Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jun 19;8(6):e67149.
doi: 10.1371/journal.pone.0067149. Print 2013.

Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines

Su-Lin Lee  1 , Chih-Chien Chou, Hsiao-Ching Chuang, En-Chi Hsu, Po-Chen Chiu, Samuel K Kulp, John C Byrd, Ching-Shih Chen
Affiliations

Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines

Su-Lin Lee et al. PLoS One. .

Retraction in

Abstract

Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA-MB-468 and PC-3 cells. Thus, we hypothesize that ILK might bestow growth advantage and metastatic potential in the course of tumor progression.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effects of mTORC1/2 inhibition on cytotoxicity and Ser-473-Akt phosphorylation in PTEN-negative cancer cell lines.
(A) Differential susceptibility of LNCaP, PC-3, and MDA-MB-468 (468) cells to Ku-0063794-mediated suppression of cell viability in 5% FBS-supplemented medium after 24 h of treatment. Cell viability was determined by MTT assays. Points, means; bars, SD (n = 6). (B) Dose-dependent inhibitory effects of Ku-0063794 on mTOR signaling, as indicated by p70SK phosphorylation, and Akt phosphorylation at Ser-473 versus Thr-308. Representative blots from three independent experiments are shown.
Figure 2
Figure 2. Evidence that ILK acts as a Ser-473-Akt kinase in PC-3 and MDA-MB-468 cells.
(A) Dose-dependent effect of T315 on the viability of LNCaP, PC-3, and MDA-MB-468 (468) cells in 5% FBS-supplemented medium after 24 h of treatment. Cell viability was determined by MTT assays. Points, means; bars, SD (n = 6). (B) Dose-dependent effects of T315 on the phosphorylation of Akt at Ser473 versus Thr308 and GSK3β. Suppression of GSK3β phosphorylation served as a marker for T315-mediated ILK inhibition. (C) Left panel, co-treatment with 3 µM T315 did not enhance the ability of Ku-0063793 to inhibit Ser473-Akt phosphorylation in LNCaP cells. Right panel, co-treatment with 0.8 µM Ku-0063793 did not enhance the ability of T315 to inhibit Ser473-Akt phosphorylation in PC-3 cells. Cells were exposed to test agents at the indicated concentrations for 24 h in 5% FBS-supplemented medium. (D) Left, representative Western blot of the phosphorylation and/or expression levels of Akt, ILK, and the components of mTORC complexes mTOR, raptor, and rictor in PC-3, LNCaP, and MDA-MB-468 (468) cancer cell lines. Right, relative expression levels of p-Ser-473- and p-Thr-308-Akt, Akt, ILK, mTOR, raptor, and rictor in PC-3 and MDA-MB-468 cells relative to those in LNCaP cells. Amounts of immunoblotted proteins from three independent experiments were quantitated by densitometry and normalized to β-actin. Signals from phosphorylated Akt were first normalized to that of total Akt and then to that of β-actin. The abundance of each protein in PC-3 and MDA-MB-231 cells was expressed as a percentage of that in LNCaP cells. Values, means; bars, S.D. (n = 3). All immunoblots are representative of three independent experiments. *P<0.05, compared to LNCaP cells.
Figure 3
Figure 3. Evidence that mTORC2 is the Ser-473-Akt kinase in LNCaP cells.
(A) Western blot analysis of the effect of si/shRNA-mediated knockdown of ILK, rictor, MK2, DNA-PK, ATM, PKCα, PKCβII, PAK1, or PAK2 on the expression of individual target proteins and the phosphorylation of Akt at Ser473 versus Thr308. (B) Effects of siRNA-mediated ILK knockdown, alone or in combination with Ku-0063794 (0.4 µM, 24 h), on Akt phosphorylation at Ser473 versus Thr308. (C) Dose-dependent effect of ectopic expression of ILK, alone (left panel) or in combination with Ku-0063794 (0.4 µM, 24 h), on the phosphorylation of Ser473-Akt and GSK3β. All immunoblots are representative of three independent experiments.
Figure 4
Figure 4. Evidence that ILK is the Ser473-Akt kinase in PC-3 and MDA-MB-468 cells.
(A) Western blot analysis of the effect of si/shRNA-mediated knockdown of ILK, rictor, MK2, DNA-PK, ATM, PKCα, PKCβII, PAK1, or PAK2 on the expression of individual target proteins and the phosphorylation of Akt at Ser473 versus Thr308. (B) Effects of shRNA-mediated rictor knockdown, alone or in combination with T315 (2 µM, 24 h), on Akt phosphorylation at Ser473 versus Thr308. (C) Western blot analysis of the effect of shRNA-mediated repression of ILK (left) or rictor (right) on the phosphorylation of Akt at Ser473 versus Thr308, GSK3β (ILK target) and p70S6K (mTOR target) in MDA-MB-468 cells. For induction of ILK shRNA, cells stably transfected with a lentiviral vector encoding tetracycline-inducible ILK shRNA (TRE-ILKi) were exposed to 2 µg/ml doxycycline for 5 days. All immunoblots are representative of three independent experiments.
