Interneuron dysfunction in humans is often associated with neurological and psychiatric disorders, such as epilepsy, schizophrenia, and autism. Some of these disorders are believed to emerge during brain formation, at the time of interneuron specification, migration, and synapse formation. Here, using a mouse model and a host of histological and molecular biological techniques, we report that the signaling molecule cyclin-dependent kinase 5 (Cdk5), and its activator p35, control the tangential migration of interneurons toward and within the cerebral cortex by modulating the critical neurodevelopmental signaling pathway, ErbB4/phosphatidylinositol 3-kinase, that has been repeatedly linked to schizophrenia. This finding identifies Cdk5 as a crucial signaling factor in cortical interneuron development in mammals. Dysfunction of cerebral cortical interneurons, which may stem from their abnormal development and migration, has been implicated in the etiology of a number of neurological and psychiatric disorders in humans, such as epilepsy, schizophrenia, and autism . In receCdk5, a versatile proline-directed serine/threonine kinase, and its main activator p35, are signaling molecules expressed in neurons of the developing Cx . p35/CdkErbB  proteins (1–4) are members of the tyrosine protein kinase family and the epidermal growth factor (EGF) receptor subfamily. ErbBs interact with ligands of the EGF superfamily, including neuregulins (NRGs), dimerize, catalytically activate each other by cross-phosphorylation, and then stimulate various signaling pathways. ErbB4, the only ErbB member with multiple isoforms , mediatep35 knockout mice (KO) leads to permanent reduction in the final number of a subtype of interneurons -positive) that may affect neuronal circuit formation, thus increasing the risk of neurodevelopmental disorders, such as schizophrenia.Here, we report that p35/Cdk5 directs tangential migration of cortical interneurons by regulating the ErbB4/PI3-kinase signaling pathway at the pallial/subpallial border (PSB). We first showed that the Cyt1 isoform of ErbB4, coupled to PI3-kinase, emerges when interneurons reach the PSB and enter the Cx streaming toward sources of ErbB4 ligands ). We then identified ErbB4 as a novel p35/Cdk5 kinase substrate, and demonstrated that Cdk5 is an upstream regulator of ErbB4/PI3-kinase signaling by positively controlling tyrosine phosphorylation of the PI3-kinase docking site Y1056. Further, we found that Cdk5-dependent ErbB4/PI3-kinase signaling cascade regulates interneuron polarity, directionality, and leading process morphology at the PSB. Finally, we showed that lack of Cdk5 activity in p35, Cdk5, GAD67-GFP (Δneo), ErbB4HER4heart, and ErbB4HETmice were used in this study . All procedures were performed under license, and in accordance to regulations of the UK Home Office, Japan Neuroscience Society and Keio University School of Medicine.GAD67-GFP transgenic mice at E13.5 and E15.5 were isolated by FACS method as described previously (GABAergic (GFP-positive) and non-GABAergic (GFP-negative) cells, from Cx and GE of eviously .COS7 cells were transfected with expression vectors using Lipofectamine 2000 reagent (Invitrogen) according to manufacturer's protocol, and collected after 48 h.Supplementary material, as well as the sources of antibodies and reagents.Standard techniques were used for these analyses and are described in detail in Phosphorylation state-specific polyclonal antibody (ab) that specifically recognizes phosphorylated ErbB4 at Thr1152 was generated and purified by Sigma-Genosys  using a rat peptide sequence CELDEEGYM[pThr]PMHDK conjugated to carrier protein KLH injected as antigen in rabbits.Supplementary material). Cyt1 (accession number AY375306.1) and Cyt2 (accession number AY375307.1), submitted to the GenBank by Gambarotta et al. in 2003, entirely matched the sequences obtained in this study. GST-ErbB4Ala1143-Tyr1262, containing T1152 (referred to as GST-T1152), was cloned using Cyt1 as a template. Nrg3 , encoding the full EGF-like domain, was cloned from a mouse E12.5 embryo cDNA library.JMa-Cyt1 (referred to as Cyt1) and JMa-Cyt2 (referred to as Cyt2) isoforms of ErbB4, as well as ErbB4ΔICD-JMa (referred to as ErbB4ΔICD), truncated for most of the intracellular domain (ICD), were cloned from a rat adult forebrain cDNA library .A single-point mutation in the Cdk5 phosphorylation [T1152 (ACT) to A (GCT)] or PI3-kinase-binding site [Y1056 (TAC) to F (TTC)] of ErbB4 or multiple point mutations within the EGF-like domain of Nrg3  were introduced using a standard QuikChangeCyt1, Cyt2, Cyt1-T1152A, Cyt1-Y1056F, Cyt2-T1152A, and ErbB4ΔICD were expressed from the mychis B (-) (Invitrogen) or the pCAG-IRES-EGFP . The surface area of the embryonic MGE or adult somatosensory Cx (including adjacent white matter) was measured with the Image J (NIH) program.t-test (Microsoft Excel). Error bars represent SEM.Data were expressed as mean ± standard error of the mean (SEM) and evaluated for significant differences by means of a 2-tailed Student's GAD67GFPWT mice by FACS , was first explored, bearing in mind that some ErbBs are Cdk5 substrates  forebrain or its GABAergic and non-GABAergic cells, isolated from the ACS Fig. A, was fibstrates , and thabstrates .Figure ErbB1, ErbB2, and ErbB4 and very little, if any, ErbB3 in the Cx . However, the GFPGAD67(+) cells, isolated from tangential cortical migratory streams at E13.5 and subjected to PCR, expressed ErbB4 and no other members of the ErbB family . This finding was confirmed in a microarray study targeting all forebrain interneurons, namely, GE and cortical GFPGAD67(+) cells, at E13.5 and E15.5 (Supplementary Table 2). In addition, immunohistochemistry with ErbB4 ab revealed that about 3 quarters of all GFPGAD67(+) cortical interneurons express ErbB4 in either the preplate  or intermediate zone  at E13.5 . In contrast, the expression of other ErbBs was found in the GFPGAD67(−) cortical cells . Finally, the expression of Cdk5 and its activators p35 and p39 was also observed in the Cx and in GFPGAD67(+) cells .End-point PCR, microarray analyses and immunoblotting studies revealed the expression of GAD67(−)) of the developing forebrain (Supplementary Table 2), and, according to the literature, their topographical distribution was distinct . Therefore, we assessed the effect of these ErbB4 ligands on the migration of interneurons in an in vitro migration assay. Subjects of our analysis were either cells derived from E13.5 medial (M) GE, the site of origin of early-born, ErbB4-expressing interneurons, or Cx. As ErbB3 is hardly expressed in the Cx  and HB-EGF does not facilitate chemotaxis via ErbB1 . ErbB4 ligands, like other members of the EGF superfamily, contain a common EGF-like domain essential for their activity. This domain has a characteristic structure with 6 cysteine residues that form 3 intramolecular disulfide bonds. Here, we used either an EGF-like domain of NRG1β and HB-EGF, or conditioned medium (CM) of COS7 cells transfected with a plasmid encoding the EGF-like domain of NRG3. Following the 6-h long assay, we noted: first, MGE cells migrated toward NRG1β and HB-EGF in a dose-dependent manner . Second, MGE cells were attracted to NRG3-CM, which is a significant novel finding, and this action was abolished using either CM of COS7 cells transfected with a plasmid encoding a mutated EGF-like domain of NRG3 (NRG3mut-CM), or NRG3-CM treated with a blocking NRG3 ab that specifically recognizes the EGF-like domain of the ligand . Finally, the chemotactic response of cortical cells to ErbB4 ligands was peculiarly more robust than that of MGE cells . In summary, GABAergic cells are attracted by NRG1, NRG3, and HB-EGF in vitro.ErbB4 ligands, namely NRG1, NRG3, and HB-EGF, were abundantly expressed in the noninterneurons . On the other, cytoplasmic isoforms, Cyt1 and Cyt2, differ by the presence (Cyt1) or absence (Cyt2) of a binding site for PI 3-kinase ; the coupling of Cyt1 with PI3-kinase/Akt pathway stimulates chemotaxis (GAD67(+); Fig. 2B,C). Importantly, GFPGAD67(+) cells in the GE, but not in the Cx, at E13.5, lacked expression of the PI3-kinase-binding/chemotaxis-mediating ErbB4 isoform . Accordingly, by using phosphorylation state-specific ab, we found that ErbB4 was significantly phosphorylated on Cyt1-specific Y1056 in the Cx, but not in the lateral (L) GE or MGE . Together, these findings suggest that ErbB4/PI3-kinase signaling may be important for migration of interneurons toward and within the Cx .The signaling mechanisms by which ErbB4 exerts its functions in cell migration are only partly understood. Unlike other members of ErbB receptor family, this gene is subject to differential promoter usage and alternative splicing . On the 2emotaxis . Here, wGFP(+) interneurons express all but Class IB (Supplementary Table 2). Class IA PI3-kinase (hereafter referred to as PI3-kinase) is the most abundant member of the family in the developing Cx. It consists of a p110 catalytic subunit bound to 1 of 5 regulatory subunits, known as p85s. Recruitment of the p85/p110 complex to receptors occurs via p85 SH2 domains that bind preferentially to phosphorylated tyrosine at the consensus YxxM motif. ErbB4 contains at least 2 YxxM motifs (Y950mvM and Y1056tpM); the Y1056tpM sequence is Cyt1-specific and undoubtedly able to bind/activate PI3-kinase  on ErbB4 ; these were identified using the motif search engine Scansite (http://scansite.mit.edu/).Earlier studies have shown the importance of NRG1/ErbB4 , PI3-kinFig. 