About

Jesse Dixon is an Associate Professor in the Gene Expression Laboratory at the Salk Institute for Biological Studies. He completed his undergraduate degree in Molecular Biology at Princeton University. He then earned both his MD and PhD at the University of California San Diego, where his PhD research in Biomedical Sciences was conducted in the lab of Bing Ren, focusing on genome-wide principles of higher-order chromatin structure. Jesse Dixon began his independent career at the Salk Institute as an independent fellow through the Helmsley Salk Fellows Program. His research centers on understanding the organization and regulation of the genome, particularly the principles governing chromatin architecture and its impact on gene expression.

Research topics

• Biology
• Cell biology
• Chemistry
• Cancer research
• Computational biology
• Genetics

Selected publications

• Scottish Open Fractures of Tibia (SOFT) audit; a multi-centre retrospective review of ortho-plastic management of tibial shaft fractures at major trauma centres (MTCs) in Scotland

  The Surgeon · 2025-01-27 · 1 citations

  article1st authorCorresponding
  PublisherDOI

• Emerging mechanisms of regulation for endoplasmic/sarcoplasmic reticulum Ca2+ stores by secretory pathway kinase FAM20C

  Current Opinion in Chemical Biology · 2023-03-24 · 2 citations

  reviewSenior authorCorresponding
  PublisherDOI

• Abstract 1615: RNA Polymerase II Recruitment of Transcriptional Regulators Promote Phase Separation during Eukaryotic Transcription

  Journal of Biological Chemistry · 2023-01-01

  articleOpen access
  PublisherDOI

• Divalent cation driven liquid‐liquid phase separation of disordered acidic proteins

  The FASEB Journal · 2022-05-01

  articleSenior author

  Divalent cations are essential to cellular processes through interaction with biological macromolecules, involvement in enzyme‐mediated catalysis, and as secondary messengers in signaling cascades. Recently, we have discovered divalent cation driven liquid‐liquid phase separation (LLPS) underlies endoplasmic reticulum Ca 2+ stores through disordered acidic calcium binding protein calsequestrin‐1 (CASQ1). CASQ1 interacts with divalent cations to enter a LLPS state via complex coacervation. CASQ1 LLPS propensity is positively regulated by FAM20C‐dependent phosphorylation that induces an order‐to‐disorder transition accompanied by dramatic structural expansion. These events increase intracellular Ca 2+ stores and regulate cellular stress response. Proteome wide analysis of disordered acidic proteins suggests divalent cation driven LLPS may be an emerging mechanism extending beyond the ER. Particularly, these proteins are highly enriched in the nucleus and cytosol where they accumulate in a number of biological condensates alongside other polyanions like RNA to regulate gene expression. We hypothesize divalent cation driven LLPS is a widespread mechanism driving both protein‐protein and protein‐nucleic acid interactions.

  PublisherDOI

• Hippo pathway regulation by phosphatidylinositol transfer protein and phosphoinositides

  Nature Chemical Biology · 2022 · 63 citations

  • Cell biology
  • Chemistry
  • Computational biology
  PublisherDOI

• The ABCs of the atypical Fam20 secretory pathway kinases

  Journal of Biological Chemistry · 2021 · 35 citations

  • Cell biology
  • Chemistry
  • Biology

  The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be phosphorylated. However, it took more than a century to identify Fam20C, which phosphorylates both casein and phosvitin under physiological conditions. This kinase, along with its family members Fam20A and Fam20B, defined a new family with altered amino acid sequences highly atypical from the canonical 540 kinases comprising the kinome. Fam20B is a glycan kinase that phosphorylates xylose residues and triggers peptidoglycan biosynthesis, a role conserved from sponges to human. The protein kinase, Fam20C, conserved from nematodes to humans, phosphorylates well over 100 substrates in the secretory pathway with overall functions postulated to encompass endoplasmic reticulum homeostasis, nutrition, cardiac function, coagulation, and biomineralization. The preferred phosphorylation motif of Fam20C is SxE/pS, and structural studies revealed that related member Fam20A allosterically activates Fam20C by forming a heterodimeric/tetrameric complex. Fam20A, a pseudokinase, is observed only in vertebrates. Loss-of-function genetic alterations in the Fam20 family lead to human diseases such as amelogenesis imperfecta, nephrocalcinosis, lethal and nonlethal forms of Raine syndrome with major skeletal defects, and altered phosphate homeostasis. Together, these three members of the Fam20 family modulate a diverse network of secretory pathway components playing crucial roles in health and disease. The overarching theme of this review is to highlight the progress that has been made in the emerging field of extracellular phosphorylation and the key roles secretory pathway kinases play in an ever-expanding number of cellular processes.