Figure 5
Figure 5. Evidence that ILK binds and phosphorylates Akt in PC-3 cells.
(A) Co-immunoprecipitation analysis reveals the association of ILK with Akt in PC-3 cells, which can be attenuated by T315. PC-3 cells were treated with 2.5 µM T315 or DMSO control for 24 h. Equal amounts of cell lysates were immunoblotted (WB) with antibodies against ILK, p-Ser473-Akt, or Akt (Input; lower panel), or were immunoprecipitated (IP) with anti-Akt antibody and protein A/G agarose followed by immunoblotting with antibodies against ILK, p-Ser473-Akt, or Akt (upper panel). (B) Effect of T315 on the kinase activity of ILK. Equal amounts of immunocomplexes obtained by immunoprecipitation of ILK from PC-3 cell lysates were incubated with bacterially expressed GST-Akt and 200 mM of ATP in the presence of DMSO or T315 at indicated concentrations for 20 min, and were then subjected to Western blotting with antibodies against p-Ser-473-Akt, Akt, or ILK. The immunoprecipitation procedure using IgG was performed as control. Left panel, Western blot analysis of the dose-dependent effect of T315 on the kinase activity of ILK. Right panel, relative expression levels of phosphorylated Ser-473-Akt after normalization to total Akt and subtraction from control (time zero). Amounts of immunoblotted proteins from three independent experiments were quantitated by densitometry. The abundance of phosphorylated Ser-473-Akt in T315-treated cells was expressed as a percentage of that in the DMSO control cells (0 µM). Values, means; bars, S.D. (n = 3). All immunoblots are representative of three independent experiments. *P<0.05, compared to DMSO control (0 µM).
Figure 6
Figure 6. ILK interacts with rictor in PC-3 and MDA-MB-468, but not in LNCaP cells.
Co-immunoprecipitation analysis reveals that ILK associates with rictor in MDA-MB-468 and PC-3 cells, which can be attenuated by T315. No such association was observed in LNCaP cells. Cells were treated with 2.5 µM T315 or DMSO control for 24 h. Equal amounts of cell lysates were immunoblotted with antibodies against ILK, rictor or β-actin (Input; lower panels), or were immunoprecipitated (IP) with anti-ILK antibody and protein A/G agarose followed by immunoblotting (WB) with antibodies against ILK or rictor (upper panels).
Figure 7
Figure 7. Pharmacologic inhibition or genetic silencing of ILK suppresses EMT and invasive phenotype.
(A) Inhibition of ILK by si/shRNA-mediated silencing or treatment with T315 reduces the expression of Snail in association with an increase in that of E-cadherin and concomitant reduction in that of vimentin in PC-3 (left panel) and MDA-MB-468 (right panel) cells. Cells were treated with T315 for 24 h. For induction of ILK shRNA, MDA-MB-468 cells stably transfected with a lentiviral vector encoding tetracycline-inducible ILK shRNA (TRE-ILKi) were exposed to 2 µg/ml doxycycline for 5 days. Immunoblots are representative of three independent experiments. (B) Images of invasive colonies after growth of PC-3 cells on basement membrane matrix for 6 days in the presence of DMSO control versus T315 at the indicated concentrations. Bars, 100 μm. (C) Dose-dependent effects of T315 and Ku-0063794 on the EGF-induced phosphorylation of Akt at Ser-473 versus Thr-308 in the PTEN-functional MDA-MB-231 cell line. Cells were co-treated with inhibitor and EGF for 24 h. Representative blots from three independent experiments are shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mora A, Komander D, van Aalten DM, Alessi DR (2004) PDK1, the master regulator of AGC kinase signal transduction. Semin Cell Dev Biol 15: 161–170. - PubMed
    1. Dong LQ, Liu F (2005) PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle. Am J Physiol Endocrinol Metab 289: E187–196. - PubMed
    1. Hresko RC, Mueckler M (2005) mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes. J Biol Chem 280: 40406–40416. - PubMed
    1. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098–1101. - PubMed
    1. Delcommenne M, Tan C, Gray V, Rue L, Woodgett J, et al. (1998) Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc Natl Acad Sci U S A 95: 11211–11216. - PMC - PubMed

Publication types

MeSH terms