ULK1·ATG13·FIP200 Complex Mediates mTOR Signaling and Is Essential for Autophagy

Abstract

Autophagy is a degradative process that recycles long-lived and faulty cellular components. It is linked to many diseases and is required for normal development. ULK1, a mammalian serine/threonine protein kinase, plays a key role in the initial stages of autophagy, though the exact molecular mechanism is unknown. Here we report identification of a novel protein complex containing ULK1 and two additional protein factors, FIP200 and ATG13, all of which are essential for starvation-induced autophagy. Both FIP200 and ATG13 are critical for correct localization of ULK1 to the pre-autophagosome and stability of ULK1 protein. Additionally, we demonstrate by using both cellular experiments and a de novo in vitro reconstituted reaction that FIP200 and ATG13 can enhance ULK1 kinase activity individually but both are required for maximal stimulation. Further, we show that ATG13 and ULK1 are phosphorylated by the mTOR pathway in a nutrient starvation-regulated manner, indicating that the ULK1·ATG13·FIP200 complex acts as a node for integrating incoming autophagy signals into autophagosome biogenesis. Autophagy is a degradative process that recycles long-lived and faulty cellular components. It is linked to many diseases and is required for normal development. ULK1, a mammalian serine/threonine protein kinase, plays a key role in the initial stages of autophagy, though the exact molecular mechanism is unknown. Here we report identification of a novel protein complex containing ULK1 and two additional protein factors, FIP200 and ATG13, all of which are essential for starvation-induced autophagy. Both FIP200 and ATG13 are critical for correct localization of ULK1 to the pre-autophagosome and stability of ULK1 protein. Additionally, we demonstrate by using both cellular experiments and a de novo in vitro reconstituted reaction that FIP200 and ATG13 can enhance ULK1 kinase activity individually but both are required for maximal stimulation. Further, we show that ATG13 and ULK1 are phosphorylated by the mTOR pathway in a nutrient starvation-regulated manner, indicating that the ULK1·ATG13·FIP200 complex acts as a node for integrating incoming autophagy signals into autophagosome biogenesis. Macroautophagy (herein referred to as autophagy) is a catabolic process whereby long-lived proteins and damaged organelles are shuttled to lysosomes for degradation. This process is conserved in all eukaryotes. Under normal growth conditions a housekeeping level of autophagy exists. Under stress, such as nutrient starvation, autophagy is strongly induced resulting in the engulfment of cytosolic components and organelles in specialized double-membrane structures termed autophagosomes. 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Cell. 2005; 16: 3438-3453Crossref PubMed Scopus (182) Google Scholar, 19Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (910) Google Scholar). Classical signaling pathways such as the cAMP-dependent kinase (PKA) pathway or the Tor kinase pathway appear to converge upon this complex, placing Atg1 at an early stage during autophagosome biogenesis (20Budovskaya Y.V. Stephan J.S. Reggiori F. Klionsky D.J. Herman P.K. J. Biol. Chem. 2004; 279: 20663-20671Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 21Budovskaya Y.V. Stephan J.S. Deminoff S.J. Herman P.K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13933-13938Crossref PubMed Scopus (183) Google Scholar, 22Carrera A.C. J. Cell Sci. 2004; 117: 4615-4616Crossref PubMed Scopus (57) Google Scholar). Atg1 phosphorylation by PKA blocks its association with the forming autophagosome (21Budovskaya Y.V. Stephan J.S. Deminoff S.J. Herman P.K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13933-13938Crossref PubMed Scopus (183) Google Scholar), while the Tor pathway hyperphosphorylates Atg13 causing a reduced affinity of Atg13 for Atg1, resulting in repression of autophagy (17Kabeya Y. Kamada Y. Baba M. Takikawa H. Sasaki M. Ohsumi Y. Mol. Biol. Cell. 2005; 16: 2544-2553Crossref PubMed Scopus (272) Google Scholar, 19Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (910) Google