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
. 2011 Oct;7(10):1212-21.
doi: 10.4161/auto.7.10.16660. Epub 2011 Oct 1.

ULK1 inhibits the kinase activity of mTORC1 and cell proliferation

Chang Hwa Jung  1 , Minchul Seo, Neil Michael Otto, Do-Hyung Kim
Affiliations

ULK1 inhibits the kinase activity of mTORC1 and cell proliferation

Chang Hwa Jung et al. Autophagy. 2011 Oct.

Abstract

ULK1 (Unc51-like kinase, hATG1) is a Ser/Thr kinase that plays a key role in inducing autophagy in response to starvation. ULK1 is phosphorylated and negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that ULK1 is not only a downstream effector of mTORC1 but also a negative regulator of mTORC1 signaling. ( 1-3) Here, we investigated how ULK1 regulates mTORC1 signaling, and found that ULK1 inhibits the kinase activity of mTORC1 and cell proliferation. Deficiency or knockdown of ULK1 or its homolog ULK2 enhanced mTORC1 signaling, cell proliferation rates and accumulation of cell mass, whereas overexpression of ULK1 had the opposite effect. Knockdown of Atg13, the binding partner of ULK1 and ULK2, mimicked the effects of ULK1 or ULK2 deficiency or knockdown. Both insulin and leucine stimulated mTORC1 signaling to a greater extent when ULK1 or ULK2 was deficient or knocked down. In contrast, Atg5 deficiency did not have a significant effect on mTORC1 signaling and cell proliferation. The stimulatory effect of ULK1 knockdown on mTORC1 signaling occurred even in the absence of tuberous sclerosis complex 2 (TSC2), the negative regulator of mTORC1 signaling. In addition, ULK1 was found to bind raptor, induce its phosphorylation, and inhibit the kinase activity of mTORC1. These results demonstrate that ULK1 negatively regulates the kinase activity of mTORC1 and cell proliferation in a manner independent of Atg5 and TSC2. The inhibition of mTORC1 by ULK1 may be important to coordinately regulate cell growth and autophagy with optimized utilization of cellular energy.

