Title: Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy
Abstract: Article17 December 2010free access Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy Hong-Wen Tang Hong-Wen Tang Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Search for more papers by this author Yu-Bao Wang Yu-Bao Wang Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Shiu-Lan Wang Shiu-Lan Wang Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Mei-Hsuan Wu Mei-Hsuan Wu Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Shu-Yu Lin Shu-Yu Lin Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan NRPGM Core Facilities for Proteomics and Glycomics, Academia Sinica, Taipei, Taiwan Search for more papers by this author Guang-Chao Chen Corresponding Author Guang-Chao Chen Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Search for more papers by this author Hong-Wen Tang Hong-Wen Tang Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Search for more papers by this author Yu-Bao Wang Yu-Bao Wang Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Shiu-Lan Wang Shiu-Lan Wang Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Mei-Hsuan Wu Mei-Hsuan Wu Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Search for more papers by this author Shu-Yu Lin Shu-Yu Lin Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan NRPGM Core Facilities for Proteomics and Glycomics, Academia Sinica, Taipei, Taiwan Search for more papers by this author Guang-Chao Chen Corresponding Author Guang-Chao Chen Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan Search for more papers by this author Author Information Hong-Wen Tang1,2, Yu-Bao Wang1, Shiu-Lan Wang1, Mei-Hsuan Wu1, Shu-Yu Lin1,3 and Guang-Chao Chen 1,2 1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan 2Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan 3NRPGM Core Facilities for Proteomics and Glycomics, Academia Sinica, Taipei, Taiwan *Corresponding author. Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Taipei 115, Taiwan. Tel.: +88 622 785 5696/extn 7010; Fax: +88 622 788 9759; E-mail: [email protected] The EMBO Journal (2011)30:636-651https://doi.org/10.1038/emboj.2010.338 There is a Have you seen? (February 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. In this study, we show that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, we found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9) trafficking when cells were deprived of nutrients. Our findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein. Introduction Autophagy is a highly conserved catabolic process in which long-lived proteins, RNA, and organelles are degraded in lysosomes. It occurs at a relatively low level during normal growth conditions but can be strongly induced under such environmental stress as nutrient starvation, hypoxia, and oxidative stress. Once autophagy is induced, cytoplasmic components are engulfed within specialized double-membrane vesicles known as autophagosomes (Mizushima, 2007). These vesicles subsequently fuse with lysosomes for degradation and recycling. Although much of our knowledge on autophagy was first obtained from morphological observation in mammalian cells, the molecular components of this process were largely identified in yeast (Klionsky, 2007). Through the genetic screening of yeast, about 30 autophagy-related (ATG) genes have been identified (Yorimitsu and Klionsky, 2005; Suzuki and Ohsumi, 2007). A subset of these Atg proteins assembles into different protein complexes, including the Atg1 protein kinase complex, the autophagy-specific phosphatidylinositol 3-kinase (PI3K) complex, the Atg8 and Atg12 ubiquitin-like conjugation systems, and Atg9 recycling system (Xie and Klionsky, 2007), and is essential for the formation of autophagosomes. Interestingly, many of these ‘core’ Atg proteins are also found in mammals, suggesting the autophagic mechanism has been conserved. One major question about autophagy is where and how autophagosomes emerge. In yeast, the pre-autophagosomal structure (PAS) is thought to be the organizing centre for formation of sequestering vesicles (Reggiori and Klionsky, 2005; Xie and Klionsky, 2007). It seems that for this formation to begin, the PAS must be targeted by Atg proteins. The molecular mechanism underlying this process is not known, though the Atg1 complex may be involved in the recruitment of Atg proteins to PAS (Kawamata et al, 2008). The serine/threonine protein kinase Atg1 acts as an important link between the nutrient-sensing target of rapamycin (TOR) kinase signalling and autophagy. It has been shown that Atg1 