The transduced and selected cells were starved for 24 h and stimulated or not with IGF for 30 min at the concentration of 40 ng/mL. The Sin1 knock out (KO) cells have been characterized as the mTORC2-deficient cells, in which rictor without its binding partner Sin1 becomes an unstable protein and does not bind mTOR (13). The retroviral expression plasmids of H-ras (plasmid#18749, pWZL hygro H-ras V12) and PI3K H1047R (plasmid#12524, pBABE puro PI3KCA H1047R) were obtained from Addgene. The mTORC2 signaling up-regulation by the ras or PI3K oncogenes is associated with the rictor T1135 phosphorylation. In addition to the PI3K pathway, the ILK has been characterized as an important regulator of Akt (33-35). We found that the rictor Thr-1135 phosphorylation takes place not only within the rictor assembled within the mTORC2 complex but also in the mTORC2-deficient cells. The serum-starved MDA-MB-435 cells with or without the IGFI stimulation were lysed and analyzed by immunoblotting for the indicated proteins and phosphorylation states by the phospo-specific rictor and Akt antibodies (left).

Deregulation of this signaling pathway is common in a variety of human cancers. The constitutively active form of the PI3K catalytic unit p110 (H1047R) has been constitutively expressed in A549, HeLa, and MDA-MB-435 cells based on the retroviral pBabe expression vector.

3D). PODNL1 promotes cell proliferation and migration in glioma via regulating Akt/mTOR pathway.

2020 Oct 8;133(19):jcs234930. Get the latest public health information from CDC: https://www.coronavirus.gov. This study was extended into three different growth factor–dependent cancer cell lines (A549, HeLa, and MDA MB-435) and revealed similar results (Fig.

A. S2B). Get the latest research from NIH: https://www.nih.gov/coronavirus. In our next experiment, we studied whether rictor phosphorylation on Thr-1135 occurs in the mTORC2-deficient cells. Julien LA, Carriere A, Moreau J, Roux PP. A second infection was done following the same protocol the next day. An unbiased proteomic screen to identify integrin-linked kinase (ILK) interactors revealed rictor as an ILK-binding protein. Rictor dephosphorylation is not associated with dephosphorylation of Akt Ser473.  |  Second, the oncogenic factors activating the ras/PI3K pathway and mTORC2 signaling cause the growth factor–independent phosphorylation of rictor on Thr-1135. J Cell Sci. The soluble fractions of cell lysates were isolated by centrifugation at 13,000 rpm for 10 minutes in a microcentrifuge. Cell. The rictor phosphorylation in total cellular extracts represents the rictor phosphorylation within the mTORC2 complex, and the phosphorylation state of Thr-1135 can be applied as the indirect activity marker of this kinase complex.

A549, HeLa, MDA-MB-435, and MCF10A were obtained from the American Type Culture Collection. Characterization of the rictor Thr-1135 phosphorylation. Rictor phosphorylation is sensitive to the inhibition of PI3K, mTOR, or expression of integrin-linked kinase.

The scraped lysates were incubated for 20 minutes at 4°C to complete lysis. We found that this rictor phosphorylation takes place in the mTORC2-deficient cells, suggesting that this modification might play a role in the regulation of not only mTORC2 but also the mTORC2-independent function of rictor. Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. Enter multiple addresses on separate lines or separate them with commas. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. The rictor phosphorylation in total cellular extracts represents the rictor phosphorylation within the mTORC2 complex, and the phosphorylation state of Thr-1135 can be applied as the indirect activity marker of this kinase complex. Rictor remains poorly characterized. These data indicate that mTOR as a central component of both mTOR complexes controls the rictor Thr-1135 phosphorylation.

The cells were lysed and analyzed as described in A. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component rictor on Thr-1135. Briefly, excised rictor gel bands were washed thrice with 50% acetonitrile/200 mmol/L ammonium bicarbonate. J Cancer.

The gene RICTOR is located on chromosome 5 at 5p13.1 with a sequence length of 5440 bp, oriented on the minus strand. 2008 Sep 15;68(18):7409-18. doi: 10.1158/0008-5472.CAN-08-1522. The translated RICTOR protein contains 1709 amino acids and is present in the cytosol. Third, the rictor phosphorylation is similar to the mTORC2-dependent Akt phosphorylation as being sensitive to inhibition of PI3K, mTOR, and ILK.

