CMTR1 contributes to mRNA cap formation by methylating the first transcribed nucleotide ribose at the O-2 position. mRNA cap O-2 methylation has roles in mRNA stabilisation and translation, and self-RNA tolerance in innate immunity. We report that CMTR1 is recruited to serine-5-phosphorylated RNA Pol II C-terminal domain, early in transcription. We isolated CMTR1 in a complex with DHX15, an RNA helicase functioning in splicing and ribosome biogenesis, and characterised it as a regulator of CMTR1. When DHX15 is bound, CMTR1 activity is repressed and the methyltransferase does not bind to RNA pol II. Conversely, CMTR1 activates DHX15 helicase activity, which is likely to impact several nuclear functions. In HCC1806 breast carcinoma cell line, the DHX15-CMTR1 interaction controls ribosome loading of a subset of mRNAs and regulates cell proliferation. The impact of the CMTR1-DHX15 interaction is complex and will depend on the relative expression of these enzymes and their interactors, and the cellular dependency on different RNA processing pathways.

Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5'-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose2,6-bisphosphate (F-2,6-P-2), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet-fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects.

Article Figures and data Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Mutations in the human kinase PINK1 (hPINK1) are associated with autosomal recessive early-onset Parkinson's disease (PD). hPINK1 activates Parkin E3 ligase activity, involving phosphorylation of ubiquitin and the Parkin ubiquitin-like (Ubl) domain via as yet poorly understood mechanisms. hPINK1 is unusual amongst kinases due to the presence of three loop insertions of unknown function. We report the structure of Tribolium castaneum PINK1 (TcPINK1), revealing several unique extensions to the canonical protein kinase fold. The third insertion, together with autophosphorylation at residue Ser205, contributes to formation of a bowl-shaped binding site for ubiquitin. We also define a novel structural element within the second insertion that is held together by a distal loop that is critical for TcPINK1 activity. The structure of TcPINK1 explains how PD-linked mutations that lie within the kinase domain result in hPINK1 loss-of-function and provides a platform for the exploration of small molecule modulators of hPINK1. https://doi.org/10.7554/eLife.29985.001 eLife digest Estimates suggest that more than 10 million people worldwide are living with Parkinson’s disease. This condition, which is incurable, is characterised by the death of brain cells leading to tremors and loss of motor control. So far scientists have been able to link mutations in nearly 20 different genes to Parkinson’s disease. This includes the gene that codes for an enzyme called PINK1. Mutations that affect PINK1 are often seen in people with early-onset Parkinson’s disease and cases that are inherited through families. Changes to PINK1 affects the ability of cells to keep producing energy. Without enough energy to power biochemical processes, affected cells will eventually die. Understanding why PINK1 has this effect and how to prevent it could be helpful in treating Parkinson’s disease. One way to understand the role of PINK1 is to study the three-dimensional structure of the protein to examine how it interacts with other molecules. The PINK1 protein from the flour beetle Tribolium castaneum is easier to produce than human PINK1; this makes it easier to study its structure in the laboratory. Using flour beetle PINK1 and a technique called X-ray crystallography, Kumar, Tamjar, Waddell et al. made a detailed three-dimensional model of the structure of the PINK1 protein. This revealed new details of the structure including a region at one end called the “C-terminal extension”, which is involved in PINK1’s enzyme activities. The structure also highlighted the purposes of two loop regions, one that controls PINK1 activity and one that affects its interactions with other proteins. Further examination of some of these features showed that they are also part of human PINK1 proteins. Using this new protein structure, Kumar et al. went on to examine 20 mutations that are found in patients with Parkinson’s disease and have been able to show the effect that these changes have on PINK1 at a molecular level. It may now be possible to begin designing drugs to prevent or reverse these changes, which could lead to new treatments for Parkinson’s disease. https://doi.org/10.7554/eLife.29985.002 Introduction Autosomal recessive inherited loss-of-function mutations in human PTEN-induced kinase 1 (hPINK1), represent the second most frequent cause of early-onset Parkinson’s disease (PD) (Valente et al., 2004). hPINK1 has been proposed to act as a master regulator of mitochondrial quality control, promoting the elimination of damaged mitochondria via autophagy known as mitophagy (McWilliams and Muqit, 2017). In response to mitochondrial depolarisation, hPINK1 is activated and phosphorylates ubiquitin at Serine65 (Ser65). Ser65-phosphorylated ubiquitin then binds to the ubiquitin E3 ligase Parkin with high affinity, serving to prime Parkin for phosphorylation by hPINK1 at an equivalent Ser65 residue that lies within its N-terminal ubiquitin-like domain (Ubl) (Kondapalli et al., 2012; Kazlauskaite et al., 2014; Kane et al., 2014; Koyano et al., 2014). This chain of phosphorylation events stimulates maximal activation of Parkin E3 ligase activity, resulting in ubiquitylation of multiple substrates at the outer mitochondrial membrane and represents a critical upstream step in the induction of mitophagy (McWilliams and Muqit, 2017). The molecular mechanisms of hPINK1 activation and substrate recognition are poorly understood. hPINK1 is distinct from other protein kinases due to the presence of three unique insertions (Ins1, Ins2 and Ins3) within the kinase domain and a C-terminal extension (CTE) of unknown function that bears no homology to any known protein domain (Figure 1A). Nearly, 30 missense and nonsense hPINK1 mutations have been