3A; . SimilarB). The role of Cdk5 activity in NRG1β/ErbB4-directed migration was confirmed in E13.5 p35 litters; the chemotactic response to NRG1β was strong in wt cells (201 ± 10), but significantly reduced in cells derived from p35 KO . Migration toward the negative control  was weak in both experimental settings . These findings support the notion that the PI3-kinase and Cdk5 signaling pathways regulate NRG1β/ErbB4-dependent cortical interneuron migration in vitro.The ability of NRG1β to induce chemotactic response in E13.5 cortical cells with altered either PI3-kinase or Cdk5 signaling was analyzed using an in vitro chemotactic assay. Pretreatment of wild-type (wt) cells with 10 μM LY294002 (PI3-kinase-specific inhibitor) or 10 μM Roscovitine (Cdk5-specific inhibitor), dissolved in dimethyl sulfoxide (DMSO), significantly decreased their migration toward NRG1β compared with untreated or DMSO-treated controls . We focused on threonine 1152 (T1152PmH/R) that lies in close proximity to Cyt1-specific PI3-kinase-binding site Y1056. To initially test whether ErbB4 is a substrate of Cdk5, we used a phosphospecific ab that recognizes a minimal Cdk consensus site, phosphorylated threonine adjacent to a proline (T(PO4)P), and discovered a stronger ErbB4 phosphorylation in COS7 cells that co-expressed p35/Cdk5 compared with other experimental conditions . To further confirm that ErbB4 is indeed phosphorylated by Cdk5, we performed an in vitro p35/Cdk5 kinase assay using recombinant GST-fused ErbB4 fragments containing either the native T1152 residue (GST-T) or an alanine point mutation (T1152A), which was obtained by site-directed mutagenesis and cannot be phosphorylated . GST and histone 1, an established substrate of Cdk5, were used as negative and positive controls, respectively. This assay revealed a robust phosphorylation by p35/Cdk5 of intact (GST-T), but not mutated [GST-T(A)] ErbB4 fragments , identifying ErbB4 as a Cdk5 substrate in vitro.A number of studies have suggested a strong correlation between Cdk5 activity and the ErbB receptor pathway , 2007. T3Cdk5 litters. Decreased phosphorylation of ErbB4Y1056 and AktS473 in Cdk5 KO animals compared with controls, confirmed our hypothesis that phosphorylation of ErbB4 by Cdk5 acts as a positive regulator of ErbB4/PI3-kinase/Akt pathway in the Cx in vivo .Next, we hypothesized that Cdk5, by phosphorylating ErbB4 at T1152 in close proximity to PI3-kinase-binding site (Y1056), could regulate ErbB4/PI3-kinase activity. To explore this premise, we looked at the levels of ErbB4 phosphorylation on residue Y1056, as well as amounts of phosphorylated Akt (S473), a downstream molecule in ErbB4/PI3-kinase signaling cascade, in protein lysates of E15.5 forebrain derived from T1152; Fig. 4E) or Cyt1-specific PI3-kinase-binding site . To test ab specificity, we co-transfected COS7 cells with plasmids encoding either 1) ErbB4 (Cyt1), ErbB4ΔICD or mutated, phosphorylation-resistant ErbB4Y1056F (pErbB4Y1056 ab) or 2) p35/Cdk5 in addition to ErbB4 (Cyt1), ErbB4ΔICD or mutated, phosphorylation-resistant ErbB4T1152A (pErbB4T1152 ab), and, after 48-h incubation, treated the cells with 50 ng/mL of NRG1β for 10 min. Immunoblotting of COS7 cells expressing intact or ICD-lacking ErbB4 showed positive or negative signal, respectively, for both phosphorylation-specific antibodies . However, while pErbB4T1152 ab did not recognize mutated ErbB4T1152A , there was a reduced signal in ErbB4Y1056F-expressing COS7 cells probed with pErbB4Y1056 ab compared with positive control (ErbB4), indicating that this ab could identify other possible PI3-kinase-binding consensus sites on the receptor . In developing forebrain, both phosphorylated forms of ErbB4 were observed during the peak period of tangential interneuron migration . Notably, pErbB4T1152 was detected in postmitotic areas of both the subpallium and Cx , while pErbB4Y1056 was prominently observed only at the PSB and in the Cx . Importantly, immunohistochemical analysis revealed the presence of both pErbB4T1152 and pErbB4Y1056 in approximately two-thirds of embryonic GFPGAD67(+) cells . In addition, pErbB4T1152 was detected in adult PV-positive interneurons . At subcellular level, phosphorylated ErbB4 proteins were found to co-localize with β-III-tubulin, a structural component of microtubules, particularly in the initial segment of the interneuron leading process . The fact that the residues that characterize the consensus site for Cdk5 phosphorylation or PI3-kinase-binding of ErbB4 have been conserved across species , and are in a state of phosphorylation in migrating interneurons, indicate that they might have an important role in directing these cells from the subpallium towards the cerebral Cx.To evaluate the state of phosphorylation of ErbB4 in cortical interneurons, we used phosphorylation-specific antibodies that recognize Cdk5 consensus site (pErbB4A), utilizing an array of different ErbB4 constructs encoding the full-length (Cyt1) or genetically modified ErbB4, with the aim of altering Cdk5 phosphorylation (Cyt1T1152A), PI3-kinase-binding (Cyt1Y1056F and Cyt2), both Cdk5 and PI3-kinase pathways (Cyt2T1152A) or entire ErbB4 intracellular signaling . As a control, we used CAG-driven EGFP vector (pCAG) that also served as a backbone for all ErbB4 constructs . To enhance the fluorescent signal, all plasmids were co-transfected with the CAG-driven tdTomato vector; the efficiency of the co-transfection, measured as a ratio between GFP(+) and tdTomato(+) cells, was close to 1 . For all analyses, images of red tdTomato fluorescence, converted to grayscale mode, were used.To test the function of phosphorylated ErbB4 in tangential migration of cortical interneurons, we performed focal electroporation of MGE of the whole telencephalic hemisphere at E13.5 , the lateral view allowed us to observe the tangential spread of MGE cells in the entire hemisphere . Images of forebrain sections, after cutting the hemispheres coronally , depicted the MGE cell migratory streams after passing the LGE; one running toward the piriform cortex (PCx) and the other in the direction of the neocortex . The latter bifurcated at the level of the PSB and gave rise to 2 streams running through the PPL and IZ . We observed a significant departure from the typical direction of migration in all experimental conditions, except control (pCAG). Strikingly, overexpression of intact ErbB4 (Cyt1) or ErbB4 modified to specifically suppress Cdk5, PI3-kinase or both pathways caused a pronounced phenotype characterized by abnormal accumulation of MGE cells in the PSB , and revealing an arcuate distribution when the hemisphere was viewed laterally . These MGE cells, on a coronal view and using a sector analysis , were largely gathered in the ventral (V) Cx (including the PSB) with fewer cells reaching the dorsal (D) Cx  compared with controls . It is worth noting that MGE cells overexpressing the intact ErbB4 receptor (Cyt1) exhibited a milder directionality defect compared with those which carried genetically modified ErbB4 . A complete removal of the ICD of ErbB4 (ErbB4ΔICD) in MGE cells led to a distinct phenotype in which numerous ErbB4ΔICD-expressing cells failed to advance from the MGE toward the LGE and Cx ; those that successfully migrated across the subpallium and into the Cx, were most likely a subpopulation that did not express ErbB4 in the first place .The positions of MGE cells were assessed in whole hemispheres and in forebrain sections 48-h postelectroporation. While the medial view of the hemispheres revealed the site of plasmid injection (MGE) Fig. B–H, the ere Fig. B′–H′. Imlly Fig. B″–H″, deErbB4 Cyt or ErbB4A). These appearances were perfectly in keeping with the descriptions reported by 6A) or branched/bifurcated  leading process. In addition, we found polar MGE cells that had a short and thick leading process with numerous protrusions emanating from the main neurite; they had a brushy appearance, hence, we termed them brush-like branched cells ; these were found in the trajectory between MGE and Cx, with the maximum accumulation at the PSB.We found that genetic manipulation of the ErbB4 signaling pathway in migrating MGE cells not only affected the direction of movement, but also altered their morphologies. For morphometric analysis, we focused on MGE cells situated at the PSB and Cx, and observed 4 types of morphologies Fig. A. These B–H). We first examined the morphology of normal  MGE cells and found a significant increase in the proportion of branched/bifurcated (type 2) cells when they overexpressed intact ErbB4  or genetically modified ErbB4  compared with control . On the other hand, cells that expressed ErbB4ΔICD and found at the PSB exhibited morphology similar to pCAG control . Interestingly, brush-like branched (type 3) and round (type 4) cells were never observed after transfection with control plasmid (pCAG) or ErbB4ΔICD. However, the proportion of brush-like branched (type 3) cells per entire polar cell population  or the proportion of round (type 4) cells per total number of examined cells  was significantly increased when MGE cells overexpressed intact or genetically modified ErbB4. The extent of phenotypic change depended on the ErbB4 signaling pathway(s) affected: mild , moderate  or severe  . In summary, abolition of ErbB4 phosphorylation by Cdk5 on residue T1152 had a profound effect on polarity and directionality of MGE cells. Furthermore, deficiency of both phosphorylation-sensitive residues (Cdk5-targeted T1152 and PI3-kinase-binding Y1056 in Cyt2T1152A) had the same great impact as loss of Y1056 alone (Cyt1Y1056F and Cyt2) , confirming that Cdk5 regulates ErbB4/PI3-kinase activity in migrating interneurons .Alterations in the morphology of MGE cells, and the extent of the transformation, depended on the specific ErbB4 signaling pathway that was targeted Fig. B–H. We fp35 and conditional heartErbB4HER4 KOs by using immunohistochemistry with calbindin (CB) ab. In both KO mice we detected significantly fewer CB+ cells in the Cx compared with control animals . However, the number of CB+ cells in the striatum at E14.5 was increased in p35- and reduced in ErbB4-lacking animals compared with control littermates . Therefore, in p35 KOs, interneurons failed to cross the PSB and exhibited a “cortical migratory defect,” while in ErbB4 conditional KOs these cells could not advance through the LGE/striatum, thus showing a “GE migratory defect” . Besides, the levels of ErbBs, ligands and associated molecules, assessed in a microarray study, remain normal in embryonic p35 KOs (Supplementary Table 2), as well as cell proliferation and cell death in the embryonic forebrain in both the p35 and heartErbB4HER4 KOs .Next, we compared interneuron phenotype in the developing Cx of p35 and heartErbB4HER4 KOs was permanent, we looked into numbers of adult cortical GABAergic neurons. Immunohistochemical analysis of mice at P21, using GFP (in GAD67GFPp35 litters) or GABA (in heartErbB4HER4 litters) ab, revealed a significant decrease in the density of GABAergic cells in both the p35 and the ErbB4 KOs compared with controls . We next wondered what specific classes of interneurons were affected by loss of p35 or ErbB4, and discovered that these 2 genes could control the development of the same, but also different interneuron subpopulations. While there was a significant reduction in density of PV-containing interneurons in both KOs compared with control littermates , we observed fewer SST+ cells only in p35 KO animals compared with controls . In addition, loss of either p35 or ErbB4 gene did not affect the calretinin  interneuron numbers  or specification of interneuron subtypes .Lastly, to determine whether reduction of interneurons in the developing Cx of