Scholar). In contrast, nutrient starvation or inhibition of Tor leads to dephosphorylation of Atg13 thus increased Atg1 complex formation and kinase activity, resulting in stimulation of autophagy (19Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (910) Google Scholar). Surprisingly, the physiological substrates of Atg1 kinase have not been identified; thus how Atg1 transduces upstream autophagic signaling is undefined. Recently, mammalian homologs of Atg1 have been identified as ULK1 and ULK2 (Unc-51-like kinase) 2The abbreviations used are: ULK, Unc-51-like kinase; DTT, dithiothreitol; PBS, phosphate-buffered saline; GFP, green fluorescent protein; IP, immunoprecipitation; MEF, mouse embryonic fibroblast; RNAi, RNA interference; MBP, myelin basic protein; mTOR, mammalian target of rapamycin. (23Chan E.Y. Kir S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, E.Y. R. S. J. J. Cell Sci. 2006; 119: PubMed Scopus Google Scholar, T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google Scholar). ULK1 and ULK2 are and to the or forming upon nutrient starvation T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google of ULK1 in cells autophagy (23Chan E.Y. Kir S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, E.Y. R. S. J. J. Cell Sci. 2006; 119: PubMed Scopus Google Scholar). The exact role of ULK1 ULK2 in autophagy is and it is the two M. T. J. F. J. PubMed Scopus Google Scholar). the of autophagy from yeast to it is to that mammalian to yeast Atg13 or been identified The protein FIP200 kinase protein of was identified as an of both ULK1 and ULK2 T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google Scholar), and it has been that FIP200 the of yeast Atg17, T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google Scholar, T. Mizushima N. 5: PubMed Scopus Google Scholar). In this we into the molecular of ULK1 to a into how mammalian signaling pathways autophagy the identification of a complex of ULK1, and the mammalian of This complex is required not only for localization of ULK1 to the membrane but for maximal kinase In both ATG13 and ULK1 are kinase substrates in the mTOR pathway and thus function to sense nutrient this the role of mammalian complex in the initial autophagic triggers and to the to the autophagic and and mouse from from mouse was from the at mouse from and mouse and mouse from was from and mouse ULK1 from mouse FIP200 in ULK1 was into containing an and ATG13 was into containing an in ULK1 was into containing a and both ATG13 and FIP200 into containing an was a from for mTOR and mTOR from of the for protein ULK1 and FIP200 into containing while ATG13 was into containing and ATG13 was into for in proteins for ULK1, and ATG13 in cells using the from Cell in 50 and over a Following proteins in containing and in and DTT, and at In the of ULK1 and protein was by over an and a by and was in and as described Y. T. X. 2007; PubMed Scopus Google Scholar). Cell mammalian cells in with and and at of autophagy, cells to and and in for as a at was in growth and cells and cells with to the and cells using a with cells used as the of of of and ULK1 or cells in PBS, and in DTT, for at by an additional at for at with with at Following proteins in containing for with for at with Following four in and one in PBS, in and proteins identified by RNA mouse FIP200 and ATG13 using to the The FIP200 used in the and The ULK1 and The ATG13 and Following and multiple and in with to protein and ATG13 cells in a using the RNA RNA was and of was used for first of the reaction as the for the for both ATG13 and to of the of both was using the of autophagy, cells in and in by in at in a at for with mouse for with at by of protein in for an additional in by one in in and by and protein at was in DTT, for at with ULK1 with for by protein in for an additional four in by in and by and ATG13 with for in and as for ULK1 at in and for 50 a in and by and ULK1 and the of FIP200 or ATG13 was in kinase 50 DTT, containing myelin basic and of for at by the of by to and by for as described K. S. Mol. Biol. Cell. 2004; PubMed Scopus Google Scholar). and using ULK1 a with ATG13 and the role of ULK1 in autophagy we for using a