associates with Atg13 and Atg17 in response to TOR regulation (Kabeya et al, 2005). Moreover, Atg1 is involved in the recycling of Atg9 between PAS and multiple peripheral structures (Reggiori et al, 2004), which may contribute to cycling of autophagosome membrane. In mammals, two Atg1 homologues have been identified: the UNC-51-like kinases, Ulk1 and Ulk2 (Kuroyanagi et al, 1998; Yan et al, 1999). It has been reported that both Ulk1 and Ulk2 can relocalize to mammalian PAS (called isolation membrane/phagophore) when cells are starved of nutrients and the formation of autophagosomes is inhibited when either Ulk1 or Ulk2 is depleted (Hara et al, 2008). These findings suggest a functional redundancy between these proteins. Like yeast Atg1, Ulk1 has been found to regulate the trafficking of mammalian Atg9 (mAtg9) from the trans-Golgi network (TGN) to forming autophagosomes under starvation conditions (Young et al, 2006). More recent studies have shown that Ulk1 also interacts with mammalian Atg13 (mAtg13), the scaffolding protein FIP200 (a functional analogue of yeast Atg17), and Atg101 (Hara et al, 2008; Jung et al, 2009; Hosokawa et al, 2009a, 2009b). These proteins form a large, stable complex and are essential for autophagosome formation. Apart from the Ulk1-mAtg13-FIP200 complex, it is unclear what other Ulk1 downstream signals are generated to trigger autophagy. In a recent study, we identified an interaction between Atg1 and the cytoskeletal protein paxillin (Chen et al, 2008). We also found that overexpression of Drosophila Atg1 induced autophagy and aberrant actin structures, suggesting a link between Atg1 and actin cytoskeleton in autophagy. As actin filaments form cables that serve as tracks guiding the movement of various cargos (Lanzetti, 2007), it may be that the actin cytoskeleton functions as structural support or as a track assisting the movement of autophagic components to PAS. In fact, in mammalian cells treated with actin-depolymerizing drugs such as cytochalasins B and D, the formation of autophagosomes has been found to be impaired (Aplin et al, 1992; Seglen et al, 1996). In yeast, actin filaments are reported to be essential for selective types of autophagy and the movement of Atg9 between the peripheral sites and the PAS (Reggiori et al, 2005; Monastyrska et al, 2008). However, the precise role that Atg1 has in the regulation of actin-dependent Atg9 cycling and autophagosome formation remains unknown. In this study, we investigated the relationship between Atg1 and actin cytoskeleton and their possible involvement in autophagy. We discovered in Drosophila a new substrate of Atg1, Sqa, a myosin light chain kinase (MLCK)-like kinase. Depletion of Sqa, its mammalian homolog zipper-interacting protein kinase (ZIPK), and the inactivation of myosin II compromised starvation-induced autophagy in both Drosophila and mammalian cells. In addition, depletion of ZIPK and inhibition of myosin II markedly inhibited the redistribution of mAtg9 from TGN to the peripheral pool in response to nutrient deprivation. Our study not only identifies a novel role of Atg1 in the regulation of actomyosin activation, but also provides some insight into the mechanism underlying Atg1 regulation of Atg9 trafficking during the early stages of autophagosome formation. Results Overexpression of Atg1 induces myosin II activation In one of our previous studies, as well as in one by Scott et al (2007), overexpression of Drosophila Atg1 was found to increase autophagy and result in apoptotic cell death (Chen et al, 2008). We also found that high levels of Atg1 led to abnormal cell morphology and F-actin reorganization (Figure 1B), as has been found previously (Chen et al, 2008). This suggests an increase in actomyosin contractility. As myosin II activity is regulated by the phosphorylating state of the myosin regulatory light chain (MRLC), we used a phospho-specific MRLC antibody that would recognize the pSer21 of Drosophila MRLC homolog Spaghetti squash (Sqh) to assess the myosin II activity of wing imaginal discs expressing Atg1 (Matsumura et al, 1998). Overexpression of Atg1 in the developing wing with ptc-GAL4 driver resulted in a dramatic increase level of phospho-MRLC and F-actin accumulation in GFP-marked Atg1-expressing cells, but not in ptc-GAL4 controls or in cells expressing the kinase-deficient Atg1, Atg1-KR (Figure 1A–C). Furthermore, the Atg1-induced MRLC phosphorylation could be suppressed by co-expressing the non-phosphorylatable form of Sqh, SqhA20A21 (Figure 1D) (Jordan and Karess, 1997). Taken together, these