reported in patients worldwide (Deas et al., 2009), located predominantly within the kinase domain or predicted to truncate the CTE. However, the molecular mechanism by which they disrupt hPINK1 catalysis and/or recognition of its physiological substrates ubiquitin and Parkin is unknown. We have previously identified a hPINK1 orthologue in Tribolium castaneum (TcPINK1), which in contrast to human hPINK1, almost entirely lacks the first kinase domain insertion (Woodroof et al., 2011) (Figure 1A). TcPINK1 exhibits constitutive catalytic activity towards ubiquitin, Parkin and generic substrates in vitro (Woodroof et al., 2011; Kazlauskaite et al., 2015). Furthermore, the motor defects of Drosophila PINK1 null flies can be efficiently rescued in vivo by crossing lines that over-express TcPINK1 (Woodroof et al., 2011). Herein, we report the crystal structure of TcPINK1, revealing structural insights into the CTE and kinase domain loop insertions. In particular, we show that the third insertion contributes to formation of a bowl-shaped binding site for ubiquitin that is critical for either TcPINK1 or hPINK1 mediated ubiquitin and Parkin Ubl phosphorylation. Furthermore, we have elaborated an important regulatory role for TcPINK1 autophosphorylation of Serine205 (Ser205), which together with the third insertion, aids in physiological substrate recognition. Overall, these findings provide molecular insights into the mechanisms of hPINK1 kinase activity, ubiquitin substrate recognition, and define the molecular basis of Parkinson’s disease-causing mutations. Figure 1 with 1 supplement see all Download asset Open asset Overall structure of TcPINK1. (A) Domain architecture of TcPINK1 showing kinase domain (150-486); location of Ins2, Ins3, catalytic motif (HRD) and activation loop motifs (DFG; APE) and CTE (487-570). The location of hPINK1 Ins1, which is largely absent in TcPINK1, is indicated. CTE, C-terminal extension; Ins, insertion. Created with IBS (Liu et al., 2015). The boundaries of the crystallisation construct with the mutations introduced is also shown, lower panel. (B) Overall structure of TcPINK1 showing the canonical N-lobe and C-lobe (grey). Catalytic and activation loop motifs are shown in magenta sticks and Mg2+ is shown as a black sphere. Ins2, Ins3 and CTE of TcPINK1 are coloured as in Figure 1A; disordered regions of PINK1 are shown in dashed line; Ins1 of hPINK1 is also shown as part of disordered loop in TcPINK1. (C) Topology diagram of the TcPINK1 structure. Core kinase domain secondary structure elements are shown in grey and indicated with numbers (β-sheet) and letters (α-helices) according to the nomenclature in (Hanks and Hunter, 1995). Non-canonical secondary structure elements (CTE, C-terminal extension; Ins, insertion) are indicated with Roman numerals for Ins2 (cyan), Ins3 (green) and numbering for CTE (blue). Created with TopDraw (Bond, 2003). (D) Ubiquitin phosphorylation assay with full-length TcPINK1 (WT), kinase-inactive D359A mutant (KI), and ATP-binding pocket mutations A194D, K196A and E217K. 2 μg of indicated enzyme was incubated with 2 μM ubiquitin (K63-linked tetraubiquitin) and [γ-32P] ATP for 10 min. Gels were analysed by Coomassie staining (lower panel) and [γ-32P] incorporation visualised by autoradiography (upper panel) followed by Cerenkov counting of substrate bands for quantification (right panel). https://doi.org/10.7554/eLife.29985.003 Results and discussion The TcPINK1 kinase fold is decorated with unique structural features The crystal structure of a catalytically active fragment of TcPINK1 was solved and refined to 2.78 Å using E. coli expression of a cDNA construct encompassing the kinase domain and CTE (Ser150 - Asp570), and possessing mutations to reduce surface entropy (Glu527Ala and Lys528Ala), a 10 amino acid deletion in a large loop (Δ261–270), and a phospho-mimetic mutation (Ser205Glu) (Figure 1A). The crystal structure of TcPINK1 reveals core secondary structure elements of a typical protein kinase domain (Hanks and Hunter, 1995) (Figure 1B,C). As described previously (Woodroof et al., 2011), the N-lobe of the TcPINK1 kinase domain contains three insertions: Lys182–Pro192 (Ins1), Glu221–Ala253 (Ins2), and Leu260–Met288 (Ins3) and in contrast to hPINK1, Ins1 exists as a loop remnant in TcPINK1 (Figure 1A,B). Ins2 forms a small domain containing a βi-strand (222-226) and an αi-helix (231-242) followed by a loop (243-253). Ins3 is disordered and is not included in the final model (Figure 1B,C). Finally, the majority of the TcPINK1 CTE (Ala487–Leu556) is α-helical in nature (Figure 1B,C), and rather than forming a separate domain, is tightly packed on the E, G and H α-helices of the C-lobe of the kinase domain (3747 Å2 buried surface area), supporting previous data on the importance of this region for catalysis (Woodroof et al., 2011). A DALI structure similarity search revealed that Ins2 and the CTE structural elements are unique to TcPINK1 and not found in the almost thousand kinase structures solved to date. Although TcPINK1 was crystallised without ATP, the kinase domain appears to adopt an active, closed conformation, based on the positioning of the αC helix, activation segment conformation, and the ‘in’ position of the DFG motif (Hari et al., 2013; Huse and Kuriyan, 2002; Nolen et al., 2004) (Figure 1B). A model of the ATP was included by superposition with the conformationally similar cAMP dependent protein kinase-ATP complex (RMSD 3.2 Å on 252 equivalent Cα atoms). This suggested that residues Ala194, Lys196, Leu344, Tyr297 and Glu217 are critical for ATP binding (Figure 1—figure supplement 1A). In agreement with this, biochemical analysis revealed that the Ala194Asp, Lys196Ala and Glu217Lys TcPINK1 mutants prevented phosphorylation of its physiological substrates, ubiquitin and the Parkin Ubl domain (Figure 1D, Figure 1—figure supplement 1B). Furthermore, inspection of the structure indicates that the conformation of residues forming two conserved hydrophobic networks (the ‘R-spine’ comprising residues Ile253, Val219, Phe360, His335 and Asp410, and the ‘C-spine’ residues