affinity a mouse embryonic a ULK1 with an and Following with and proteins identified by proteins identified FIP200 and protein the of this two FIP200 as a protein T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google and a protein to yeast Atg13 E.Y. A. Mol. Cell. Biol. PubMed Scopus Google Scholar), which been identified in a as a to yeast Atg13 Veenhuis M. 2007; PubMed Scopus Google Scholar). This protein to the of the mouse Further, and yeast Atg13 is we and ATG13 not and the functional described we now to as FIP200 and ATG13 have been identified as proteins of ULK1, how they ULK1 is not in this we to the of FIP200 and ATG13 ULK1 that ULK1 a complex with both to the yeast complex. cells we the ULK1 and of with to not only the of ULK1 but to ULK1 or FIP200 we that both ULK1 and as multiple and that upon starvation is a of both proteins to a with This is of a starvation-induced dephosphorylation and of with in a of ULK1 and ATG13 and In ULK1 has been to phosphorylated in cells T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google Scholar, E.Y. A. Mol. Cell. Biol. PubMed Scopus Google Scholar). of autophagy by starvation to the of ULK1 or FIP200 with with that ATG13 is with ULK1 and FIP200 to autophagy and ATG13 are to ULK1 phosphorylated autophagy cells in for by with or by to the of ATG13 is upon autophagy from cells or with to and ULK1 and ATG13 are upon autophagy cells in containing or by and with or in mTOR ATG13 cells with ATG13, or in with or mTOR as cells in containing or for in and to and with The of both ULK1 and FIP200 with that ATG13 with both proteins or that are two of one that with ULK1 and one that with In yeast, Atg1 is to with through Atg13 (17Kabeya Y. Kamada Y. Baba M. Takikawa H. Sasaki M. Ohsumi Y. Mol. Biol. Cell. 2005; 16: 2544-2553Crossref PubMed Scopus (272) Google Scholar). the of with ULK1 is by FIP200 we the of ULK1 in that of ULK1 was with in cells of FIP200 by with cells and with and the and FIP200 was by of ULK1 and with and that ATG13 can with both ULK1 and FIP200 in the of we ULK2 as a It that though of ULK1 or FIP200 is of the protein is of FIP200 to the of ULK1 with this is as the is to starvation-induced autophagy in as by and of and that all proteins are required for autophagy, we in cells of ATG13, and to a of ATG13 we used and to ATG13 is in ATG13 in two was used in the experiments described other experiments using two with the indicating of ULK1 and FIP200 was by and and of cytosolic to the the autophagosomal with its as the with lysosomes is a of autophagy. in was of the to the and an in upon indicating autophagy However, in ATG13, FIP200 and the of to the or the in was indicating a of autophagy in to This is by in cells in cells is an in upon starvation, indicating a in autophagosome formation and well with the in In contrast, in was in the ATG13, or the autophagy in ULK1, ATG13, and FIP200 in it is to that they by ULK1 and FIP200 have been to cytosolic in cells but with the membrane upon stimulation of autophagy and T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google Scholar). In the report the of ATG13, a a with which both the membrane and upon starvation E.Y. A. Mol. Cell. Biol. PubMed Scopus Google Scholar). ATG13 is to the we with Both ULK1 and a cytosolic normal growth However, they to structures upon starvation the of we cells with and or and in the of and with other and the of and while in normal proteins a cytosolic not a of ULK1, ATG13, and FIP200 at the membrane upon starvation, the that all proteins in a complex. that ULK1, ATG13, and FIP200 can and a complex, we in vitro using proteins in cells that in ULK1 ULK1 with FIP200 and ATG13 both and in of FIP200 to the reaction not the of ATG13 with ULK1 and ATG13 not the of FIP200 with ULK1 indicating that ULK1 with FIP200 and ATG13 at a was in all and to with ULK1 in ATG13 ATG13 with FIP200 and ULK1, both and in ULK1, ATG13, and FIP200 a complex, we a using the proteins ULK1 at and well with the of ULK1 in of indicating ULK1 is a in the of other ATG13 in a to and indicating a or a in the of other FIP200 at the molecular of FIP200 is FIP200 is a in the of other all proteins are and by a complex containing