data suggested that Atg1 mediated the activation of actomyosin in a kinase-dependent manner. In addition to wing imaginal discs, we also found a robust increase in phopho-MRLC in response to Atg1 expression in fat-body cells (Supplementary Figure S1A) and in cultured S2R+ cells (Supplementary Figure S1B), suggesting that the Atg1-induced actomyosin activation is not tissue specific. Figure 1.Atg1 induces myosin II activation and spaghetti-squash activator (Sqa) identification. (A–F) Atg1-induced myosin II activation depends on the kinase activity of Atg1. Third-instar wing imaginal discs from ptc-GAL4 UAS-GFP controls or flies expressing indicated transgenes were stained with phospho-MRLC (blue) and TRITC-labelled phalloidin (red). Low level of phospho-MRLC staining was observed in controls (A) and cells overexpressing kinase-deficient Atg1-KR (C), whereas a robust increase in phospho-MRLC was found in cells overexpressing Atg1 (B). Co-expression of Atg1 and spaghetti-squash (SqhA20A21) exhibited low level of phospho-MRLC staining (D). Atg1-induced high level of phospho-MRLC staining was not suppressed by expression of caspase inhibitor p35 (E) or by depletion of Atg12 (Atg12RNAi) (F). Bar, 20 μm. (G) Schematic diagram of Drosophila and mammalian MLCK family. The quantities within the kinase domains indicate the degree of amino acid identity to the kinase domain of Sqa (CG1776). Ank, ankyrin repeats; Death, death domain; Fn, fibronectin domain; Ig, immunoglobulin domain; NLS, nuclear localization signal. (H) Sqa, but not Atg1, directly phosphorylated Sqh in vitro. Flag–Atg1, Flag–Atg1-KR, HA–Sqa and HA–Sqa-KA were immunoprecipated from lysate of transfected cells and incubated in an in vitro kinase reaction mixture containing [γ-32P]ATP and bacterially expressed recombinant wild-type Sqh or SqhA20A21. As shown on the autoradiogram (top panel), wild-type but not the kinase-deficient Atg1 (Atg1-KR) and Sqa (Sqa-KA) was autophosphorylated. No phosphorylation was seen with SqhA20A21. The equal input of His-fusion proteins is shown on the Coomassie staining. Anti-Flag and anti-HA immunoblottings (IBs) were used as controls to quantify the amount of proteins precipitated. Download figure Download PowerPoint We next examined whether the increased MRLC phosphorylation in Atg1-expressing cells was caused by Atg1-induced cell death or autophagy. We found that neither expression of the caspase inhibitor p35, nor RNAi-mediated downregulation of Atg12 suppressed the Atg1-induced MRLC phosphorylation (Figure 1E and F), indicating that Atg1-induced myosin II activation occurred through a caspase and autophagy-independent process. We further investigated whether actomyosin activation had a mediating role in Atg1-induced cell death and autophagy. We found the Atg1-induced caspase activation and autophagosome formation to be significantly suppressed by co-expressing SqhA20A21 (Supplementary Figure S2), suggesting that myosin II activation is required for Atg1-induced autophagy and cell death. CG1776 encodes a novel MLCK-like protein To investigate the possibility of a direct relationship between Atg1 and Sqh, we first tested whether Sqh could be a direct phosphorylation target of Atg1. Using His-tagged Sqh as a substrate in our in vitro kinase analysis, we did not find Atg1 to directly phosphorylate Sqh (Figure 1H). Therefore, it might be possible that Atg1 induces MRLC phosphorylation through myosin II activators, as phosphorylation of MRLC has been reported to be regulated by a spectrum of stimulating kinases, including MLCK, Rho-associated protein kinase (ROK), and the death-associated protein kinase (DAPK) family proteins (Vicente-Manzanares et al, 2009). In Drosophila, Stretchin-Mlck (Strn-Mlck) is the MLCK most closely related to the vertebrate smooth muscle/non-muscle MLCKs (sm/nmMLCK) and skeletal MLCKs (skMLCK). In a BLAST homology search against the BDGP database using the kinase domain of Strn-Mlck, we found a new MLCK-like protein, CG1776. The kinase domain of CG1776 is 50 and 49% identical with that of Strn-Mlck and smMLCKs, respectively (Figure 1G). However, unlike Strn-Mlck and smMLCK, CG1776 does not contain a Ca2+/calmodulin regulatory domain. We also found the kinase domain of CG1776 to be highly homologous to that of the DAPK family proteins such as DAPK1 and DAPK3/ZIPK (Figure 1G). Interestingly, a recent study has implicated a role of CG1776 in Drosophila S6 kinase (S6K) phosphorylation (Findlay et al, 2007). As Atg1 has been found to inhibit TOR/S6K-dependent cell growth via a negative feedback regulation (Lee et