  PublisherOA PDFDOI

• Author response: POMK regulates dystroglycan function via LARGE1-mediated elongation of matriglycan

  2020-09-24 · 1 citations

  peer-reviewOpen access

  Article Figures and data Abstract Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Matriglycan [-GlcA-β1,3-Xyl-α1,3-]n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetyl-glucosaminyltransferase 1 (LARGE1) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNAc-β1,3-GlcNAc-β1,4-Man) preceding matriglycan synthesis, is required for LARGE1-mediated generation of full-length matriglycan on α-DG (~150 kDa). In the absence of Pomk gene expression in mouse skeletal muscle, LARGE1 synthesizes a very short matriglycan resulting in a ~ 90 kDa α-DG which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE1 directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG, thereby preventing muscular dystrophy. Introduction The extracellular matrix (ECM) is essential for development, regeneration and physiological function in many tissues, and abnormalities in ECM structure can lead to disease (Rowe and Weiss, 2008; Hudson et al., 2003). The heteropolysaccharide [-GlcA-β1,3-Xyl-α1,3-]n (called matriglycan) is a scaffold for ECM proteins containing laminin-G (LG) domains (e.g. laminin, agrin, and perlecan) (Yoshida-Moriguchi and Campbell, 2015; Hohenester, 2019; Michele et al., 2002; Ohtsubo and Marth, 2006) and has the remarkable capacity to be tuned during skeletal muscle development and regeneration (Goddeeris et al., 2013). Over 18 genes are involved in the synthesis of the post-translational modification terminating in matriglycan (Figure 1), and defects in this process cause dystroglycanopathies, i.e. congenital and limb-girdle muscular dystrophies that can be accompanied by brain and eye defects. Like-acetyl-glucosaminyltransferase 1 (LARGE1) synthesizes matriglycan on the cell-surface glycoprotein, α-dystroglycan (α-DG) (Inamori et al., 2012). Addition of matriglycan enables α-DG to serve as the predominant ECM receptor in skeletal muscle and brain (Yoshida-Moriguchi and Campbell, 2015; Hohenester, 2019; Jae et al., 2013; Yoshida-Moriguchi et al., 2010; Yoshida-Moriguchi et al., 2013). Crystal structure studies have shown that a single glucuronic acid-xylose disaccharide (GlcA-Xyl) repeat binds to laminin-α2 LG4 domain (Briggs et al., 2016; Hohenester et al., 1999), and there is a direct correlation between the number of GlcA-Xyl repeats on α-DG and its binding capacity for ECM ligands (Goddeeris et al., 2013; Inamori et al., 2012). During skeletal muscle differentiation, LARGE1 elongates matriglycan to its full length for normal skeletal muscle function (Goddeeris et al., 2013). However, little is known about the mechanisms that control matriglycan elongation. Figure 1 Download asset Open asset Synthesis of the α-DG Laminin-Binding Modification and Enzymes Involved. Synthesis of the laminin-binding modification begins with the addition of the core M3 trisaccharide (GalNAc-β3-GlcNAc-β4-Man) on α-DG by the sequential actions of Protein O-Mannosyltransferase 1 and 2 (POMT1/2), Protein O-linked Mannose N-Acetyl-glucosaminyltransferase 2 (POMGNT2), and β1,3-N-Acetylgalactosaminyltransferase 2 (B3GALNT2), in the ER. POMK phosphorylates the C6 hydroxyl of mannose after synthesis of core M3. The phosphorylated core M3 is further elongated in the Golgi by Fukutin (FKTN), Fukutin related protein (FKRP), Transmembrane Protein 5 (TMEM5), β1,4-Glucuronyltransferase 1 (B4GAT1), and Like-acetyl-glucosaminyltranserase 1 (LARGE1). Isoprenoid synthase domain-containing (ISPD) produces