Val176, Leu301, Leu343, Leu344, Leu345, ILe421 and Ile417) is compatible with an active conformation (Kornev et al., 2006, 2008). Ins3 forms the ubiquitin binding pocket hPINK1-dependent phosphorylation of Ser65 on ubiquitin and the Ubl domain of Parkin is critical for maximal Parkin activation (Wauer et al., 2015; Kumar et al., 2017). However, the regions of hPINK1 that recognize and bind its substrates remain unknown. Via DALI structure similarity search, we identified the LIM kinase-cofilin complex (Hamill et al., 2016) as being conformationally similar to TcPINK1 (RMSD 2.8 Å on 298 equivalenced Cα atoms). Using the phosphopeptide in this complex as an anchor (Figure 2A), we superimposed Ser65 of ubiquitin onto the TcPINK1 active site, resulting in a model of the TcPINK1-ubiquitin complex, free of steric clashes (Figure 2B). Using this model, we explored the key structural elements in TcPINK1 that may contribute to recognition of ubiquitin. Although Ins3 in the TcPINK1 kinase domain is mostly disordered and could not be built as part of the structure; its predicted location places it proximal to the ubiquitin in the model of the complex and therefore we hypothesized that it is likely to contribute a critical role towards substrate recognition and binding. To test this, we deleted 10 residues (residues 261–270, equivalent to the deletion used in the crystallisation construct) of Ins3 and measured TcPINK1 catalytic activity using multiple physiological readouts, including TcPINK1 catalysed phosphorylation of ubiquitin and the Parkin Ubl domain, and transphosphorylation of a kinase-inactive (D359A) TcPINK1 fragment (residues 125–570; His-SUMO cleaved) (Figure 2C,D, Figure 2—figure supplement 1A). Strikingly, deletion of Ins3 resulted in complete loss of ubiquitin or Parkin Ubl phosphorylation, while preserving transphosphorylation activity towards kinase-inactive TcPINK1 (Figure 2C,D, Figure 2—figure supplement 1A). We also found that mutation of key residues Leu371, Tyr375, Lys382 and Lys435 within the canonical substrate binding pocket of kinases resulted in loss of activity against ubiquitin (Figure 2B, Figure 2—figure supplement 1B). Figure 2 with 1 supplement see all Download asset Open asset Identification of the PINK1 substrate binding bowl. (A) Crystal structure of the LIM kinase substrate complex (pdb id: 5HVK, 18) showing LIM kinase (coloured as TcPINK1 in Figure 1B) and bound substrate cofilin (yellow) with Ser3 (yellow stick) poised in acceptor position. (B) Ubiquitin (yellow) was modelled in TcPINK1 (coloured as in Figure 1B) by superposing Ser3 of cofilin with Ser65 (shown as sticks) of ubiquitin after superposing the TcPINK1 and LIM kinase domains; disordered Ins3 is marked by dashed green line; 2Fo-Fc map, contoured at 1.5σ, corresponding to phosphorylated Ser207 region is shown highlighting the Glu205 position in respect to Asn287 (tail of disordered Ins3), inset. (C) Ubiquitin phosphorylation assay with full-length TcPINK1 (WT), kinase-inactive D359A mutant (KI), insertion three deletion mutant (Δ261–270), S205A and/or S207A mutants in the autophosphorylation sites. 2 µM ubiquitin (K63-linked tetraubiquitin) was used as substrate; assay conditions were similar to Figure 1D. Gels were analysed by Coomassie staining (lower panel) and [γ-32P] incorporation visualised by autoradiography (middle panel) followed by Cerenkov counting of substrate bands for quantification (upper panel). (D) TcPINK1 transphosphorylation assay with full-length TcPINK1 (WT) or various mutants as in Figure 2C. 2 µM of kinase inactive TcPINK1 (125-end) was used as substrate; assay conditions were similar to Figure 1D. Gels were analysed by Coomassie staining (lower panel) and [γ-32P] incorporation visualised by autoradiography (middle panel) followed by Cerenkov counting of substrate bands for quantification (upper panel). https://doi.org/10.7554/eLife.29985.005 We have previously identified multiple autophosphorylation sites for TcPINK1 by mass spectrometry, including Ser205 (equivalent to Ser228 in hPINK1) and Ser207 (equivalent to Ser230 in hPINK1) (Woodroof et al., 2011). Ser207 phosphorylation was also observed in the crystal structure (Figure 2B). Inspection of the structure revealed that Glu205 (the phosphomimetic of Ser205) lies only 4 Å away from the tail (Asn287) of the disordered Ins3 (Figure 2B). We therefore hypothesized that autophosphorylation of Ser205 may influence and/or contribute to ubiquitin recognition and binding mediated by Ins3. Therefore, to test the effect of Ser205 phosphorylation in substrate recognition, we performed parallel assays with ubiquitin, Parkin Ubl domain and transphosphorylation of kinase-inactive TcPINK1 as performed for the Ins3 deletion. Critically Ser205Ala, but not Ser207Ala, prevented ubiquitin and Parkin Ubl phosphorylation while preserving transphosphorylation activity towards kinase-inactive TcPINK1 (Figure 2C,D, Figure 2—figure supplement 1A). Overall, these findings indicate that Ins3, unique to the PINK1 kinases, together with autophosphorylation at Ser205, is critical for ubiquitin and Parkin Ubl phosphorylation and may form part of the ubiquitin/Ubl fold recognition pocket. Role of novel regulatory features of TcPINK1 catalysis Multiple lines of evidence suggest that hPINK1 is autoinhibited under basal conditions, including lack of significant activity of hPINK1 in vitro (Woodroof et al., 2011) and the detection of robust activity of hPINK1 in cells only under conditions of mitochondrial depolarization triggering hPINK1 activation via an unknown mechanism (Kondapalli et al., 2012). In contrast to hPINK1, insect orthologues isolated from Drosophila melanogaster, Pediculus humanus corporis and Tribolium castaneum exhibit constitutive catalytic activity in vitro (Woodroof et al., 2011; Wauer et al., 2015). Since the TcPINK1 kinase domain adopts an active conformation in our crystal structure, we sought to exploit this knowledge to probe possible mechanisms of hPINK1 activation. Activation loop phosphorylation is the most common mechanism of protein kinase activation (Hanks and Hunter, 1995), with phosphorylation