all proteins was and in and the the molecular of this protein complex is over However, are molecular additional experiments are in the for of the molecular of this complex. It is that a of both ULK1 and ATG13 to and complex ULK1 both ATG13 and all proteins can in a complex, we both FIP200 and ATG13 required for ULK1 first at the localization of ULK1 upon autophagy stimulation. ULK1 has been to to the membrane upon starvation T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google Scholar). this by both ULK1 and ULK1 from a cytosolic to a upon starvation, localization while both ULK1 and a cytosolic normal growth conditions not It is that only one of the two of ULK1 is required for its cellular upon However, we that of ATG13 or FIP200 blocks the localization of ULK1 or to the membrane in cells and with a ULK1 complex, containing both FIP200 and ATG13, is required for ULK1 localization to membrane upon autophagic In to of ULK1 FIP200 and ATG13 the kinase activity of we have proteins for ULK1, ATG13, and for the first of activity can in with yeast Atg1, the kinase substrates of ULK1 are we as the kinase In the of ULK1, ATG13 or FIP200 not not while ULK1 phosphorylation of ATG13 ULK1 kinase activity in the of FIP200 and in an ATG13 FIP200 is to the kinase activity of ULK1 in the of The of both ATG13 and FIP200 in an stimulation of ULK1 activity maximal ULK1 kinase activity both ATG13 and FIP200 in cellular that the role of ATG13 and FIP200 in the kinase activity of ULK1 has been T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google Scholar, E.Y. A. Mol. Cell. Biol. PubMed Scopus Google Scholar, J. H. A. Y. H. T. T. M. PubMed Scopus Google Scholar), and this is by experiments in cells ULK1 with a in the kinase or the Both in ULK1, and the ULK1 to the and not this as the we the cellular activity of ULK1 in both and cells and that ULK1 in or cells that in cells This is of but is in cells and with and and and It that ULK1 kinase activity is in and the in vitro that both proteins are required for ULK1 maximal kinase It is to that ULK1 in and cells are reduced to in cells It has been that ULK1 is in cells T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google Scholar), and now it that ATG13 is that ULK1 is in a complex with both ATG13 and and that the complex formation is required for ULK1 critical for how autophagy physiological substrates unknown. In this we that FIP200 is a of ULK1 In cells of ULK1, FIP200 as a in with of a of FIP200 In of ULK1 in cells in with FIP200 in a of FIP200 This in is ULK1 kinase activity, as of the kinase or ULK1 to Further, of with to the level in cells ULK1 can as well as ATG13, in vitro It that upon autophagy we not to of FIP200 in the role of FIP200 phosphorylation by ULK1 in autophagy has not been the other it is that only a of FIP200 is to ULK1 phosphorylation thus the FIP200 is a protein B. Cell PubMed Scopus Google Scholar). of the ULK1 by that ULK1 in multiple phosphorylation in the and of autophagy in an in ULK1 with and with and ULK1 is increased in and cells of is increased upon starvation the of ULK1 from of cells can increased by indicating ULK1 is by multiple In that ATG13 as a in with the of the upon autophagy stimulation. this ATG13 is a phosphorylated as of with its In all the pathway acts as a of autophagy and inhibition of with is to autophagy. In yeast it has been that of cells with in dephosphorylation of Atg13 and this well with autophagy a in mammalian of cells with or in dephosphorylation of and ULK1 This that mTOR is in the of the mammalian ULK1·ATG13·FIP200 complex in a to Tor of the yeast complex. we that of mTOR in an of ATG13 phosphorylation and with and This in ATG13 phosphorylation was upon starvation or with and this in ATG13 phosphorylation was mTOR kinase activity as of a of mTOR in of the ATG13 and mTOR can ULK1 phosphorylation not ATG13, and ULK1, are substrates of the mTOR kinase identification and of a complex, essential for mammalian autophagy. This complex of at least ULK1, ATG13, and of in vitro and in that all proteins are in a complex proteins can with indicating proteins from the only indicating that they are of the complex. proteins at the