PubMed Disclaimer

Figures

Figure 1
Figure 1
ULK1, ULK2 and Atg13, but not Atg5, negatively regulate mTORC1 signaling. (A) Knockdown of ULK1, ULK2 and Atg13, but not Atg5, increased mTORC1 signaling. 293T cells were transduced by Atg13, ULK1, ULK2 or scrambled shRNA (control). The phosphorylation of s6K1 (p-Thr389) and the expression levels of proteins were analyzed by protein gel blotting. β-actin was a loading control. (B) Overexpression of ULK1 inhibited S6K1 phosphorylation. Cell lysate was obtained from 293T cells overexpressing myc-tagged ULK1 or cells transduced by mock vector. The phosphorylation state of endogenous S6K1 (p-Thr389) was analyzed by protein gel blotting. (C) ULK1 deficiency increased leucine-stimulated S6K1 phosphorylation. ULK1 MEFs were cultured in RPMI medium deprived of leucine for 40 min before cells were supplemented with leucine (52 µg/ml) for different periods of time. The phosphorylation state (p-Thr389) and expression level of S6K1 were analyzed by protein gel blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a loading control. (D) ULK1 deficiency increased insulin-stimulated S6K1 phosphorylation. ULK1 MEFs were incubated in DMEM without serum overnight and treated with insulin (10 nM) for different periods of incubation time. The phosphorylation states of S6K1 (p-Thr389) and Akt (p-Ser473) and their expression levels were analyzed by protein gel blotting. β-actin was monitored as a loading control. (E) ULK1 knockdown increasesd S6K1 phosphorylation in 293T cells. 293T cells transduced by ULK1 shRNA or scrambled shRNA (control) were treated with insulin (100 nM) as described in (D). The phosphorylation states and expression levels of proteins were analyzed by protein gel blotting. (F) ULK2 knockdown increased S6K1 phosphorylation in 293T cells. The cells transduced by ULK2 shRNA or scrambled shRNA (control) were treated with insulin, and the phosphorylation state of S6K1 was analyzed as described (D). (G) Atg5 deficiency did not increase S6K1 phosphorylation. Atg5 MEFs were treated with insulin as described in (D).
Figure 2
Figure 2
ULK1, ULK2 and Atg13, but not Atg5, negatively regulate cell proliferation and cell mass accumulation. (A) Knockdown of ULK1, ULK2 or Atg13 in HeLa cells increased cell proliferation rate. HeLa cells were stably transduced by shRNA for ULK1, ULK2, Atg13 or scrambled sequence (control) using lentiviral vectors. Cell number was counted on day 1, 2 and 3 (see Materials and Methods for details). Values are means ± std. *p < 0.01 between control cells and each of the shRNA transduced cells. (B) Knockdown of ULK1 in 293T cells increased cell proliferation rate. (c) ULK1 deficiency in MEFs enhanced cell proliferation. (D) ULK1 overexpression in 293T cells reduced cell proliferation rate. 293T cells were transduced by either plasmid encoding myc-tagged ULK1 or empty vetor. (e) Atg5 deficiency in MEFs did not have a significant effect on cell proliferation. (F) Knockdown of ULK1 or Atg13 in HeLa cells reduced cell size. shRNA-transduced HeLa cells were analyzed for their cell sizes (see Materials and Methods for details). Average diameters of cells were 16.23 ± 0.11, 15.02 ± 0.07 and 14.99 ± 0.04 µm for scrambled, ULK1- and Atg13-shRNA transduced cells, respectively. (G) Knockdown of ULK1, ULK2 and Atg13 in 293T cells reduced cell size. Average diameters of cells were 14.72 ± 0.08, 14.35 ± 0.11, 14.25 ± 0.09 and 14.10 ± 0.13 µm for scrambled, ULK1, Atg13 and ULK2 shRNA transduced cells, respectively. (H) ULK1 deficiency in MEFs reduced cell size. Average diameters of cells were 17.42 ± 0.14 and 17.17 ± 0.09 µm for ULK1+/+ and ULK1−/− MEFs, respectively. (I) Atg5 deficiency in MEFs increased cell size slightly. Average diameters of cells were 17.55 ± 0.12 and 17.86 ± 0.10 µm for Atg5+/+ and Atg5−/− MEFs, respectively. (J) Knockdown of ULK1, ULK2 or Atg13 in HeLa cells increased total cell mass. Total cell mass was calculated using cell number and cell volume on day 3. Values of mean ± std for total cell mass were converted to percent values relative to the mass of cells from the control group. *p < 0.05 between control cells and each of shRNA-transduced cells. (K) Knockdown of ULK1 in 293T cells increased total cell mass. Data were analyzed as described in (J). (L) ULK1 deficiency in MEFs increased total cell mass. (M) Atg5 deficiency in MEFs marginally increased total cell mass.
Figure 3
Figure 3
ULK1 inhibits mTORC1 signaling independently of TSC2. TSC2+/+ and TSC2−/− MEFs were stably transduced by ULK1 or scrambed shRNA using lentiviral vectors. The shRNA-transduced cells were cultured in the absence of serum overnight and treated with insulin (10 nM) for 30 min. The phosphorylation states and the expression levels of S6K1 and Akt were analyzed by protein gel blotting.
Figure 4
Figure 4