al, 2007; Scott et al, 2007), we wanted to study the relationship between Atg1 and CG1776. We renamed CG1776 as sqa for the following reasons. First, we performed in vitro kinase assays using Sqh as a substrate to assess whether the sequence homology of Sqa and MLCK extends to their biochemical activity. We found that wild-type Sqa could phosphorylate itself and Sqh (Figure 1H), but the catalytically inactive form, Sqa-KA, could not. Second, when Sqa was clonally expressed in wing imaginal discs using the flip-out GAL4 system (Ito et al, 1997), a dramatic increase of Sqh phosphorylation was detected in GFP-positive Sqa-expressing cells (Figure 3C). Thus, both our biochemical and immunostaining analyses indicated that Sqa is a bona fide MLCK-like protein capable of activating Sqh. Sqa genetically interacts with Atg1 To determine whether Sqa is involved in the Atg1-mediated activation of myosin II, we examined the effects of RNAi-mediated inhibition of Sqa on the Atg1-induced wing defects. Overexpression of Atg1 with ptc-GAL4 resulted in disappearance of anterior cross-veins in adult wings (Figure 2B), as was found in a previous study (Chen et al, 2008). Importantly, we found that ablation of Sqa expression by Sqa-RNAi strongly reversed the Atg1-induced disappearance of the cross-veins (Figure 2G). Moreover, similar to that of Atg1, overexpression of Sqa in wing discs using ptc-GAL4 resulted in an anterior cross-vein missing phenotype (Figure 2C). The Sqa-induced wing vein defects could be suppressed by co-expressing either Sqa-RNAi or SqhA20A21 (Figure 2I and J), suggesting the observed phenotype is Sqa specific and depends on myosin II activation. However, epistasis analysis revealed that depletion of Atg1 did not suppress the Sqa-induced wing vein defects (Figure 2K), suggesting that Sqa might function as a downstream target of Atg1. To determine whether Sqa is required for Atg1-induced activation of myosin II, we investigated whether ablation of Sqa expression could suppress the activation of myosin II and the disorganization of actin cytoskeleton induced by Atg1. Using anti-phospho-MRLC antibody in our immunofluoresence analysis, we found a marked decrease in MRLC phosphorylation and normal actin organization in the regions where Atg1 and Sqa-RNAi were co-expressed (Supplementary Figure S3A and B). Furthermore, consistent with our findings in Supplementary Figure S2, we found that inhibition of myosin II activation by depletion of Sqa strongly suppressed the Atg1-induced cell death and autophagy (Supplementary Figure S3C–F). These results together indicate that Sqa functions as an important downstream target of Atg1 in the regulation of myosin II activation and is required for Atg1-induced autophagy and cell death. Figure 2.Genetic and biochemical interactions between Atg1 and spaghetti-squash activator (Sqa). (A–K) Genetic interactions between Atg1, Sqa, and spaghetti-squash (Sqh). Compared with the ptc-GAL4 controls (A), expression of Atg1 or Sqa by ptc-GAL4 resulted in missing anterior cross-vein phenotypes (B, C). However, RNAi-mediated downregulation of Atg1 (Atg1RNAi) or Sqa (SqaRNAi) did not cause wing vein defects (D, E). Depletion of Atg1 and Sqa suppressed Atg1 and Sqa-induced wing vein defects, respectively (F, I). Atg1-induced wing defects were modulated by depletion of Sqa (G) or by co-expression of SqhA20A21 (H). Nevertheless, Sqa-induced wing vein defects were suppressed by co-expression of SqhA20A21 (J) but not Atg1RNAi (K). (L) Schematic presentation of the domain structures of Sqa and deletion mutants. (M–N) Atg1 physically interacted with Sqa. (M) 293T cells were transfected with HA-tagged Sqa WT (wild-type) or KA, together with Flag-tagged Atg1 WT or KR. The cells were lysed 48 h after transfection and immunoprecipitated (IP) with anti-Flag antibodies. The immunoprecipitated proteins and the total cell lysates (TCL) were analysed by immunoblotting (IB) with antibodies as indicated. (N) 293T cells transfected with Flag–Atg1-KR and various HA-tagged Sqa contracts were subjected to immunoprecipitations with anti-HA antibody. The immunoprecipitated proteins and the total cell lysates were analysed by immunoblotting with antibodies as indicated. (O) Atg1 directly phosphorylated Sqa in vitro. Flag-tagged Atg1 WT or KR immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant Sqa-K1, Sqa-K2, and Sqa-C in an in vitro kinase assay. The lower panels represent equal input of His-fusion proteins and Atg1 immunoprecipitates. Download figure Download PowerPoint Sqa is a substrate of Atg1 To further