Of these sites, T1135 has been shown to undergo growth factor-responsive …

It results in the autophosphorylation of the receptor cytoplasmic domains and the tyrosine phosphorylation of docking proteins. Signal Transduct Target Ther. Fig. Rictor phosphorylation induced by serum, insulin and insulin-like growth factor is blocked by rapamycin. Tiemann K, Garri C, Lee SB, Malihi PD, Park M, Alvarez RM, Yap LP, Mallick P, Katz JE, Gross ME, Kani K. Oncogene. The MDA-MB-435, A549, and HeLa cell lines were grown in the growth medium containing 10% serum with or without rapamycin at 100 nmol/L concentration for the indicated periods of time. Nevertheless, the rictor Thr-1135 phosphorylation site is conserved in vertebrates and most likely carries an important role in regulation of mTORC2 by providing the phosphorylation-dependent binding site to 14-3-3 proteins (41, 43). The day prior transfection, human embryonic kidney 293T cells (1.2 × 106) were plated on 6-cm plates in 3 mL DMEM supplemented with 10% fetal bovine serum. Deregulation of the rictor Thr-1135 phosphorylation by the oncogenic forms of ras and PI3K NLM doi: 10.1126/scisignal.aav3249. One potential mechanism of this regulation might be linked to the mTORC1-dependent phosphorylation of the rictor Thr-1135 site that points out on S6K1 as a kinase candidate. All of the above cell lines were cultured at a density that allowed cell division throughout the course of the experiment. This complex functions as a nutrient-sensitive kinase complex that regulates protein synthesis by phosphorylating its two substrates, S6K1 and 4EBP1.

2020 Oct 7;5(1):230. doi: 10.1038/s41392-020-00324-2.

Epub 2018 Dec 21. Sarbassov) and NIH grant AI104389 (D.M. eISSN: 1557-3125

2019 Jun 15;21(1):74. doi: 10.1186/s13058-019-1155-7.

The PI3K-dependent signaling kinase complex defined as mammalian target of rapamycin complex 2 (mTORC2) functions as a regulatory Ser-473 kinase of Akt. The cells were lysed and the equal amount of protein lysates were analyzed by Western blotting by the indicated antibodies. 4B). Rictor is an essential component of mTORC2. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. NIH

Wang X, Yue P, Kim YA, Fu H, Khuri FR, Sun SY. Following the confirmation of rictor phosphorylation on the Thr-1135 site, we studied its functional role on the growth factor–dependent activation of mTORC2. The mTORC2 signaling complex functions as the regulatory Ser-473 kinase of Akt; therefore, the main known function of this kinase complex is to mediate a signaling between PI3K and its important downstream effector Akt. 2012 Jan 2;287(1):581-8. doi: 10.1074/jbc.M111.304337. This finding indicates that activation of the mTORC1/S6K1 signaling coordinates a rictor function by phosphorylating it specifically at least within one site that explains a high sensitivity of this rictor phosphorylation to rapamycin.

HHS Anderson Cancer Center Breast Specialized Programs of Research Excellence, American Cancer Society (RSG-09-026-01CCG01), and NIH grant CA133522 (D.D. Copyright © 2020 by the American Association for Cancer Research. Based on our characterization of the rictor Thr-1135 phosphorylation, we identified that this phosphorylation is coupled to growth factor signaling and is also a rapamycin-dependent site of rictor. The retroviral vectors used as plasmids were propagated in and purified from XL-10 Gold bacterial cells and cotransfected together with the Δ VPR and VSVG plasmids into actively growing cells as previously described (18). Epub 2009 Dec 7. Clipboard, Search History, and several other advanced features are temporarily unavailable. Multiple site acetylation of Rictor stimulates mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt protein. No potential conflicts of interest were disclosed.

Based on the alterations of MS readings of the distinct rictor's peptides, Thr-1135 and Ser-1177 have been identified with a high probability as rictor phosphorylation sites (Supplementary Fig. B, the mTORC2 kinase assay. Following the IGFI stimulation of the transfected cells, the phosphorylation of Akt on the Ser-473 site occurred in a growth factor–dependent manner.

The rictor immunoprecipitates (IP) from the serum-starved or IGFI-stimulated MDA-MB-435 cells were used in a kinase assay with a full-length wild-type Akt1 as a substrate.

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