We discovered that cortical interneurons utilize specific ErbB4-mediated signaling pathways to regulate their motility while coursing toward and through forebrain areas enriched in NRG1, NRG3, and HB-EGF. Explicitly, we found that p35/Cdk5 plays an important role in their directed migration by phosphorylating ErbB4 and subsequently modulating one of the canonical ErbB4 signaling pathways, PI3-kinase/Akt.We found that ErbB4 is the only ErbB family member expressed by migrating cortical interneurons. This gene encodes 2 distinct cytoplasmic isoforms that can selectively regulate growth factor responses by activating different signaling pathways . Publish+ cells . On the other hand, early-born migrating interneurons (E12.5/13.5) do not express Cyt1 until they reach the PSB, a cortical signaling center also known as antihem . Interneurons with altered ErbB4/PI3-kinase activity, as shown in this study, can pass through the MGE/LGE permissive corridor, but their migration is random, the leading process anomalous, and many cannot traverse the PSB. Yet, in the event of complete loss of ErbB4 or NRG1 signaling in the developing forebrain, a proportion of these cells fail to advance and stay confined to the MGE . To further address the exact role of different ErbB4 isoforms and accompanied signaling pathways in interneuron migration, electroporation of heartErbB4HER4 animals with either Cyt1 or Cyt2 should be performed.Interneurons born in the MGE migrate through the subpallium to reach the Cx and differentiate predominantly into PV- and SST-positive subtypes . We show antihem , the “an antihem  or the v antihem , where t antihem  and NRG3 antihem . TherefoWe provide the first evidence that Cdk5 phosphorylates ErbB4 and facilitates cell migration by modifying ErbB4 signaling. Other ErbBs are known substrates of Cdk5; specifically, Cdk5 phosphorylates ErbB2 and ErbB3 at the neuromuscular junction NMJ; , 2005 anSupplementary Fig. 8).Cdk5 phosphorylates ErbB4 at T1152, situated in close proximity to the PI3-kinase-binding site (Y1056), and in turn promotes ErbB4 tyrosine phosphorylation. Loss of Cdk5 function suppresses ErbB4 Y1056 phosphorylation and subsequently PI3-kinase activity. Recent studies have also shown that p35/Cdk5 positively controls ErbB2  and PI3-p35 KOs alters leading process morphology in migrating cortical interneurons. Specifically, p35-deficient interneurons display a significant increase in branching while entering the Cx from ventral telencephalic sources. Similarly, motor axon projections at the NMJ  which may promote the formation of aberrant neuronal circuitry underpinning clinically-defined neurodevelopmental disorders. Indeed, interference in NRG1/ErbB4 and p35/Cdk5 signaling has been associated with epilepsy , the Strategic Research Program for Brain Sciences , the Grant-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science, and Technology of Japan (K.N.) and grants from Breakthrough Breast Cancer (B.H.). Funding to pay the Open Access publication charges for this article was provided by the Wellcome Trust.This work was supported by


The galactomannan Davanat® was more active, albeit not a strong inhibitor . Pure synthetic oligosaccharide fragments found in the side chains and backbone of pectins and galactomannans were additionally tested. The most commonly found galactan configuration in pectins had no inhibition of the galectins tested. Galactosylated tri- and pentamannosides, representing the structure of Davanat®, had an inhibitory effect of galectins comparable with that of free galactose. Further evaluation using cell-based assays, indirectly linked to galectin-3 inhibition, showed no inhibition of galectin-3 by the polysaccharides. These data suggest that the physiological effects of these plant polysaccharides are not due to inhibition of the canonical galectin carbohydrate-binding site.Some complex plant-derived polysaccharides, such as modified citrus pectins and galactomannans, have been shown to have promising anti-inflammatory and anti-cancer effects. Most reports propose or claim that these effects are due to interaction of the polysaccharides with galectins because the polysaccharides contain galactose-containing side chains that might bind this class of lectin. However, their direct binding to and/or inhibition of the evolutionarily conserved galactoside-binding site of galectins has not been demonstrated. Using a well established fluorescence anisotropy assay, we tested the direct interaction of several such polysaccharides with physiological concentrations of a panel of galectins. The bioactive pectic samples tested were very poor inhibitors of the canonical galactoside-binding site for the tested galectins, with IC GCS-100 and PectaSol-C®, and fractionated pectin powder (FPP) , but they can be further organized into different classes with respect to the exact composition and substitution of the polysaccharide backbone  or substituted to various degrees with saccharides such as 2-O-methylxylose, 2-O-methylfucose, aceric acid, and several more in RG-II , d-apiose in apiogalacturonan, and d-xylose in xylogalacturonan  residues, depending on the species, with chains composed of either β-d-galactose  , α-l-arabinose (arabinans), or a mixture of both . The different classes of pectins should, however, not be considered as being separate molecules; on the contrary, they can be covalently attached to each other in many cases. It should also be pointed out that the exact composition of a pectin can vary considerably between different plant species, between plant tissue, and during development, making the pectins a highly complex family of polysaccharides  or to enrich for certain pectic domains, side chains, or monosaccharides  (28–d-mannose (Man) substituted with mono α-d-Gal via 1,6-linkage to the backbone  ,r (FPP) 124. One or (FPP) 1. Pectinsbackbone . The twocturonan , 26. RG-charides –27. Becalactans) , 28–30. und 1.7) –34.d-Gal residues . Moreover, galectins are involved in a number of cellular functions, such as regulation of intracellular glycoprotein trafficking, cell adhesion, cell signaling, and apoptosis with consequent effects in cancer, inflammation, and immunity  , 18, 32 residues –37 as foimmunity –37, makiity –1–9–, 18, 19.ity –1–9–, 21, 38 ity –1–9–, 38 wher9–et al. , 39, 40 To test the interaction of these compounds with physiological concentrations of galectins in solution, we have now employed a fluorescent anisotropy (FA) assay that permits evaluation of their occupancy and affinity for the canonical galectin carbohydrate-binding site. Some of the compounds are further tested in cell-based assays for indirect measurement of galectin-3 inhibition in extra- and intracellular environments. Both the biochemical analysis using the FA assay and the cell-based assays show that, in contrast to expectations based on previous publications in the field, pectins and galactomannans are in fact poor inhibitors of galectin/carbohydrate interactions and thereby contradicting the notion found in the literature stating that pectins and galactomannans are potent and selective inhibitors of the galectin CRD.l-arabinose and Davanat® was from Sigma, Lot 042K7277 (custom-made product D7065). One preparation of MCP was kindly provided by MacKinnon et al. . Another preparation of MCP as well as RG-I (ginseng), RG-I-4 (ginseng), and pectic galactan preparations from potato named p-oligo were as described in Gao et al.  was kindly provided by Galecto Biotech AB . NDP52 conjugated to fluorescein, FITC-aminohexanoic acid-PGLAYGNPYSGIQE, was purchased from Peptron Inc. . Hen blood in Alsever's solution was purchased from Håtunalab AB . ID-Cards “NaCl, enzyme test, and cold agglutinins” ) and ID-CellStab solution were received from Svenska Labex AB , and 2.5% trypsin in phosphate-buffered saline (PBS) was bought from VWR International AB . Glycyl-l-phenylalanine 2-naphthylamide (GPN) was purchased from Santa Cruz Biotechnology, Inc. . The human breast carcinoma cell line JIMT-1 was purchased from American Type Culture Collection . DMEM/Ham's F-12 medium (Biochrom GmbH), fetal bovine serum (FBS) (Biochrom GmbH), penicillin/streptomycin solution (Biochrom GmbH), nonessential amino acids (Biochrom GmbH), and insulin were bought from VWR International AB.Chemicals including fluorescent probes and galectins were as described before –44, unle–n et al.   is given in units of mA, where the theoretical maximum is 400, and the value for free fluorescein typically 23. All conditions were measured in at least two duplicate wells. This showed that the anisotropy measurement is highly reproducible, with between well difference of >5 mA occurring less than 10% of the time and >10 mA less than 1% of the time.0 typically about 35 mA) to the value when all probe is bound by galectin  . In this study, the effect of inhibitors of the saccharide probe/galectin interaction was tested in two ways. In the first, a fixed low concentration of fluorescein-tagged probe (0.1 μm) and galectin were mixed with a range of concentrations of inhibitor. An optimal probe was chosen for each galectin to permit use of as low as possible concentration of galectin, while still achieving a sufficient rise in anisotropy for a reliable measuring range . With structurally defined inhibitors  the molecular mass, molar concentrations, and number of binding sites (in this case one binding site per ligand) were known and their dissociation constants (dK) could therefore be directly calculated, using the data from the FA assay as described in Sörme et al. (50 in mg/ml) giving 50% reduction of the measured end point (anisotropy) was calculated, where 0% inhibition is anisotropy in the absence of inhibitor, and 100% inhibition is estimated as the anisotropy of the free probe . Even if the molar concentration of the inhibitor is not known, the anisotropy value at a given condition permits calculation of the molar concentration of free galectin, as described below, and from this what fraction of the galectin is bound by a given mass per volume concentration of inhibitor. From this, the ratio of the molar concentration of the free inhibitor binding sites to their dK value can be calculated, even if the FA assay cannot distinguish between the case when this is due to a few high affinity binding sites in the polysaccharide fraction or many of low affinity.In the presence of increasing amounts of galectin, the FA increases from the value for unbound probe  was mixed with a fixed concentration of inhibitor and a range of concentrations of galectin (C). Without inhibitor, this gives a curve going from anisotropy for free fluorescent