membrane upon autophagy stimulation. The stability of ULK1 the of both FIP200 and ATG13, as of one in reduced ULK1 at This is with proteins that a and not of FIP200 in or FIP200 is believed to function in multiple pathways B. Cell PubMed Scopus Google Scholar), and it is only a of cellular FIP200 with maximal ULK1 kinase activity in vitro is only both ATG13 and FIP200 are in that all proteins are complexed the ULK1 and the Atg1 it was the mammalian of autophagy the yeast with to the In yeast, it is well that inhibition of Tor kinase, through nutrient starvation or in dephosphorylation of Atg13 to the formation of a complex Atg1, and of this complex stimulation of Atg1 kinase activity, and this is to the mechanism by which Tor signaling autophagy (19Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (910) Google Scholar). In mammalian ULK1 was as the yeast to Atg1 (23Chan E.Y. Kir S. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google of Atg13 or was from the mammalian ULK1 function in autophagy with yeast the of FIP200 as a protein essential for autophagy T. Mizushima N. 5: PubMed Scopus Google Scholar), to yeast though was T. A. C. S. T. Mizushima N. J. Cell Biol. PubMed Scopus Google Scholar, T. Mizushima N. 5: PubMed Scopus Google Scholar). and E.Y. A. Mol. Cell. Biol. PubMed Scopus Google Scholar), have now identified mammalian show that this protein with FIP200 as well as ULK1, that the mammalian ULK1·ATG13·FIP200 complex is the of the yeast complex. are yeast Atg1 and mammalian ULK1 in yeast it is believed that the dephosphorylation of Atg13 triggers the formation of the complex, while in mammalian cells the ULK1·ATG13·FIP200 complex is to autophagy in ULK1·ATG13·FIP200 complex formation of autophagy by starvation, dephosphorylation of ATG13 ULK1 can both phosphorylated and of ATG13 to a that ULK1 is by mTOR in a to with or starvation, in dephosphorylation of ULK1 as well as Additionally, of mTOR the of ULK1 in of Atg1 has been in yeast upon stimulation of autophagy, though this was to of (16Matsuura A. Tsukada M. Wada Y. Ohsumi Y. Gene. 1997; 192: 245-250Crossref PubMed Scopus (387) Google Scholar). now show that this is the at least in and acts as level of in to ATG13 both ULK1 and ATG13 upon starvation or we that it is this the formation of the complex that is the for autophagy in mammalian It is to that this dephosphorylation of ULK1 and ATG13 in cells of complex and that localization to the which is in the of dephosphorylation of ULK1 and ATG13, we that the yeast mTOR and the ULK1 kinase complex normal growth thus the mechanism by which the mTOR pathway autophagy in eukaryotes. it that one protein the the ULK1·ATG13·FIP200 complex. In yeast it has been that the Atg1 and Atg13 is by Atg17, as in cells Atg1 and Atg13 to by (17Kabeya Y. Kamada Y. Baba M. Takikawa H. Sasaki M. Ohsumi Y. Mol. Biol. Cell. 2005; 16: 2544-2553Crossref PubMed Scopus (272) Google Scholar, 18Cheong H. Yorimitsu T. Reggiori F. Legakis J.E. Wang C.W. Klionsky D.J. Mol. Biol. Cell. 2005; 16: 3438-3453Crossref PubMed Scopus (182) Google Scholar). Here we that in the of ATG13 can ULK1 in to and ATG13 can FIP200 in the of we in vitro using proteins that two of ULK1, ATG13, and FIP200 can with other of the protein. to the exact function of the components of the ULK1 kinase complex, with to autophagy. of the major of ATG13 and FIP200 is to ULK1 kinase can in the of the maximal stimulation of ULK1 activity the other both ATG13 and FIP200 are required for ULK1 localization to the and of of correct localization of it the formation of the ULK1·ATG13·FIP200 complex is essential for ULK1 In the complex formation to for ULK1 protein a function of ATG13 and FIP200 is to ULK1 kinase activity, the and are are the physiological substrates of ULK1 and are the of The work to the to not only it appear that ULK1 can both ATG13 and FIP200 in vitro but that ULK1 can FIP200 in cells are in the process of the FIP200 and ATG13 phosphorylated by ULK1 to functional autophagy. to maximal ULK1 kinase activity, both ATG13 and FIP200 to and two proteins in of ULK1 in vitro for ULK1 substrates to the ULK1·ATG13·FIP200 complex to the of of the X. J. for critical and with