stabilising the active conformation via electrostatic interaction between the phospho-serine/threonine residue and a basic pocket consisting of the arginine in the HRD motif (Nolen et al., 2004). Previous mass spectrometry analysis of TcPINK1 did not identify activation loop phosphorylation, and furthermore, alanine mutation of a candidate T-loop phosphorylation site residue, Ser377, did not impair TcPINK1 catalytic activity (Woodroof et al., 2011). Consistent with this, we do not observe phosphorylation of Ser377 in the TcPINK1 structure and furthermore, Ser377 is located too distant (12.2 Å) from Arg336 of the HRD motif to form an electrostatic interaction (Figure 3A). Instead the TcPINK1 structure reveals that a highly conserved aspartate (Asp381) within the activation loop, an arginine residue (Arg336) of the HRD motif and an arginine residue (Arg216) in the αC-helix, form an equivalent interaction (Figure 3A), which is further stabilized via interaction with basic residues (Lys212 on the αC-helix; Arg240 and Arg241 in Ins2) and another conserved aspartate (Asp366). To explore which residues were important for TcPINK1 catalysed phosphorylation of its substrates, we investigated point mutants of residues lying within this region. The Asp366Ala TcPINK1 mutant was unstable under our expression conditions and was not assessed. Importantly, our data revealed that mutants in this region, particularly the Asp381Ala and Arg240Ala mutants, were associated with a significant reduction in TcPINK1 catalytic kinase activity towards ubiquitin (Figure 3B). Although Asp381 and Asp366 are conserved in hPINK1, Lys212 and Arg216 form a basic patch on the αC-helix that is conserved among the insect orthologues but not in hPINK1, where the equivalent residues are Asn235 and Gln239. Furthermore, Arg240 and Arg241 of TcPINK1 are replaced by Gly264 and Pro265 in hPINK1, respectively (Figure 3—figure supplement 1A). We hypothesized that these differences in basic residues between TcPINK1 and hPINK1 could explain the lack of recombinant hPINK1 activity. To test this, we generated a mutant of hPINK1 incorporating Asn235Lys/Gln239Arg/Gly264Arg/Pro265Arg substitutions, however, this did not lead to any significant enhancement in hPINK1 activity (data not shown). Figure 3 with 1 supplement see all Download asset Open asset Ins2 of TcPINK1 adopts a unique conformation. (A) Close up view of active site of TcPINK1 (coloured as in Figure 1B) showing key ionic interactions involving Asp381 of the activation loop with Arg336 of the HRD motif and Arg216 of the αC helix; and Asp366 with Lys212 of the αC helix and Arg240 of Ins2. (B) Ubiquitin phosphorylation assay with full-length TcPINK1 (WT), kinase-inactive D359A mutant (KI), D381A, R216A, K212A and R240A mutants. 2 µM of Ubiquitin (K63-linked tetraubiquitin) was used as substrate; conditions were similar to Figure 1D. Gels were analysed by Coomassie staining (lower panel) and [γ-32P] incorporation visualised by autoradiography (upper panel) followed by Cerenkov counting of substrate bands for quantification (right panel). (C) Structural comparison of TcPINK1 and the CDK-Cyclin complex (pdb id: 5IF1) showing intramolecular interactions between Ins2 (cyan) and αC helix (grey) of TcPINK1 that resemble the intermolecular interactions between CDK (grey) and Cyclin (cyan). The salt bridge between Glu and Lys (shown in sticks) for kinase activation is represented by a dashed line. Interactions between the C-lobe and the distal Ins2 loop are highlighted (Inset). (D) Ubiquitin phosphorylation assay with full-length TcPINK1 (WT), kinase-inactive D359A mutant (KI) and TcPINK1 Ins2 mutants with βi-strand and αi-helix deletion (∆222–242), αi-helix deletion (∆231–242), or distal loop deletion (∆243–253). 2 µM of Ubiquitin (K63-linked tetraubiquitin) was used as substrate, conditions were similar to Figure 1D. Gels were analysed by Coomassie staining (lower panel) and [γ-32P] incorporation visualised by autoradiography (middle panel) followed by Cerenkov counting of substrate bands for quantification (upper panel). https://doi.org/10.7554/eLife.29985.007 Inspection of the TcPINK1 structure suggests another potential regulatory mechanism of activation through interaction of Ins2 with the αC-helix (1120 Å2 buried surface area) (Figure 3C). This interaction is reminiscent of the activation mechanism of cyclin dependent kinases (CDKs), whereby intermolecular stabilization of the CDK αC-helix occurs due to interactions with the cyclin α5-helix (Figure 3C) (Jeffrey et al., 1995). The TcPINK1 αi-helix (residues 231–242) of Ins2 (residues 221–253) occupies a similar position to cyclin, inducing a displacement of the αC-helix and allowing formation of the key salt bridge between the conserved Glu217 on the αC-helix and the conserved Lys196 on the N-lobe, maintaining the kinase in an active conformation (Figure 3C). Akin to the intermolecular CDK-cyclin interaction, the TcPINK1 αC-helix-αi-helix intramolecular interface also consists of hydrophobic interactions (Figure 3C). Whilst the overall conservation of Ins2 (residues 221–253) is low, the C-terminal/distal region (243-253) of Ins2 remains highly conserved (Figure 3—figure supplement 1A), forming a loop (Ins2 loop) which tethers the proximal region of Ins2 (βi-strand and αi-helix) with the rest of the C-lobe mediated by interactions between His247 and Asn249 of Ins2 and Ser545 of CTE and Gln320 of the C-lobe, respectively. Furthermore, a hydrophobic core is formed between Leu244 and Ile250 of Ins2 with Phe360 of the ‘DFG motif’ and Ile357 (Figure 3C). Interestingly, deletion of the αi-helix (residues 231–242) alone or in combination with the βi-strand (residues 222–242) region of Ins2 does not affect the catalytic activity of TcPINK1. However, deletion of the Ins2 loop (residues 243–253) abolishes the kinase activity of TcPINK1 (Figure 3D, Figure 3—figure supplement 1B). Therefore, our analysis suggests a critical role for the Ins2 loop (243-253) as an anchor, to maintaining the kinase domain in an active state, although the function of the proximal region of Ins2 (222-242) remains to be fully