probe (A0) to the value for all probes bound by galectin (Amax) as described above. In the presence of inhibitor, the curve shifts to the right and may or may not change slope, and it was compared with model binding curves as described under “Results.” The data were also used to estimate concentration and affinity of binding sites on saccharide inhibitor using the following derivations.In the next assay, a fixed low concentration of fluorescent probe , where [GP] is the concentration of fluorescent probe bound by galectin. This can be calculated from the measured anisotropy because [GP]/[P] = (A − A0)/(Amax − A), and galectin affinity for the probe, dKP, is determined by analysis in the absence of inhibitor as described by Sörme et al. (dKI = [IG]/[G] = ([Gtot]/[G]) − 1 − ([GP]/[G]), because [IG] = [Gtot] − [G] − [GP]; the last term can also be calculated from the measured anisotropy value and the equations given above, even if it is insignificantly small compared with the other terms when the concentration of probe is small compared with the galectin concentrations used. The ratio [I]/dKI derived in this way was plotted (y axis) against total added galectin (x axis) as shown in Fig. 1E, in an attempt to estimate the average affinity of the binding sites found in Davanat® and PectaSol-C®.From the law of mass action for galectin interaction with the fluorescent probe (P), it follows that the concentration of free galectin [G] = Ke et al. . The valdK in the low micromolar range). By using this peptide conjugated to fluorescein as probe in the FA assay  was prepared and incubated with 1 mg/ml trypsin at 37 °C for 1 h. The cells were then washed with 0.15 m NaCl. The erythrocytes were diluted and preserved as a 10% solution in ID-CellStab solution. For the hemagglutination in V-shaped microplates, 1 volume of erythrocyte suspension in PBS , 1 volume of galectin solution, 1 volume of 0.15 m NaCl with or without the galectin inhibitor, and 1 volume of 1% bovine serum albumin (BSA) in 0.15 m NaCl were added to each well of the microplate. The plate was then shaken vigorously for 90 min at room temperature and then left standing still for at least 90 min before observing the hemagglutination status in each well. For the hemagglutination in gel cards, 1 volume of the inhibitor dissolved in 1% BSA in 0.15 m NaCl, 1 volume of galectin dissolved in 0.15 m NaCl, and 1 volume of erythrocyte suspension  in 0.15 m NaCl were mixed and shaken for a short period in U-shaped microplates at room temperature. Each mixture was then transferred to individual gel card wells, whereupon the cards were centrifuged for 10 min at 85 × g in an ID-Centrifuge (DiaMed GmbH) and thereafter photographed and analyzed for hemagglutination.Hemagglutination by galectin-3 was investigated in two assays, hemagglutination in V-shaped microplates (96 wells) and hemagglutination in gel cards described by Nowak et al.  and Lapi  of a galectin, G, is governed by its binding to a receptor, R, to form the complex RG. We asked how much galectin inhibitor, I, competing with this receptor will be needed to prevent the effect. In a first case we assume, for simplicity, that >50% saturation of the receptor is required to see an effect. Hence, we asked what concentration of added inhibitor, [Itot], is required to bring down receptor saturation to this threshold of 50% that is minimal inhibitory concentration (MIC) of the effect, under different conditions of added galectin concentration [Gtot], available receptor concentration [Rtot], and affinities of the galectin/receptor interaction (dKR) and galectin/inhibitor interaction (dKI). Please note that MIC defined this way is not the same as IC50 mentioned above, which instead is the inhibitor concentration reducing initial galectin-induced anisotropy (probe binding) by half, which is of some value less than 100%.We assume that the effect (tot] = [G] + [IG] + [RG] and The interactions will be governed by tot] = MIC (y axis) as a function of concentration of added galectin [Gtot] (x axis) will give a straight line with a slope of 1 + (dKI/dKR). The weaker the inhibitor, i.e. higher dKI, the steeper the line will be. The line will cross the x axis at the point where just the added galectin by itself gives 50% receptor saturation; hence, [Itot] = 0 and [Gtot] = 1/2[Rtot] + dKR (from i.e. high dKI).Thus plotting [IKdR from . An intei.e. [R]/[RG] = Q, where Q = 1 only in the special case of 50% receptor saturation. Then dKR/Q = [G], and tot]/(1 + Q), which after insertion into Equations 5In a more general analysis, one can consider cases when the receptor saturation required for an effect is a number other than 50%, tot] versus [Gtot] will be steeper for cases where lower receptor saturation is required for effect, with Q > 1. Second, if two relatively weak inhibitors are compared, the relative slopes of the two lines will be independent of Q, when the Q(dKI/dKR) term in the slope is large enough so the 1 can be ignored. At the border of effect, when [Itot] = 0, now [Gtot] = [Rtot]/(1 + Q) + (dKR/Q). Thus a higher Q will give an intercept of the x axis for at lower value of [Gtot].This has two interesting consequences. First, the slope of the line /dKI was calculated from the anisotropy values, as described under “Experimental Procedures,” and plotted against the added galectin concentration in Fig. 1E. As expected, the values in the absence of inhibitor were ∼0. With 10 mg/ml Davanat®, the values were about 3.3 for all galectin concentrations used in the analysis. This shows that there was no significant saturation of any galectin-binding site on the inhibitor even at the highest galectin concentration. Had there been saturation, the curve would have turned down and approached the zero level, as illustrate by the model curves (dashed lines) in Fig. 1E. The ratio of about 3 for [I]/dKI also agreed with the inhibitory potency of Davanat® in Fig. 1A and the size of the shift between the curves in Fig. 1C, and no evidence for higher affinity sites was evident in the latter as illustrated by the steeper dotted model curve that does not fit the data. Thus, 10 mg/ml Davanat® contains at least 1 mm galectin-binding sites with dK values of at least 300 μm, but we cannot distinguish this from cases with even higher concentrations of binding sites with correspondingly lower affinity (higher dK). With the proposed molecular mass of Davanat® of 40–60 kDa  gave an [I]/dKI of about 1.2, which shows much weaker binding of PectaSol-C® compared with Davanat®, and no sign of saturation at the highest galectin concentration tested. With the proposed molecular mass of PectaSol-C® of 10–20 kDa, 24 mg/ml of the compound would correspond to around 1.2–2.4 mm with, for example, 5–10 binding sites per molecule with average dK values of 10–20 mm. The other polysaccharides did not have enough inhibitory potency, at the highest concentrations available, to make this type of analysis possible. They all had no or < 20% inhibition, as a safe reliably detectable minimum inhibition level, in the assay type exemplified in Fig. 1A and 2A. From this one can calculate that they had an [I]/dKI ratio of <1 at the highest concentration tested.As explained under “Experimental Procedures,” the FA assay described above cannot distinguish between the case when inhibitory potency is due to few high affinity binding sites in the polysaccharide fraction, or many of low affinity. To look for high affinity binding sites, we instead titered increasing concentrations of galectin into a fixed high concentration of polysaccharide, with a small concentration of fluorescent probe as a tracer to determine concentration of free galectin , C–E. In0–60 kDa , 10 mg/mdK) of known structural components of the plant polysaccharides, made synthetically or obtained as defined fragments, and also included some reference compounds , erythrocytes are treated with agglutinin (or antibodies) in V-shaped microtiter wells and incubated for about an hour. With no agglutination , erythrocytes sink to the bottom of the well and are seen as a distinct button, whereas with agglutination, the aggregated erythrocytes get sticky and smear on the walls resulting in a mat covering the well walls or a less clear button . In this assay, erythrocytes, agglutinin, and inhibitors are loaded on top of small gel-containing columns . After centrifugation, large aggregates stay at the top, and free erythrocytes go to the bottom.This classical assay was originally developed for antibody-induced hemagglutination in blood group serology. As the readout depends on physicochemical interaction between the agglutinate and the well surface, it may be disturbed and difficult to read in the presence of complex large saccharides. Therefore, we applied gel cards as another hemagglutination assay, now used extensively in blood group serology  because m and 1.7–5 μm of galectin-3 were needed to induce clear hemagglutination in V-shaped plates and gel cards, respectively (A and B). No agglutination in these assays was seen with galectin-3 mutant (R186S) (A and B), with severely reduced affinity for LacNAc structures , lacking the N-terminal part of the protein and therefore having a reduced cross-linking ability  , A and B ability .C and D), to be used as reference values for the pectic samples and Davanat®. Results show . The MIC for β-Lac was between 12.5 and 25, 200 and 500, 1000 and 2000, or >1500 μm for 1, 2, 5, and 10 μm galectin-3, respectively     , was tesd assay) .y axis and total added galectin on the x axis, this gives a straight line with a slope of 1 + (dKI/dKR), where the two dK constants are affinities of the galectin for inhibitor (I) and receptor (R).The hemagglutination data clearly showed that with a higher concentration of galectin-3, more inhibitor was needed to prevent the hemagglutination. To understand this better, we derived an equation that relates a MIC value, defined as the inhibitor concentration bringing a fraction of putative receptors bound by galectin under 50%, with total added galectin concentration  and receptor concentration and affinity (dKR) represented by the fluorescent saccharide probe were known, and % receptor saturation could be easily measured as % bound probe from the anisotropy value. Note that the inhibitor concentration bringing total bound probe to a 50% “threshold” is a MIC value as defined for the FA assay, and it is not the same as IC50 value described above, at which binding with and without inhibitor was compared. The data agreed very well with the equation, as a weak inhibitor, like β-Lac, gave a very steep slope of about 100 (A), consistent with dKI for