cytidine diphosphate (CDP)-ribitol in the cytosol, and this serves as a sugar donor for the reactions catalyzed by FKTN and FKRP. LARGE1 synthesizes matriglycan, which directly interacts with the LG domains of matrix ligands. Complete loss-of-function mutations in the dystroglycanopathy genes abrogate synthesis of the post-translational modification terminating in matriglycan. Such mutations preclude addition of matriglycan and, thereby, cause the most severe form of dystroglycanopathy, Walker-Warburg syndrome (WWS), which is lethal in utero or within a day or two of birth (Yoshida-Moriguchi and Campbell, 2015; Hohenester, 2019; Michele et al., 2002; Ohtsubo and Marth, 2006). Protein O-Mannose Kinase (POMK) is a glycosylation-specific kinase that phosphorylates mannose of the core M3 trisaccharide (GalNAc-β1,3-GlcNAc-β1,4-Man) during synthesis of the O-mannose-linked polysaccharide ending in matriglycan (Yoshida-Moriguchi and Campbell, 2015; Hohenester, 2019; Jae et al., 2013; Yoshida-Moriguchi et al., 2013; Zhu et al., 2016). Interestingly, unlike with other dystroglycanopathy genes there are patients with complete loss-of-function mutations in POMK who suffer from mild forms of dystroglycanopathy (Di Costanzo et al., 2014; von Renesse et al., 2014), suggesting some expression of matriglycan without POMK. Here, we have used a multidisciplinary approach to show that phosphorylation of core M3 by POMK is not necessary for the LARGE1-mediated synthesis of a short, non-extended form of matriglycan on α-DG (~90 kDa) with reduced laminin-binding capacity; however, POMK activity is required for LARGE1 to generate full-length matriglycan on α-DG (~150 kDa). In the absence of the phosphorylated core M3, the non-extended matriglycan on ~90 kDa α-DG binds laminin and maintains specific force but cannot prevent eccentric contraction-induced force loss or skeletal muscle pathology. Furthermore, solution NMR studies demonstrated that LARGE1 directly interacts with core M3, binding preferentially to the phosphorylated form. Therefore, our study shows that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG. Collectively, our work demonstrates a requirement for POMK in the LARGE1-mediated synthesis of full-length matriglycan and proper skeletal muscle function. Results To determine if matriglycan can be expressed in the absence of POMK function, and therefore better understand the role of POMK in matriglycan synthesis, we studied skeletal muscle from a patient (NH13-284) with a homozygous POMK (D204N) mutation (Figure 2A) and congenital muscular dystrophy (CMD) accompanied by structural brain malformations. D204 serves as the catalytic base in the phosphorylation reaction catalyzed by the kinase (Figure 2A; Figure 2—figure supplement 1), and its mutation is predicted to eliminate POMK activity (Figure 2—figure supplement 1; Zhu et al., 2016). POMK activity from skin fibroblasts and skeletal muscle of patient NH13-284 (POMK D204N) was undetectable when compared to control fibroblasts and muscle, respectively (Figure 2B). Fibroblast LARGE1 activity and skeletal muscle B4GAT1 activity of patient NH13-284 were similar to those of a control (Figure 2—figure supplement 2A and B). Immunofluorescence analyses of POMK D204N muscle demonstrated partial immunoreactivity to IIH6 (anti-matriglycan), while the transmembrane subunit of DG, β-DG, was expressed normally in POMK D204N muscle (Figure 2C). Flow cytometry using IIH6 also demonstrated partial immunoreactivity in POMK D204N fibroblasts (Figure 2—figure supplement 2C). To test the effect of the POMK mutation on ligand