elucidated. Translating structural insights to hPINK1 Sequence alignment of hPINK1 and TcPINK1 reveals an overall high degree of conservation with the exception of the relative absence of Ins1 in TcPINK1 (Figure 4—figure supplement 1). Therefore, we next explored the structural and functional conservation between insect and human PINK1 in cells expressing hPINK1 whose kinase activity can be stimulated by carbonyl cyanide m-cholorophenylhydrazone (CCCP) that induces mitochondrial depolarization (Kazlauskaite et al., 2015). We transiently expressed wild type hPINK1 or mutants of key ATP-binding residues predicted from the TcPINK1 structure (Figure 1D, Figure 1—figure supplement 1A) into HeLa hPINK1 knockout cells generated by TALEN technology (Narendra et al., 2013). Cells were treated with 10 μM CCCP or DMSO for 3 hr and extracts immunoblotted with phospho-specific antibodies raised against hPINK1 substrates Ser65 Parkin and Ser65 ubiquitin. Consistent with our analysis of TcPINK1 (Figure 1D), we found that Parkin and ubiquitin phosphorylation was abolished in cells expressing mutant A217D, K219A, E240K and L369P forms of hPINK1 upon CCCP stimulation (Figure 4A). This indicates that the ATP-binding pocket is well conserved between TcPINK1 and hPINK1. We next explored whether Ins3, which is well conserved between TcPINK1 and higher species, is critical for ubiquitin and Parkin Ubl recognition by hPINK1. Consistent with TcPINK1, we observed abrogation of phosphorylation of ubiquitin and Parkin in cells expressing hPINK1 harbouring a deletion of Ins3 (Δ285–294 – corresponding to deletion in TcPINK1 Δ261–270) in striking contrast to cells expressing hPINK1 containing a deletion in Ins1 (Δ180–209) or a deletion in the proximal region of Ins2 (Δ245–265 - corresponding to βi-strand and αi-helix of TcPINK1). Overall, these findings indicate that hPINK1 and TcPINK1 share similar mechanisms for substrate recognition and kinase activity, suggesting that the TcPINK1 structure will provide an important framework to direct future studies aimed at structural studies of the hPINK1 enzyme. Figure 4 with 1 supplement see all Download asset Open asset Conservation of ATP and substrate binding mechanism in hPINK1. (A) hPINK1 knock out HeLa cells transiently co-expressing WT human Parkin and 3xFLAG tagged hPINK1 WT, KI or hPINK1 ATP-binding mutants A217D, K219A, E240K, L369P. A217, K219, E240 and L369 correspond to A194, K196, E217 and L344 of TcPINK1, respectively. Cells were stimulated with 10 μM CCCP or DMSO for 3 hr. Lysates were subjected to immunoblotting as follows: pSer65 Parkin (anti-phospho-Parkin); Parkin (anti-Parkin), pSer65 ubiquitin (anti-phospho-ubiquitin), hPINK1 (anti-FLAG), OPA1 (anti-OPA1) and GAPDH (anti-GAPDH). (B) hPINK1 knock out HeLa cells transiently co-expressing WT human Parkin and 3xFLAG tagged hPINK1 WT, KI or hPINK1 Ins mutants ∆Ins1 (180–209 deletion), ∆Ins2 (245–265 deletion), ∆Ins3 (285-294). Cells were stimulated with 10 μM CCCP or DMSO for 3 hr. Lysates were subjected to immunoblotting as follows: pSer65 Parkin (anti-phospho-Parkin); Parkin (anti-Parkin), pSer65 ubiquitin (anti-phospho-ubiquitin), hPINK1 (anti-FLAG), OPA1 (anti-OPA1) and GAPDH (anti-GAPDH). https://doi.org/10.7554/eLife.29985.009 Molecular mechanisms of inactivating Parkinsonism mutations in hPINK1 Mutations in hPINK1 are a leading cause of familial early-onset PD and whilst previous studies have demonstrated that mutations disrupt hPINK1-dependent activity and mitophagy in cells (McWilliams and Muqit, 2017; Deas et al., 2009), very little is known about how these impact hPINK1 structure. Of the approximately thirty reported homozygous or compound heterozygous pathogenic mutations (Deas et al., 2009), twenty are conserved in TcPINK1 (Supplementary file 1). Mapping of these pathogenic mutations onto the structure of TcPINK1 (Figure 5, Supplementary file 1), reveals that they cluster within functionally distinct regions of hPINK1. A major group of PD-associated mutations lie within the ATP-binding pocket including Ala217Asp, Glu240Lys, Ala244Gly and Leu369Pro and perturb interactions with ATP. A second group of mutations that lie in the catalytic motif or activation loop include Gly386Ala, Pro416Arg/Leu and Glu417Gly (Figure 5A,C) result in catalytically inactive hPINK1 (Deas et al., 2009). The His271Gln mutation lies within the critical distal Ins2 loop of hPINK1 (Figure 5A,C), that anchors the proximal Ins2 to the C-lobe (Figure 3C), and has previously been found to abolish catalytic activity in vitro (Woodroof et al., 2011) as well as hPINK1 activity in cells (Okatsu et al., 2012). In contrast, the Gly309Asp (G309D) PD mutation located within Ins3 (Figure 2) would be predicted to perturb hPINK1 and substrate (ubiquitin/Ubl domain of Parkin) interaction as our analysis suggests that Ins3 is required for ubiquitin phosphorylation (Figure 2C, Figure 2—figure supplement 1A). Consistent with this, a previous study showed that the G309D hPINK1 mutant fails to phosphorylate Parkin Ubl in cells (Iguchi et al., 2013) whereas the equivalent TcPINK1 G285D mutant continues to exhibit catalytic activity towards generic and peptide substrates in vitro (Woodroof et al., 2011). The remainder of mutations mainly affects residues critical for structural integrity of hPINK1. In particular, the CTE of TcPINK1 forms a hydrophobic core against the E, G and H helices of the C-lobe (Figure 5B), and a cluster of PD mutations in hPINK1 including Leu539Phe, Leu347Pro and Leu489Pro located in this vicinity suggest the importance of this hydrophobic core (Figure 5B). Attempts to purify soluble protein for a number of point mutants targeting this hydrophobic interface were unsuccessful, underscoring the importance of this hydrophobic core which explains loss of kinase activity of TcPINK1 upon CTE deletion in previous studies (Woodroof et al., 2011). Although it has previously been suggested that CTE may be important for dimerization (Okatsu et al., 2013), we did not find evidence for a functional dimer in our TcPINK1 purification procedures or crystal structure. Figure 5 Download asset Open