β-Lac of about 90 μm and for the probe (dKR) of about 0.9 μm. With a much more potent inhibitor (33DFTG), with a dKI ∼10 nm, the slope was close to 1 as expected (B). In both cases, the x axis intercept was about 1.2, in good agreement with its predicted value from the formula of [Gtot] = 1/2[Rtot] + dKR, when[Itot] = 0. After this validation, we plotted the MIC for β-Lac with galectin-3-induced hemagglutination. Here, the MIC was not as precisely defined as for the FA assay, as it is somewhere between the last dilution of β-Lac with inhibition and next without inhibition. Nevertheless, using estimates of these values, the data again were clearly consistent with a straight line. This now had slope of about 180. With known dKI for β-Lac of about 90 μm, as mentioned above, this suggests that the average affinity for receptors on the erythrocyte surface (dKR) is <1 μm and/or the threshold receptor saturation may be less than 50%, as explained in the more generalized formula  as dKI is expected to be much smaller than dKR. Besides this, the equation, deduced under “Experimental Procedures,” also explains why it may be possible to inhibit a threshold-dependent (e.g. highly cooperative) effect, like hemagglutination, with very low concentrations of inhibitor, if the concentration of galectin added is just enough to raise receptor saturation over the threshold, and the concentration of inhibitor is just enough to bring it under the threshold again. In our case, for example, only 25 μm β-Lac was enough to inhibit hemagglutination induced by 1 μm total galectin, hence about 3 times lower than the dK value of β-Lac for galectin-3. With a dK value of 90 μm for β-Lac, this would give about 0.8 μm free galectin available for inducing hemagglutination (but failing to do so), showing how close to the threshold the assay was in this case.To test this relationship experimentally, we first used the FA assay, where galectin affinity for inhibitor  used as a positive control. With 0.5 or 5 mg/ml FPP or Davanat®, however, no inhibition of the GPN-induced galectin-3 accumulation could be seen for either compound (A and C). FPP, however, was shown to induce galectin-3 accumulation by itself at the highest concentration , which is not seen for Davanat®, indicating damage to a vesicular compartment in the cells treated with 5 mg/ml FPP (A).The anti-cancer effects of the different plant polysaccharides have sometimes been proposed to be mediated by extracellular galectin activities such as cell adhesion 1–12––19, 38 b–57–lectin-3 . If the lectin-3 , B and Dcompound , A and Cg/ml FPP A.50 values were in the same range or slightly lower compared with the IC50 values for inhibition of the galectin probe binding. This suggests that the galectin/ASF interaction is fully explained by the availability of galectin with free carbohydrate-binding sites and indicates the absence of other mechanisms for this inhibition.Davanat® was additionally tested for its inhibitory potency of galectin-1- or -3-induced ASF precipitation , as a moA and B). Moreover, the activity of galectin-1, as measured by the FA value, gradually decreased over time after mixing sample components, the rate of the decrease correlated with the concentration of Davanat® present, and the presence of β-mercaptoethanol prevented most, but not all, of the galectin-1 inactivation . This is in striking contrast to the case with galectin-3 and galectin-9N, where no further change in the FA value read at 5 min was seen after the longer incubation times (up to 3 h), and the presence or absence of β-mercaptoethanol made no significant difference (B and 2B); this also provides evidence that the effect of β-mercaptoethanol is not due to a change of the Davanat®, but instead involves galectin-1, as expected. Galectin-1 contains six cysteines, and C3 has been found to be most prone to initiate oxidative inactivation, as the mutant C3S is relatively (but not completely) oxidation-resistant in some environments (C and D). Monomeric galectin-1 is most prone to oxidative inactivation, and dimeric galectin-1 is relatively resistant , where the monomer accounts for a significant part.The interaction of Davanat® with galectin-1 was also examined in further detail . As expefference B and 2B;ronments , 62. Thiesistant , 63. ConC and D) showed that Davanat® or PectaSol-C® did not induce type-C self-association of galectin-3. Had they done so, the binding curve in the presence of polysaccharide would have had a lower slope, which would not have reached saturation of the probe because more galectin would bind the ligand than expected from its number of binding sites .In a previous report , we showng sites . There wE), and Davanat® showed no influence on the Anginex effect on galectin-1/probe interaction. In contrast, as shown here for comparison, Anginex potentiated the inhibition of galectin-1 by ASF by a factor of about 500, and this occurred equally with or without β-mercaptoethanol .We also tested the interaction of galectin-1 with Davanat® in the presence of the anti-angiogenic peptide Anginex , becauseFig. 7et al. . This is the case for Galβ1–4Gal, which is the most common galactose-containing disaccharide moiety in pectin-derived glycans, and Galα1–6Man, which is most common in galactomannans .There is a large body of evidence that the defining canonical galactose-binding site of galectins plays a central role in their function . Inhibit1–9–et al. ) becausee et al. ) in a ste et al. . Howevere et al. , 34, 39.e et al. , where t. red in  does notet al.  and higher temperature (about 23 °C compared with 4–17 °C). The more sensitive “tuning” of the assay delimitation between agglutination and non-agglutination may have made it more sensitive to lower concentrations of pectin fragments even if these have low affinity for the galectin, as we show in our theoretical consideration , the polysaccharides analyzed here show no inhibition of the carbohydrate binding activity of purified galectins in solution. There are several possible reasons for this discrepancy.The poor galectin inhibitory potency of the polysaccharides analyzed here is not, as mentioned above, consistent with the biological effects proposed to be due to galectin inhibition. At the concentrations typically used to induce cancer- or inflammation-related effects in cell culture or The higher galectin inhibitory potencies of the plant polysaccharides reported by others are mainly based on indirect assays that include incubation with cells over extended times. Neither of these show that the measured effect is due to binding of the plant polysaccharide to galectin-3, because there are many other effects that could have occurred during the long incubation and in turn affected the localization of the galectin. The externalization and cell surface exposure of galectin-3 are regulated by lipopolysaccharides , 70, forPlant polysaccharides such as modified citrus pectin and galactomannan are not expected to be taken up into cells across membranes, but they may very well be endocytosed, as for example is well known for dextran , which o57–dK ∼10 μm for galectin-1. Conversely, it was concluded that Davanat® at 0.35 and 0.7 mg/ml did not compete with canonical binding of β-Lac to 70 μm galectin-1 as analyzed by chemical shift perturbation .The possible interaction of a plant polysaccharide with another binding site on a galectin has been most clearly demonstrated by NMR for the binding of Davanat® to high concentrations galectin-1 and -3 , 39, 40.urbation , which ired here . In anotTo assess the possible contribution of such non-canonical sites in galectin-mediated glycoprotein cross-linking, we analyzed aggregation of ASF induced by galectin-1 and galectin-3. The concentration of Davanat® that inhibited the ASF aggregation by 50%  where the galectin-1 monomer is a significant component, it may be significant in a physiological situation. It may not have been seen in the NMR studies mentioned above, where much higher concentration was used, where galectin-1 is almost exclusively dimeric. Thus enhanced oxidative inactivation of galectin-1 or other proteins is another possible mechanism of action of plant polysaccharides.et al. , or interaction with other types of molecules altogether . Giin vitro vesicle damage studies. M. A., R. A. F., and A. A. K. produced some of the pectin and galactomannan samples analyzed in the study. U. J. N. and co-workers synthesized the fluorescent saccharide probes used in the FA assay. H. L. designed and supervised the project. The writing of the manuscript was mainly done by J. S., A. L., and H. L. with contributions and constructive feedback from all authors.Fluorescence anisotropy and turbidimetry studies were performed by B. K.-K., J. S., and A. L. Galectin-3-induced hemagglutination studies were done by J. S. and B. K.-K. J. S. and S. O. performed the


Atopic dermatitis is a multifactorial disease associated with elevated production of IgE and sensitization to multiple food and inhalant allergens.The aim of this study was to assess the levels of total serum IgE (IgE) and specific IgE (sp IgE ) for Dermatophagoides pteronyssinus (Dp), Blomia tropicalis (Bt), egg white, casein, alpha-lactalbumin and beta-lactoglobulin in patients with atopic dermatitis (AD); and the influence of gender, age and severity of disease in these parameters.We conducted a retrospective, cross-sectional study with patients with AD, between 2-18 years old and who had regular follow-up. Data were collected from medical records and the LAPIA Database. IgE and sp IgE measurements were performed by ImmunoCAP.We analyzed 30 patients, 50% (15) males, mean age = 10.3 years (SD = 10.6). IgE levels were elevated in 79% of the patients, 55% had sp IgE sensitization to mites; 36% to egg white; 34.6% to alpha-lactalbumin; 26.9% to beta-lactoglobulin and 30.76% to casein. In children, the percentage of sensitization to mites was 35.29% and 76.9% in teenagers (p = 0.02). The percentage of sensitization to mites, egg, casein, alpha-lactalbumin and beta-lactoglobulin in patients with mild AD was, respectively, 25%; 11.1%; 12.5%; 11.1% and 14.3%; in those with moderate / severe AD was: Dp and Bt (75%), egg white (88.9%), casein (87.5%), alpha-lactalbumin (88.9%) and beta-lactoglobulin (85.7%) [p <0.05].We observed a high percentage of patients with elevated IgE levels, a large proportion with sp IgE sensitization to mites and a third with sensitization to food. Our data indicate that sensitization to mites is more common in adolescents and that patients with moderate / severe AD have a higher risk of sp IgE sensitization to mites, egg and milk proteins.