ULK1 and ULK2 bind to raptor. (A) ULK1, ULK2 and Atg13 bind to raptor. Myc-tagged constructs were expressed with HA-tagged raptor in 293T cells. Forty-eight hours post-transfection, the amount of HA-raptor isolated by immunoprecipitation using anti-myc antibody was analyzed by protein gel blotting. HA-tagged S6K1 was used as a negative control. (B) HA-tagged ULK1 was co-immunoprecipitated with myc-tagged raptor from 293T cells. Myc-tagged tubulin and S6K1 were used as negative controls. (C) Confirmation of the ULK1-raptor interaction at endogenous levels. Raptor and rictor immunoprecipitates were obtained from 293T cells using anti-raptor and anti-rictor antibodies, respectively. The amount of ULK1 in the immune complex was assayed by protein gel blotting. The asterisk symbol (*) indicates a nonspecific band near 150 kDa. (D) Confirmation of the raptor-ULK1 interaction at endogenous levels in 293T cells. Pre-immune serum was used as a negative control. (E) Atg13 is not required for the interaction between raptor and ULK1. 293T cells transduced by shRNA were subjected to raptor immunoprecipitation. The amounts of ULK1, Atg13 and raptor in raptor immunoprecipitates were analyzed by protein gel blotting. (F) Raptor requires its full length for binding to ULK1. The full length (aas 1–1,335) or fragments derived from raptor were expressed as HA-tagged versions in 293T cells together with myc-tagged ULK1. The amount of myc-ULK1 in HA immunoprecipitates was analyzed by protein gel blotting.
Figure 5
Figure 5
ULK1 regulates the mTOR-raptor interaction in response to insulin and induces raptor phosphorylation. (A) ULK1 deficiency destabilized the mTOR-raptor interaction in vitro in response to insulin, and the destabilization correlated with higher levels of S6K1 phosphorylation. ULK1 MEFs were treated with insulin as described in Figure 1D. mTOR immunoprecipitate was obtained using anti-mTOR antibody in cell lysis buffer containing 0.3% CHAPS, and the amount of raptor was analyzed by protein gel blotting. (B) ULK2 knockdown in 293T cells destabilized the mTOR-raptor interaction in response to insulin. shRNA-transduced 293T cells were treated with insulin as described in Figure 1D. The amount of raptor in mTOR immunoprecipitate was analyzed by co-immunoprecipitation and protein gel blotting. (C) ULK1 knockdown suppressed the mobility shift of raptor on SDS-PAGE. Raptor from shRNA-transduced 293T cells, which were treated with insulin for 30 min, was analyzed on SDS-PAGE. (D) ULK1 deficiency suppressed the mobility shift of raptor. Raptor from ULK1 MEFs treated with insulin for 30 min was analyzed. (E) ULK1 induces phosphorylation of raptor. HA-tagged raptor was expressed with myc-tagged ULK1 wild-type (wt) or M92A kinase dead mutant (kd) in 293T cells. HA-raptor and myc-ULK1 were isolated by immunoprecipitation using anti-myc antibody. Migration patterns of raptor and ULK1 on SDS-PAGE were analyzed by protein gel blotting after the myc immunoprecipitate was treated with or without lambda phosphatase.
Figure 6
Figure 6
ULK1 inhibits the kinase activity of mTORC1. (A) ULK1 has a negative effect on the kinase activity of mTORC1. mTOR was isolated from ULK1 MEFs. The kinase activity of mTOR was analyzed using S6K1 as substrate. The phosphorylation state of S6K1 at Thr389 was analyzed by protein gel blotting. The level of phosphorylation was quantitatively analyzed in the bar graph on the right-hand side. Values are means ± std from three independent experiments. *p < 0.05. (B) The inhibitory effect of ULK1 on mTORC1 kinase activity was assessed in vitro using 4E-BP1 as substrate. The bar graph represents quantitative assessment of the phosphorylation state of 4E-BP1 at Thr37/46. Values are mean ± std from two independent experiments. *p < 0.05. The mobility shift of raptor was unclear because we used a high perent of acrylamide in SDS-PAGE to analyze 4E-BP1. (C) Raptor immunoprecipitate isolated from ULK1 MEFs was analyzed for the kinase activity toward phosphorylation of 4E-BP1. (D) Raptor immunoprecipitate isolated from scrambled (sc) or ULK1 shRNA-transduced 293T cells was analyzed for the kinase activity toward phosphorylation of 4E-BP1. (E) Rictor immunoprecipitate isolated from ULK1 MEFs was analyzed for the kinase activity toward phosphorylation of Akt at Ser473. (F) Model for the reciprocal regulation between the ULK1 complex and mTORC1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Lee SB, Kim S, Lee J, Park J, Lee G, Kim Y, et al. ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase. EMBO Rep. 2007;8:360–365. doi: 10.1038/sj.embor.7400917. - DOI - PMC - PubMed
    1. Scott RC, Juhasz G, Neufeld TP. Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr Biol. 2007;17:1–11. doi: 10.1016/j.cub.2006.10.053. - DOI - PMC - PubMed
    1. Jung CH, Jun CB, Ro SH, Kim YM, Otto NM, Cao J, et al. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol Biol Cell. 2009;20:1992–2003. doi: 10.1091/mbc.E08-12-1249. - DOI - PMC - PubMed
    1. Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6:463–477. doi: 10.1016/S1534-5807(04)00099-1. - DOI - PubMed
    1. Vellai T, Bicsak B, Toth ML, Takacs-Vellai K, Kovacs AL. Regulation of cell growth by autophagy. Autophagy. 2008;4:507–509. - PubMed

Publication types

MeSH terms

Substances