elucidate the interaction between Atg1 and Sqa, we investigated whether Sqa physically interacted with Atg1. HEK 293T cells were transfected with Flag-tagged wild-type Atg1 together with HA-tagged Sqa. Immunoblotting of the anti-Flag immunoprecipates from cell lysates revealed no co-precipitation between wild-type Atg1 and Sqa (Figure 2M). It has been reported that protein kinases generally interact with substrates transiently and with relatively low affinity (Manning and Cantley, 2002). To overcome this problem for our binding assay, we employed a kinase substrate-trapping approach in which the catalytically inactive kinase variants formed a stable interaction with their substrates (Deminoff et al, 2006). We found that Sqa specifically co-immunoprecipitated with the kinase inactive form of Atg1, Atg1-KR, but not with the wild-type Atg1 (Figure 2M). Conversely, the kinase inactive form of Sqa, Sqa-KA, did not interact with wild-type Atg1. These results indicated that Sqa may be a substrate of Atg1. Next, to determine the region of Sqa responsible for its interaction with Atg1, two HA-tagged Sqa mutants, HA–Sqa-K and HA–Sqa-C (Figure 2L), were constructed and tested for their ability to pull down Flag-tagged Atg1-KR. Co-immunoprecipitation assays revealed that Atg1-KR specifically interacted with Sqa-K, which contains the N-terminal kinase domain (amino acids 1–301), but not with Sqa-C, which contains only the C-terminal region (amino acids 291–446) (Figure 2N). Taken together, these results suggest that Sqa is an Atg1-interacting protein, and the interaction is mediated through the kinase domain of Sqa. Both our genetic and biochemical data suggested that Sqa might be a direct substrate of Atg1. To find out, we performed in vitro kinase assays using Atg1 isolated from 293T transfectants and various recombinant Sqa mutants as substrates. As shown in Figure 2O, Atg1 phosphorylated the kinase domain region of Sqa, Sqa-K1 (amino acids 1–189) and Sqa-K2 (amino acids 190–301), but not the C-terminal region of Sqa, Sqa-C, suggesting that there are multiple Atg1 phosphorylation sites within the N-terminal kinase domain of Sqa. Furthermore, the kinase-inactive Atg1-KR was not able to phosphorylate either region of the Sqa (Figure 2O). Therefore, consistent with the binding assay, our data strongly suggest that Sqa is a direct substrate of Atg1. Phosphorylation of Sqa at Thr-279 is required for Atg1-mediated myosin II activation It has been shown that MLCK activity is regulated by multisite phosphorylation through various protein kinases, including cAMP-dependent protein kinase (PKA), protein kinase C, and CaM kinase II (Stull et al, 1993; Gallagher et al, 1997). However, these kinases phosphorylate MLCK at sites in the Ca2+/calmodulin regulatory domain, which is not found in Sqa. A recent study by Graves et al (2005) revealed that phosphorylation of Thr-180, Thr-225, and Thr-265 in the kinase domain of the ZIPK (also known as DAPK3) is essential for full enzymatic activity to occur. Our sequence analysis showed Sqa to be highly homologous to ZIPK and to exhibit three corresponding phosphorylation sites at Thr-194, Thr-239, and Thr-279 (Supplementary Figure S4A). We further determined the Atg1 phosphorylation sites of Sqa using mass spectrometry-based analyses. Notably, MS/MS analysis revealed a single phosphorylation site at Thr-279 in a peptide corresponding to residues 273–285 of Sqa (Supplementary Figure S4B). We thus mutated Thr-194, Thr-239, and Thr-279 of Sqa-K2 to alanine and assayed their ability to be substrates for Atg1 in an in vitro kinase assay. Compared with the wild-type Sqa-K2, the substitution of Ala for Thr-279, but not for Thr-194 or Thr-239 strongly attenuated the phosphorylation of Sqa-K2 by Atg1 (Figure 3A). On the basis of these findings, it is likely that Thr-279 of Sqa is a major phosphorylation site of Atg1 and an additional site(s) may be present within the stretch of residues spanning from amino acids 1 to 301. Figure 3.Phosphorylation of spaghetti-squash activator (Sqa) at Thr-279 is required for Atg1-mediated myosin II activation. (A) Characterization of Atg1-dependent phosphorylation sites on Sqa. 293T cells transfected with Flag-tagged Atg1 or Atg1-KR were subjected to immunoprecipitation with anti-Flag antibodies, followed by in vitro kinase assays with bacterially expressed Sqa-K2, Sqa-K2-T194A, Sqa-K2-T239A, and Sqa-K2-T279A as substrates. Atg1 but not Atg1-KR was autophosphorylated (top panel). Relative phosphorylation levels of substrates were quantified. Data are represented as mean±s.e. of