Autophagy is a fundamental adaptive response to amino acid starvation orchestrated by conserved gene products, the autophagy (ATG) proteins. However, the cellular cues that activate the function of ATG proteins during amino acid starvation are incompletely understood. Here we show that two related stress-responsive kinases, members of the p38 mitogen-activated protein kinase (MAPK) signaling pathway MAPKAPK2 (MK2) and MAPKAPK3 (MK3), positively regulate starvation-induced autophagy by phosphorylating an essential ATG protein, Beclin 1, at serine 90, and that this phosphorylation site is essential for the tumor suppressor function of Beclin 1. Moreover, MK2/MK3-dependent Beclin 1 phosphorylation (and starvation-induced autophagy) is blocked in vitro and in vivo by BCL2, a negative regulator of Beclin 1. Together, these findings reveal MK2/MK3 as crucial stress-responsive kinases that promote autophagy through Beclin 1 S90 phosphorylation, and identify the blockade of MK2/3-dependent Beclin 1 S90 phosphorylation as a mechanism by which BCL2 inhibits the autophagy function of Beclin 1.DOI:http://dx.doi.org/10.7554/eLife.05289.001 Cells keep themselves healthy by breaking down unneeded or damaged internal structures via a process called autophagy. This process also helps a cell to survive if it is starved of nutrients. For example, if a cell does not receive enough amino acids, it cannot make new proteins. Autophagy can break down existing non-essential proteins so that their amino acids can be re-used to build other proteins that the cell needs to survive.Autophagy is performed by a set of proteins that is found in many different species, ranging from yeast to humans and plants. How these proteins are activated when a cell is starved of amino acids is not fully understood. However, evidence suggests that activating one of these proteins, called Beclin 1, by adding phosphate groups to it controls the extent to which autophagy occurs. It is also known from previous work that less autophagy occurs when Beclin 1 binds to another protein called BCL2.Wei, An et al. identified two enzymes that attach a phosphate group to a specific site on Beclin 1 to activate it, and revealed that autophagy is defective in cells that lack these enzymes. Furthermore, Wei, An et al. found the BCL2 protein prevents autophagy by binding to Beclin 1 in such a way that stops these two enzymes from activating Beclin 1.Beclin 1 is also known to prevent the growth of malignant tumors. Wei, An et al. found that to do so, Beclin 1 must have a phosphate group added to the same site that activates the protein during autophagy. This suggests that drugs that enhance the addition of this phosphate group to Beclin 1 could help activate autophagy and have anti-cancer effects.DOI:http://dx.doi.org/10.7554/eLife.05289.002 When eukaryotic cells are deprived of amino acids, they activate autophagy, a conserved lysosomal degradation pathway that enables cells to survive short-term periods of starvation by recycling nutrients and maintaining energy homeostasis . The autDespite extensive research over the past several years into the molecular mechanisms of autophagy, very little is understood about stress-responsive signals that regulate activation of autophagy protein complexes during amino acid deprivation. Inactivation of the autophagy repressor kinase mTOR (mechanistic target of rapamycin) in response to amino acid starvation contributes to autophagy through phosphorylation of ULK1/2 and ATG13 (resulting in their dissociation) and of ATG14 . The lowThe autophagy protein, Beclin 1, is a central regulator of autophagy and functions through its interaction with the Class III PI3K VPS34, VPS15, and the autophagy protein ATG14 in the initial stages of autophagosome formation . It is aHowever, it remains unknown whether stress-activated kinases directly phosphorylate Beclin 1 (or other ATG proteins) to positively regulate autophagy in response to amino acid starvation. Here we describe a novel role for members of the stress-activated protein kinase (p38 MAPK) signaling pathway, MAPKAPK2 (MK2) and MAPKAPK3 (MK3) , in mediOur findings reveal a crucial link between an evolutionarily conserved family of stress-activated protein kinases, MK2 and MK3, and a component of the autophagy machinery, Beclin 1, that plays an essential role in starvation-induced autophagy. Moreover, we demonstrate that blockade of MK2/MK3-dependent Beclin 1 phosphorylation is a mechanism by which BCL2 family proteins inhibit autophagy. These finding have important implications for understanding how cells respond to amino acid starvation and turn on autophagy.Activation of the Beclin 1/VPS34 complex is an essential event for starvation-induced autophagy . To gain33P, immunoprecipitation with an anti-Flag antibody and autoradiography, minimal phosphorylation was detected during nutrient-rich conditions, whereas Beclin 1 phosphorylation increased in response to starvation . Following metabolic labeling with We next evaluated whether endogenous Beclin 1 undergoes S90 phosphorylation in response to nutrient (amino acid) starvation using the Beclin 1 S90 phosphospecific antibody. Beclin 1 S90 phosphorylation was detected in HeLa cell lysates within 30 min after starvation and gradually increased over a 2 hr period . This inbeclin 1 , but chose to focus on starvation, given the crucial physiological importance of starvation in autophagy induction.In both MCF7 cells and U2OS cells, we observed that bafilomycin A1 treatment resulted in Flag-Beclin 1 S90 phosphorylation in the absence of starvation. To determine whether bafilomycin A1 also induces phosphorylation of endogenous Beclin 1 S90 and whether such effects are due to reactive oxygen species (ROS) generation that occurs as a consequence of inhibition of vacuolar-type-H-ATPases , we meascysteine . Our resTo address the mechanism by which Beclin 1 S90 phosphorylation induces autophagy, we compared U2OS cells expressing wild-type Beclin 1, the non-phosphorylatable Beclin 1 S90A mutant, and the phosphomimetic Beclin 1 S90E mutant with respect to (1) Beclin 1-associated VPS34 lipid kinase activity; (2) Beclin 1 immunoprecipitation of ATG14 and VPS34, and (3) the membrane localization of Beclin 1, ATG14, and VPS34. Our results show that the amounts of VPS34 and ATG14 that co-immunoprecipitate with Beclin 1 are not altered by starvation or by mutation of the Beclin 1 S90 site . In addinu/nu mice implanted with slow release estrogen tablets; and monitored for their rate of tumor growth. Levels of Beclin 1 expression were comparable in MCF7 cells transduced with both Beclin 1-expressing viruses  that mediate starvation-induced phosphorylation of Beclin 1 S90. Therefore, we performed an in vitro kinase screen using a 15 amino acid peptide corresponding to Beclin 1 amino acid residues 83–97 as a substrate. Among a pool of 190 serine/threonine kinases, MAPKAPK3, also known as and herein referred to as MK3, was the only kinase with significant activity against the Beclin 1 peptide substrate . Since tMK3 is highly homologous to MK2, another MAP kinase-activated protein kinase, and shares ∼75% sequence identity and displays functional redundancy . We founTo study the effects of MK2 and MK3 on autophagy regulation, we first evaluated autophagy in HeLa cells transfected with dominant negative MK2 (which blocks the kinase activity of both MK2 and MK3) or constitutively active MK2. As measured by LC3-II conversion, p62 degradation, and numbers of GFP-LC3 puncta in the presence and absence of bafilomycin A1, dominant-negative MK2 decreased starvation-induced autophagy . ConversThe best-characterized kinase upstream of MK2/MK3 is p38 MAPK . TherefoMapkapk2−/−/Mapkapk3−/− (herein referred to as MK2−/−/MK3−/−) MEFs retrovirally transduced with empty vector compared to MK2−/−/MK3−/− MEFs retrovirally transduced with MK2 , Beclin 1 S90E expression was sufficient to markedly increase basal autophagy in cells lacking both MK2 and MK3 , or Beclin 1 S90E (a predicted phosphomimetic mutant) on autophagy in  and MK3 . The magTo more directly test the hypothesis that MK2/3 regulate autophagy through the Beclin 1 S90 phosphorylation site, we investigated whether active MK2 can induce autophagy in cells that lack detectable endogenous Beclin 1 and only express an shRNA-resistant mutant Beclin 1 S90A construct . U2OS cebeclin 1 shRNA U2OS cells that express shRNA-resistant wild-type Beclin 1, dominant negative MK2 nearly completely suppressed starvation-induced autophagy, as assessed by p62 degradation and LC3-II conversion. However, in cells expressing shRNA-resistant Beclin 1 S90E, starvation induced a decrease in p62 levels and conversion of LC3-I to LC3-II even in the presence of dominant negative MK2 expression. This suggests that Beclin 1 S90 phosphorylation may be sufficient to partially bypass the inhibitory effects of MK2/3 inhibition on starvation-induced autophagy.Conversely, we asked whether expression of a Beclin 1 S90E phosphomimetic mutant in cells lacking detectable endogenous Beclin 1 could bypass the inhibitory