Immunolocalization of phospho-S6 kinases: a new way to detect mitosis in tissue sections and in cell culture

Abstract

During a study on the mTor pathway in the rat kidney we observed a striking increase of the phosphorylation of the S6 kinase in mitosis. In cryostat sections of perfusion-Wxed tissue mitotic cells appeared as bright spots in immunoXuorescence using an antibody speciWc for the phosphorylation site Thr421/Ser424. They were easily spotted in overviews with the objective 4£ and 10£. ImmunoXuorescence was weak during the interphase. During the prophase it increased in both the nucleus and the cytoplasm and it remained bright during the subsequent phases of mitosis. All mitotic cells which were found in tubules and in the interstitium of the kidney using a chromatin stain displayed the bright immunoXuorescence for phospho-S6 kinase. The same phenomenon was observed in rat liver and in mouse kidney as well as in a human cell line. Provided a rapid Wxation, mitotic cells could be identiWed with the immunoperoxidase technique in paraYn sections of immersion-Wxed tissue. This is the Wrst report of phosphorylation of S6 kinase during mitosis in vivo. Thus, immunohistochemistry with antiphospho-S6 kinase (Thr421/Ser424) appears to provide a convenient way to detect mitotic cells at low magniWcation.

Ser2481-autophosphorylated mTOR colocalizes with chromosomal passenger proteins during mammalian cell cytokinesis

“…[26][27][28][29] Phosphorylation at Thr421/Ser424 of p70S6K1, a downstream effector of mTOR, is also strongly upregulated during mitosis, and the S6 kinase p54S6K2 has been identified as a centrosome-located kinase throughout the cell cycle. [30][31][32] Our current findings confirm that mTOR Ser2481 autophosphorylation, a recently described pharmacodynamic biomarker that directly monitors the effects of rapamycin on the assembly and function of mTORC complexes, should be added to the growing list of phospho-active forms of proteins of the AMPK/mTOR/S6K1 signaling axis that reside at the mitotic and cytokinetic apparatus.…”

Loss of Lkb1 Provokes Highly Invasive Endometrial Adenocarcinomas

“…Although we did observe small foci of p-S6K-positive cells (Fig. 3B, inset), this seems to be secondary to focal increases in mitotic rate because S6K is phosphorylated during mitosis (23) and p-S6K is also a welldocumented marker of mitotic cells (24). We also did not observe significant alterations in the patterns or levels of phosphorylation of other putative mTOR substrates including p-4EBP1(Thr 70 ) or p-Akt(Ser 473 ) (data not shown).…”

The Death Effector Domain-containing DEDD Supports S6K1 Activity via Preventing Cdk1-dependent Inhibitory Phosphorylation

“…Shah et al (27) demonstrated that phosphorylation of those residues (featured by the Thr-421/ Ser-424 site) during mitosis pursued by Cdk1 inactivates S6K1 to terminate protein synthesis prior to cell division (28). A recent report by Schmidt et al (29) demonstrating that phosphorylation of Thr-421/Ser-424 is specifically increased during the G 2 /M phase may also support the finding, whereas during the G 1 phase, there is consensus that the phosphorylation at the autoinhibitory domain is requisite for S6K1 activation (26), as also recently demonstrated by Hou et al (30), where the Cdk5 phosphorylates the Ser-411 site, leading to activation of S6K1. In contrast to such inhibitory regulation of S6K1 during mitosis, however, a recent report by Boyer et al (31) sharply demonstrated that the activity of S6K1 peaks at mitosis, suggesting that S6K1 may also have some roles during the mitotic phase.…”