triplicates. (B–D) Phosphorylation of Sqa at Thr-279 is critical for the kinase activity of Sqa. (B) HA-tagged Sqa or Sqa-T279A immunoprecipated from lysate of transfected cells was used to phosphorylate bacterially expressed recombinant spaghetti-squash (Sqh) WT and A20A21, in an in vitro kinase assay. (C) Clonal expression of Sqa but not Sqa-T279A (GFP-positive cells) in the larval wing imaginal discs resulted in a marked increase in phospho-MRLC staining (blue) and actin reorganization (red). Bar, 20 μm. (D) The larval fat body of denoted genotypes were dissected, lysed, and subjected to western blot analysis using anti-phospho-MRLC and anti-Sqh antibodies. For quantification, the levels of MRLC phosphorylation in each genotype were measured with ImageJ and normalized to the Sqh levels. Data are expressed as a fold change compared with the wild-type controls. Each value represents mean±s.e. from triplicate experiments. See Supplementary data for genotypes. Download figure Download PowerPoint To assess the importance of Thr-279 on the function of Sqa, we generated the T279A mutation in the full-length Sqa and determined its enzymatic activity both in vivo and in vitro. Compared with the wild-type Sqa, T279A mutant showed markedly reduced autophosphorylation in an in vitro kinase assay (Figure 3B). Consistent with the autophosphorylation data, T279A mutant dramatically reduced the catalytic activity of Sqa in phosphorylating Sqh in vitro (Figure 3B). Furthermore, both immunofluorescence and immunoblotting analyses showed that T279A mutant failed to stimulate MRLC phosphorylation in vivo (Figure 3C and D). Notably, co-expression of T279A mutant also reduced Sqa-induced wing vein defects (Supplementary Figure S5E). These findings indicated that T279A mutant could behave as a dominant negative mutation. We further investigated whether it would suppress wing vein defects caused by Atg1. We found that co-expression of Sqa-T279A mutant markedly suppressed the Atg1-induced wing phenotypes and the activation of myosin II (Supplementary Figure S5F and G), indicating that Atg1-mediated phosphorylation of Thr279 is required for Sqa activation, which subsequently stimulates the activation of myosin II downstream. Myosin II activation is required for starvation-induced autophagy It has been reported that a robust autophagic response is induced in Drosophila larval fat body in response to nutrient deprivation (Scott et al, 2004). This process depends on the downregulation of TOR signalling and activation of Atg1 kinase. To investigate the physiological role of myosin II in autophagy, we first tested whether the myosin II activity of larval fat body was altered during starvation conditions, and found a robust increase in MRLC phosphorylation (Figure 4A). Importantly, starvation-induced myosin II activation was markedly abolished in Atg1 null but not in Atg1 heterozygous animals (Figure 4A), suggesting that the observed MRLC phosphorylation occurred in an Atg1-dependent manner. As the Atg1-induced autophagy could be modulated by the TOR pathway, we tested whether increased TOR activity affected starvation-induced activation of myosin II. As shown in Figure 4B, overexpression of TOR activator, the Rheb GTPase, in the starved larval fat body strongly suppressed myosin activation, compared with GFP controls. Moreover, we found that expression of either Sqa-RNAi or Sqa-T279A in larval fat body significantly blocked the upregulation of myosin activity under starvation conditions (Figure 4B). However, neither ablation of Atg7 nor Atg12 expression affected MRLC phosphorylation (Figure 4B, data not shown). These results suggest that the starvation-induced myosin II activation is regulated by TOR activity and through the Atg1–Sqa-mediated pathway. Figure 4.Atg1–spaghetti-squash activator (Sqa)-mediated myosin II activation is required for starvation-induced autophagy. (A, B) Activation of myosin II on nutrient deprivation. The larval fat body of denoted genotypes under fed or starved conditions were dissected, lysed, and subjected to western blot analysis using antibodies specific for phospho-myosin regulatory light chain (MRLC) and total MRLC. The Rheb, SqaRNAi, Sqa-T279A, spaghetti-squash (SqhA20A21), and Atg7RNAi transgenes were expressed under the control of hs–GAL4 driver (B). For quantification, the relative phosphorylation levels of MRLC were quantified as in Figure 3D. Data are represented as mean±s.e. of triplicates. (C–J) Starvation-induced autophagosome formation was compromised by inhibition of myosin II activation. Compared