effects of dominant negative MK2 . In doxyMK2−/−/MK3−/− MEFs and in doxycycline-inducible beclin 1 shRNA U2OS cells  strongly suggest that MK2/3 regulate autophagy through a mechanism involving Beclin 1 S90 phosphorylation.Taken together, these data in BCL2 inhibits autophagy through a direct interaction with the BH3 domain of Beclin 1, which consists of amino acid residues 105–128 . Given tTo directly test the hypothesis that BCL2 binding to Beclin 1 blocks MK2-mediated Beclin 1 S90 phosphorylation, we performed an in vitro kinase assay to assess the effects of MK2 on Beclin 1 S90 phosphorylation in the presence of recombinant GST-BCL2. We found that with increasing amounts of GST-BCL2, there was decreased in vitro MK2-mediated phosphorylation of Beclin 1 S90 . These dTo better understand how binding of BCL2 family proteins to Beclin 1 may inhibit MK2-mediated phosphorylation, we constructed a structure-based model  to deterNext, we assessed whether BCL2 binding to Beclin 1 in cultured cells regulates Beclin 1 S90 phosphorylation . In HeLaBcl2AAA mice that contain a T69A/S70A/S84A knock-in mutation in Bcl2 (S84 is homologous to human S87)  . PreviouAAA mice . FollowiAutophagy is a fundamental cellular survival response during nutrient starvation, permitting cells to maintain nutrient and energy homeostasis when external food supply is limited . The negbeclin 1-deficient cells using multiple different autophagic flux assays. The structurally and functionally-related p38 MAPK-activated protein kinase family members, MK2 and MK3  , but did and MK3 , but notMK2 knockout mice show increased resistance to endotoxic shock and increased susceptibility to infections  may have evolved to integrate autophagy induction with diverse other cellular functions required for cellular adaption to stress.These findings describe a previously unidentified direct link between specific members of the MAPK signaling pathway and the positive regulation of a core component of the autophagy machinery during starvation. MK2 was originally discovered as an ERK1/2 activated protein kinase that phosphorylates HSP25 and HSP27 , and wasfections  and thisons .Another implication of our finding relates to the potential relationship of MK3 deletion and Beclin 1 S90 phosphorylation in cancer development. Given the requirement of the Beclin 1 S90 site for the tumor suppressor activity of Beclin 1, it will be interesting to determine whether the lack of MK3-dependent Beclin 1 S90 phosphorylation contributes to the pathogenesis of small cell lung carcinomas that commonly harbor deletions in MK3  or of otPI3KC3-C1 consists of the lipid kinase, VPS34; the scaffolding protein, VPS15; and two autophagy proteins containing coiled-coil domains, ATG14 and Beclin 1. Our data show that Beclin 1 S90 phosphorylation increases the VPS34 lipid kinase activity of this complex, without altering its composition or membrane localization. These data are consistent with predictions from a recent EM reconstitution of the ordered domains of the PI3KC3-C1 complex . The arcBeclin 1 was originally identified as a BCL2-interacting protein , and BCLOur data uncover a previously undescribed mechanism by which BCL2 inhibits the autophagy function of Beclin 1. BCL2 binding to the BH3 domain of Beclin 1 blocks an essential step in the positive regulation of starvation-induced autophagy—MK2/MK3-dependent Beclin 1 S90 phosphorylation. Our structure-based model predicts that this may occur via steric hindrance of MK2/MK3 binding to Beclin 1 when BCL2 is bound to the BH3 domain of Beclin 1, which is in close proximity to Beclin 1 serine 90. While further studies will be required to test this structural model, our data provide strong functional evidence that BCL2 binding to Beclin 1 blocks Beclin 1 S90 phosphorylation, both in vitro and in vivo. BCL2 inhibits MK2-dependent in vitro phosphorylation of Beclin 1 S90 in a dose-dependent manner; BCL2 binding to Beclin 1 in cultured cells prevents starvation and active MK2-dependent Beclin 1 S90 phosphorylation; and Bcl2 AAA mice have deficient starvation-induced Beclin 1 S90 phosphorylation despite starvation-induced activation (phosphorylation) of MK2.Taken together, our findings describe a central regulatory loop underlying starvation-induced activation of autophagy. This loop involves two distinct arms of the MAPK signaling pathway, which act in parallel to activate the autophagy function of Beclin 1 in response to starvation . Previoubeclin 1 shRNA U2OS cells  were grown in DMEM supplemented with 10% FBS and 1 μg/ml puromycin. For autophagy assays, cells were grown in either the DMEM media described above for each cell line supplemented with 2× non-essential amino acids and 2 mM glutamine (nutrient-rich conditions) or in HBSS (starvation conditions) for the indicated time period. The HBSS used in starvation conditions contained glucose.HeLa, HEK293T, and MCF7 cells and SV40 immortalized murine embryonic fibroblasts (MEFs) retrovirally transduced with pMMP-IRES empty vector or pMMP-IRES MK2  were groOS cells  were grobeclin 1 gene was inserted into the EcoRI/BamH1 restriction site of the pBICEP-CMV2 vector. pBICEP-CMV2-Beclin 1 S90 mutants and pBABE-puro-Beclin 1 S90 mutants were constructed using a QuickChange site mutagenesis kit . The shRNA non-targetable Beclin 1 constructs harbor silent mutations (mutated sequence: GGAAGCAAAACTAGTAACA) in the region of beclin 1 targeted by shRNA (GGGTCTAAGACGTCCAACA) (sense strand) in order to rescue the effects of knockdown of endogenous Beclin 1. All of the constructs were confirmed by sequencing. Other plasmids used in this study have been previously described, and include plasmids expressing GFP-LC3  . The phosphospecific Beclin 1 S90 antibody was produced by PhosphoSolutions . Briefly, synthetic peptides corresponding to phosphorylated and dephosphorylated S90 of Beclin 1 were injected to two rabbits and sera were purified using a phosphopeptide affinity column followed by a dephosphopeptide affinity column. The phosphospecific Beclin 1 S90 antibody was used at a concentration of 1:500. Beclin 1, HSP27, p-HSP27, Actin, MK2, p62, LC3, TOM20, PDI, and GAPDH were detected using a rabbit or goat anti-Beclin 1 antibody , a rabbit anti-HSP27 antibody , a rabbit anti-p-HSP27 antibody , an anti-β-Actin HRP antibody , a rabbit anti-MK2 antibody , a mouse anti-p62 antibody  a rabbit anti-LC3 antibody , a rabbit anti-TOM20 antibody , a rabbit anti-PDI antibody , and a mouse anti-GAPDH antibody .beclin 1 shRNA U2OS cells, shRNA non-targetable Beclin 1 (or Beclin 1 S90 mutants) and MK2 mutants were co-transfected at a molecular ratio of 1:1.Lipofectamine 2000  was used for cell transfection according to the manufacturer's protocol. For the GFP-LC3 assay, GFP-LC3 and Beclin 1 (or Beclin 1 S90 mutants) were co-transfected at a molecular ratio of 1:2. For western blot analyses in Cells were lysed in lysis buffer  with protease inhibitors  and Pierce phosphatase inhibitors  for 30 min. Cell lysates were separated on 4–15% TGX gels , transferred to PVDF membranes (Bio-Rad Laboratories), and probed with the indicated antibodies. For immunoblotting levels of proteins in subcellular fractions, cytsolic, mitochondrial, and microsomal fractions were separated with a Qiagen Qproteome Mitochondrial Isolation Kit , and resuspended in an equal volume of storage buffer.33P-orthophosphate for 2 hr. The cells were washed three times with PBS, and lysed with lysis buffer  for 30 min. Lysates were centrifuged for 10 min at 13,000 rpm at 4°C. The supernatants were collected and adjusted to equal protein concentrations using the Bradford assay. 9 μl α-Flag (M2) agarose (Sigma-Aldrich) was added to each sample and samples were rotated at 4°C overnight. The agarose was washed three times with PBS. 30 μl SDS loading buffer was added to each sample and the samples were boiled at 95°C for 5 min. 10 μl of each sample were loaded on 4–15% TGX gels (Biorad Laboratories).HeLa cells were plated on 6 cm dishes, and transfected the following day with pBICEP-CMV2, pBICEP-CMV2-Beclin 1, or pBICEP-CMV2-Beclin 1 S90A using lipofectamine 2000. On the second day after transfection, cells were grown in phosphate-free medium (phosphate free DMEM + 10% FBS) for 3 hr. The cells were then washed with phosphate-free EBSS three times and were grown in phosphate-free EBSS or phosphate-free normal medium supplemented with 40 μCi Peptides based on amino acid sequences of human Beclin 1 83–97 were synthesized  and used as substrates for an in vitro protein kinase screen. The peptides were dissolved in 50 mM HEPES, pH 7.5 at a concentration of 200 μM. A radiometric Streptavidin-FlashPlate-based protein kinase assay was performed to measure the kinase activity of 190 serine/threonine kinases . For mutational analysis, peptides (wild-type and S90A) based on amino acid sequences of human Beclin 1 83–103 were used.Immunoprecipitation of endogenous Beclin 1 was performed using a polyclonal goat anti-Beclin 1 antibody  and immunoprecipitation of Flag-tagged Beclin 1 was performed using a monoclonal anti-Flag M2 antibody