Interleukin-1 (IL-1) signaling induces the formation of Lys63-linked ubiquitin (K63-Ub) chains, which are thought to activate the ‘master’ protein kinase TGFβ-activated kinase 1 (TAK1) by interacting with its TAK1-binding 2 (TAB2) and TAB3 subunits. Here, we report that IL-1β can also activate the TAB1–TAK1 heterodimer present in TAB2/TAB3 double knockout (DKO) IL-1 receptor-expressing cells. The IL-1β-dependent activation of the TAB1–TAK1 heterodimer in TAB2/3 DKO cells is required for the expression and E3 ligase activity of tumor necrosis factor receptor-associated factor 6 (TRAF6) and is reduced by the small interfering RNA (siRNA) knockdown of ubiquitin conjugating 13 (Ubc13), an E2-conjugating enzyme that directs the formation of K63-Ub chains. IL-1β signaling was restored to TAB1/2/3 triple KO cells by the re-expression of either TAB1 or TAB2, but not by an ubiquitin binding-defective mutant of TAB2. We conclude that IL-1β can induce the activation of TAK1 in two ways, only one of which requires the binding of K63-Ub chains to TAB2/3. The early IL-1β-stimulated, TAK1-dependent activation of p38α mitogen-activated protein (MAP) kinase and the canonical IκB kinase (IKK) complex, as well as the NF-κB-dependent transcription of immediate early genes, was similar in TAB2/3 DKO cells and TAB2/3-expressing cells. However, in contrast with TAB2/3-expressing cells, IL-1β signaling was transient in TAB2/3 DKO cells, and the activation of c-Jun N-terminal kinase 1 (JNK1), JNK2 and p38γ was greatly reduced at all times. These observations indicate a role for TAB2/3 in directing the TAK1-dependent activation of MAP kinase kinases that switch on JNK1/2 and p38γ MAP kinases. These observations and the transient activation of the TAB1–TAK1 heterodimer may explain why IL-1β-dependent IL-8 mRNA formation was abolished in TAB2/3 DKO cells.

