosphorylation of Ser 2481 in both complexes was sensitive to wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor.This finding indicates that insulin promotes  Ser2481 autophosphorylation in both complexes -mTORC1 and mTORC2 [17].
Our immunofluorescent analysis revealed that mTOR phosphorylated at Ser2481 localized at the specific compartments of the mitotic cells and concentrated to the midzone and midbody during anaphase and telophase.The previous data showed that at cytokinesis mTOR, phosphorylated at Ser2481, appeared as a doublet at the apical ends of two daughter cells [10].These data suggest that phospho-mTOR (Ser2481) could play an important but yet unknown role in mitosis and cytokinesis regulation.
The recent studies have shown that phospho-mTOR (Ser2481) co-localizes with chromosomal passenger proteins (CPPs) during cell cytokinesis [11].It was revealed that phospho-mTOR (Ser2481) and CPPs INCENP and Aurora B overlaped during anaphase, telophase and cytokinesis.Therefore, it was hypothesized that phosphorylated mTOR may behave as a CPP-like protein at the spindle midzone and at the cleavage furrow [11].Our findings strongly support this hypothesis.We also detected mTOR at the midbody and the cleavage furrow, but unlike the above data we demonstrated for the first time that phospho-mTOR (Ser2481) localized at the chromosomes of the metaphase plate in mitotic MCF-7 and HepG2 cells.The differences between our studies and previous work could be explained by possible discrepancies of the immunostaining protocols and technique of image acquisition.
It is known that CPPs localizes initially along chromosome arms, but progressively concentrates at inner centromeres through prometaphase and metaphase.There they regulate the chromosome structure and correction of kinetochore-microtubule attachment [18].The discovered phospho-mTOR (Ser2481) localization at the metaphase plate shows the pattern similar to CPPs, but the real role and function of Ser2481 phosphorylation during mitosis are still unknown.It suggests that it participates in the formation of a niche for future midbody.From the other hand, our finding also raises a question about phospho-mTOR (Ser2481) co-localization with centromere proteins and its possible participa-tion in the chromosomal passenger complex functioning.

Fig. 2 .
Fig. 2. Immunofluorescent detection of mTOR kinase in proliferating MCF-7 cells.A -antibodies against central region of mTOR molecule (clone F11, Merck Millipore), B -antibodies against C terminal part of mTOR (7C10, Cell Signaling Technology), Cantibodies against N-terminus of mTOR kinase (N19, Santa Cruz Biotechnology).DNA was counterstained with Hoechst 33342.Cells in the metaphase are pointed with arrows.