pre-conjugated to agarose . For co-immunoprecipitation of Beclin 1 and BCL2, immunoprecipitation was performed using a polyclonal rabbit anti-Beclin 1 antibody  and BCL2 was probed with a monoclonal HRP antibody .2, 3 mM MnCl2, 3 mM Na3VO4, 1.2 mM DTT). For each in vitro kinase reaction, 15 μl Flag-agarose was mixed with 15 μl kinase buffer (with 1 μM ATP). The kinases were added to a final concentration of 0.16 μM. The reactions were incubated at 30°C for 30 min. 30 μl SDS loading buffer was added to each reaction and the samples were incubated at 95°C for 2 min. After brief centrifugation, the supernatants were loaded onto 4–15% TGX gels (Bio-Rad Laboratories), transferred to PVDF membranes and incubated with the indicated antibodies. To assess whether BCL2 blocks MK2-mediated phosphorylation of Beclin 1 in vitro, 5 μg GST (negative control) or various amounts of GST-BCL2 recombinant protein were added to each reaction tube.For MK2/3 in vitro kinase assays, HEK293T cells were transfected with pBICEP-CMV2-Beclin 1 or pBICEP-CMV2-Beclin 1 S90A using lipofectamine 2000 ; 6 μg DNA was transfected into each 10 cm dish. After 24 hr, each dish of cells was lysed with 500 μl lysis buffer  for 30 min at 4°C. Flag-Beclin 1 was immunoprecipitated using 75 μl anti-Flag (M2) agarose (Sigma-Aldrich) for 3 hr at 4°C. The beads were washed twice with lysis buffer and another two times with phosphatase buffer. The samples were then treated with λ-protein phosphatase  according to the manufacturer's protocol. For 60 μl Flag-agarose, 4 μl λ-protein phosphatase was used. The agarose was washed twice with lysis buffer with phosphatase inhibitors, and another two times with kinase reaction buffer  were injected into the upper right mammary fat pad of each mouse. Tumor width and length were measured twice a week for a total experimental duration of 8 weeks. Each group contained 11 mice. All animal experiments were performed in accordance with institutional guidelines and approved by the UT Southwestern Medical Center Institutional Animal Care and Use Committee.A 60-day slow-release 1.7 mg estrogen pellet  was injected in the neck region of 7-week old female http://imagej.nih.gov/ij).Mouse tumor xenografts were fixed in 4% PFA, embedded in paraffin, and sectioned (∼5 microns in thickness). Immunohistochemical staining of paraffin-embedded tumor tissues was performed using anti-p62  or anti-Ki67  primary antibodies and the ABC Elite immunoperoxidase kit  according to the manufacturer's instructions. TUNEL staining was performed according to the manufacturer's instructions, using Sigma FAST 3, 3′-diaminobenzidine (DAB) tablets as the peroxidase substrate. Reciprocal intensity of p62 was quantified using the ImageJ 1.47v software (Bcl2AAA knock-in and littermate control C57/BL6 mice (8-week-old BL6 mice  were sta review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers. Vivek Malhotra also acted as Reviewing editor.Thank you for sending your work entitled “Stress-Responsive MAPK Family Members, MK2/MK3, Activate Starvation-Induced Autophagy through Beclin 1 Phosphorylation” for consideration at The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.The reviewers agreed on the significance of the findings and the high quality of the data presented. But how starvation activated MK2/MK3 and how activation of Beclin2 by MK2 initiated autophagy was not clear from the data presented. It was generally agreed that addressing some aspect of this would strengthen the conclusions.We suggest that you address the following three major issues:1) How activation of Beclin 1 by MK2 results in the initiation of autophagy? Results shown in 2) MK2 and MK3 have not been previously implicated in amino acid starvation stress responses. Moreover, the best-characterized kinase that activates MK2/MK3, p38 MAPK, is thought to negatively regulate starvation-induced autophagy . It would be very interesting and important to provide some new insight into how amino acid starvation results in MK2/MK3 activation.3) Why bafilomycin A1 treatment alone induces Beclin1 phosphorylation as seen in  The reviewers agreed on the significance of the findings and the high quality of the data presented. But how starvation activated MK2/MK3 and how activation of Beclin2 by MK2 initiated autophagy was not clear from the data presented. It was generally agreed that addressing some aspect of this would strengthen the conclusions.We are delighted that the reviewers agreed on the significance of our findings and the high quality of the data presented. In the revised manuscript, we have added additional experiments to address how starvation activates MK2/MK3. In addition, we have performed the experiments suggested by the reviewers to further address how MK2-mediated phosphorylation of Beclin 1 initiates autophagy. We thank the reviewers for their suggestions, and believe these additional experiments significantly strengthen our manuscript.We suggest that you address the following three major issues:1) How activation of Beclin 1 by MK2 results in the initiation of autophagy? Results shown insuggest a possible model, as cells expressing S90A display reduced VPS34 lipid kinase activity. However, additional mechanistic insight is needed. Might Beclin 1 S90 phosphorylation stabilize the Beclin 1/VPS34/ATG14 complex? The authors should blot for VPS34 in their immuno-precipitation experiments to monitor the interaction of VPS34 with wild type Beclin 1 and the S90A and S90E mutant proteins.As requested, we have performed immunoblots for VPS34  in the Flag-Beclin 1 immunoprecipitates in In an alternative model, does S90 phosphorylation stabilize interaction of the Beclin 1/VPS34/ATG14 complex with membranes, similar to a previously suggested model for ATG14 regulation ? The authors should monitor membrane binding of wild type Beclin 1 and the S90A and S90E mutant proteins using cellular fractionation approaches and in vivo localization experiments.As requested by the reviewers, we have performed cellular fractionation experiments to examine the membrane localization of wild-type Beclin 1, Beclin 1 S90A, and Beclin 1 S90E, as well as ATG14 and VPS34 in basal and starvation conditions. These results show that the amounts of the different Beclin 1 constructs in each subcellular fraction  are similar and do not vary in response to starvation. We observed similar findings for ATG14. For VPS34, although there are some differences in the different lanes, we did not observe any consistent pattern in several repeat experiments with respect to amounts of VPS34 in the microsomal fraction during starvation or in cells expressing the different Beclin 1 constructs. Thus, we do not believe that Beclin 1 S90 phosphorylation increases the membrane binding of the Beclin 1/VPS34/ATG14 complex. These new experiments are shown in In yet another possible model, might Beclin 1 S90 phosphorylation induce autophagy by stimulating VPS34 lipid kinase activity, similar to ULK1-mediated phosphorylation of Beclin 1 ?eLife publication by Baskaran et al. (2015) which described the EM reconstitution of the ordered domains of the Beclin 1/ATG15/VPS15/VPS34 complex, and predicted that signaling input from the disordered N-terminal region of Beclin 1 was likely to result in allosteric regulation of the VPS34 lipid kinase domain.Our results in 2) MK2 and MK3 have not been previously implicated in amino acid starvation stress responses. Moreover, the best-characterized kinase that activates MK2/MK3, p38 MAPK, is thought to negatively regulate starvation-induced autophagy . It would be very interesting and important to provide some new insight into how amino acid starvation results in MK2/MK3 activation.It is true that MK2 and MK3 have not been previously implicated in amino acid starvation stress responses, and indeed, this observation represents a novel element of our findings. The best-characterized kinase that activates MK2/MK3 is, as stated, p38 MAPK. There have also been some claims that MK2/MK3 can be activated by JNK. For these reasons, we evaluated the effects of both dominant-negative p38α and dominant-negative JNK1 on starvation-induced MK2 activation , as well as on Beclin 1 S90 phosphorylation. Our results, shown in We recognize that this finding is in apparent contradiction to the report of Webber and Tooze  that p38α negatively regulates starvation-induced autophagy. We also note that other studies report a positive role for p38α MAPK in autophagy induction, including in response to glucose starvation , IFNγ stimulation (3) Why bafilomycin A1 treatment alone induces Beclin1 phosphorylation as seen inWhile this manuscript focuses on starvation-induced autophagy and starvation-induced Beclin 1 S90 phosphorylation, we have also found that Beclin 1 undergoes S90 phosphorylation in response to several other autophagy-inducing stress stimuli. Therefore, we postulated that bafilomycin A1 might induce Beclin 1 S90 phosphorylation as a consequence of generation of reactive oxygen species (ROS), presumably as a consequence of its inhibition of vacuolar-type H(+)-ATPases (