Mutations in the human kinase PINK1 (hPINK1) are associated with autosomal recessive early-onset Parkinson's disease (PD). hPINK1 activates Parkin E3 ligase activity, involving phosphorylation of ubiquitin and the Parkin ubiquitin-like (Ubl) domain via as yet poorly understood mechanisms. hPINK1 is unusual amongst kinases due to the presence of three loop insertions of unknown function. We report the structure of Tribolium castaneum PINK1 (TcPINK1), revealing several unique extensions to the canonical protein kinase fold. The third insertion, together with autophosphorylation at residue Ser205, contributes to formation of a bowl-shaped binding site for ubiquitin. We also define a novel structural element within the second insertion that is held together by a distal loop that is critical for TcPINK1 activity. The structure of TcPINK1 explains how PD-linked mutations that lie within the kinase domain result in hPINK1 loss-of-function and provides a platform for the exploration of small molecule modulators of hPINK1.

Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

Mutations in the human kinase PINK1 (hPINK1) are associated with autosomal recessive early-onset Parkinson’s disease (PD). hPINK1 activates Parkin E3 ligase activity, involving phosphorylation of ubiquitin and the Parkin ubiquitin-like (Ubl) domain via as yet poorly understood mechanisms. hPINK1 is unusual amongst kinases due to the presence of three loop insertions of unknown function. We report the structure of Tribolium castaneum PINK1 (TcPINK1), revealing several unique extensions to the canonical protein kinase fold. The third insertion, together with autophosphorylation at residue Ser205, contributes to formation of a bowl-shaped binding site for ubiquitin. We also define a novel structural element within the second insertion that is held together by a distal loop that is critical for TcPINK1 activity. The structure of TcPINK1 explains how PD-linked mutations that lie within the kinase domain result in hPINK1 loss-of-function and provides a platform for the exploration of small molecule modulators of hPINK1. DOI: https://doi.org/10.7554/eLife.29985.00

Significance TRAF6 is essential for many biological processes, including the operation of the innate immune system and bone formation. The enzymatic activity of TRAF6, an E3 ubiquitin ligase, is thought to have a pivotal role in triggering these processes. In this paper, we reexamined this assumption by generating mice expressing a catalytically inactive mutant of TRAF6. The bone structure of the mice was normal and a major intracellular signaling pathway of the immune system could still be switched on. We found that other E3 ligases, Pellino1 and Pellino2, could generate the ubiquitin chains needed to switch on immune signaling in human cells expressing catalytically inactive TRAF6. Our findings identify essential roles of TRAF6 that are independent of its enzymatic activity.

Maturation and translation of mRNA in eukaryotes requires the addition of the 7-methylguanosine cap. In vertebrates, the cap methyltransferase, RNA guanine-7 methyltransferase (RNMT), has an activating subunit, RNMT-Activating Miniprotein (RAM). Here we report the first crystal structure of the human RNMT in complex with the activation domain of RAM. A relatively unstructured and negatively charged RAM binds to a positively charged surface groove on RNMT, distal to the active site. This results in stabilisation of a RNMT lobe structure which co-evolved with RAM and is required for RAM binding. Structure-guided mutagenesis and molecular dynamics simulations reveal that RAM stabilises the structure and positioning of the RNMT lobe and the adjacent α-helix hinge, resulting in optimal positioning of helix A which contacts substrates in the active site. Using biophysical and biochemical approaches, we observe that RAM increases the recruitment of the methyl donor, AdoMet (S-adenosyl methionine), to RNMT. Thus we report the mechanism by which RAM allosterically activates RNMT, allowing it to function as a molecular rheostat for mRNA cap methylation.

The mRNA cap recruits factors essential for transcript processing and translation initiation. We report that regulated mRNA cap methylation is a feature of embryonic stem cell (ESC) differentiation. Expression of the mRNA cap methyltransferase activating subunit RAM is elevated in ESCs, resulting in high levels of mRNA cap methylation and expression of a cohort of pluripotency-associated genes. During neural differentiation, RAM is suppressed, resulting in repression of pluripotency-associated factors and expression of a cohort of neural-associated genes. An established requirement of differentiation is increased ERK1/2 activity, which suppresses pluripotency-associated genes. During differentiation, ERK1/2 phosphorylates RAM serine-36, targeting it for ubiquitination and proteasomal degradation, ultimately resulting in changes in gene expression associated with loss of pluripotency. Elevated RAM expression also increases the efficiency of fibroblast reprogramming. Thus, the mRNA cap emerges as a dynamic mark that instructs change in gene expression profiles during differentiation and reprogramming.

Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau–tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo , and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis.

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin activators that mimic the effect of ubiquitin(Phospho-Ser65).

Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser111) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.

Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser(65)) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser(65) phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser(65) by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser(65) phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin's ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser(65) is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson's disease.

Significance NF-κB and IFN Regulatory Factor 5 (IRF5) are required for the transcription of many proinflammatory cytokines in myeloid cells. The protein kinase IKKβ is the major activator of NF-κB but how IRF5 is activated has been unclear. This paper demonstrates that IKKβ also activates IRF5 by catalyzing the phosphorylation of Ser462. The phosphorylation of this serine induces the dimerization of IRF5 and its translocation to the nucleus. The activation of the master transcription factors of the innate immune system by the same protein kinase provides a mechanism for the coordinated control of IRF5 and NF-κB in response to inflammatory stimuli.

IKKβ {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase β} is required to activate the transcription factor NF-κB, but how IKKβ itself is activated in vivo is still unclear. It was found to require phosphorylation by one or more ‘upstream’ protein kinases in some reports, but by autophosphorylation in others. In the present study, we resolve this contro-versy by demonstrating that the activation of IKKβ induced by IL-1 (interleukin-1) or TNF (tumour necrosis factor) in embryonic fibroblasts, or by ligands that activate Toll-like receptors in macrophages, requires two distinct phosphorylation events: first, the TAK1 [TGFβ (transforming growth factor β)-activated kinase-1]-catalysed phosphorylation of Ser177 and, secondly, the IKKβ-catalysed autophosphorylation of Ser181. The phosphorylation of Ser177 by TAK1 is a priming event required for the subsequent autophosphorylation of Ser181, which enables IKKβ to phosphorylate exogenous substrates. We also provide genetic evidence which indicates that the IL-1-stimulated, LUBAC (linear ubiquitin chain assembly complex)-catalysed formation of linear ubiquitin chains and their interaction with the NEMO (NF-κB essential modulator) component of the canonical IKK complex permits the TAK1-catalysed priming phosphorylation of IKKβ at Ser177 and IKKα at Ser176. These findings may be of general significance for the activation of other protein kinases.