binding, we performed a laminin overlay using laminin-111. Control human skeletal muscle showed the typical broad band of α-DG laminin binding centered at ~150 kDa range; in contrast, laminin binding at ~90 to 100 kDa range with reduced intensity was observed in POMK D204N skeletal muscle (Figure 2D). Figure 2 with 2 supplements see all Download asset Open asset Characterization of a Patient with a Loss-of-Function Mutation in POMK. (A) (above) Human POMK consists of a transmembrane domain (TM) and a kinase domain (N-lobe and C-lobe). The kinase domain contains the catalytic loop (orange) and activation segment (green). (below) Alignment of protein sequences flanking the D204N mutation. The mutation alters a highly conserved aspartate that is the catalytic base of the phosphorylation reaction catalyzed by the kinase. (B) POMK activity in control and patient NH13-284 (POMK D204N) fibroblasts (left) and skeletal muscle (right). n = 3 experiments were performed in fibroblasts. Triple asterisks: statistical significance with Student’s unpaired t-test (p-value<0.0001). Due to limited skeletal muscle, n = 1 experiment was performed. (C) Histology and immunofluorescence of control and POMK D204N skeletal muscle using IIH6 (anti-matriglycan) and a β-DG antibody. (Scale bars: Control- 200 µM, POMK D204N- 75 µM). (D) Laminin overlay of control and POMK D204N skeletal muscle. To understand the biochemical basis of the ~90 to 100 kDa laminin binding in the absence of POMK activity, we targeted Pomk using LoxP sites and Cre driven by the muscle creatine kinase (Mck) promoter, or both the Mck promoter and the paired box 7 (Pax7) promoter (Figure 3—figure supplements 1 and 2; Brüning et al., 1998; Cohn et al., 2002; Han et al., 2009; Keller et al., 2004) to generate muscle-specific Pomk-null mouse models. Histologic analyses of MckCre; Pax7Cre; PomkLoxP/LoxP (M-POMK KO) quadriceps muscles revealed hallmarks of a mild muscular dystrophy (Figure 3A). Quadriceps muscle extracts of MckCre; PomkLoxP/LoxP mice showed reduced POMK activity compared to PomkLoxP/LoxP muscle but had similar levels of LARGE1 activity (Figure 3B and C). M-POMK KO mice also showed reductions in 2-limb grip strength and body weight, and elevations in post-exercise creatine kinase (CK) levels compared to littermate control PomkLoxP/LoxP mice (Figure 3D; Figure 3—figure supplement 3). Immunofluorescence analysis of M-POMK KO muscle showed that β-DG is expressed at the skeletal muscle sarcolemma (Figure 3A); however, like patient NH13-284 IIH6 immunoreactivity persisted in M-POMK KO muscle, but at a reduced intensity (Figure 3A). Figure 3 with 4 supplements see all Download asset Open asset Mice with a Muscle-Specific Loss of Pomk Develop Hallmarks of a Mild Muscular Dystrophy. (A) H&E and immunofluorescence analyses using IIH6 (anti-matriglycan) and an anti-β-DG antibody of quadriceps muscles of 4–6 week-old PomkLoxP/LoxP (Control) and MckCre; Pax7Cre; PomkLoxP/LoxP (M-POMK KO) mice. Scale bars: 100 µM. (B) POMK and (C) LARGE1 activity in extracts of MckCre; PomkLoxP/LoxP and PomkLoxP/LoxP quadriceps skeletal muscles. Triple asterisks indicate statistical significance using Student’s unpaired t-test (p-value<0.0001, three replicates). (D) Creatine kinase levels of 8-week-old M-POMK KO and Control mice. p-values were calculated with Student’s unpaired t-test. Triple asterisks: statistical significance with p-value<0.05 (p-value=0.0008), n = 12 Control and 14 M-POMK KO mice. We next examined ex vivo force production in extensor digitorum muscles (EDL) muscles of 18–20- week-old Control and M-POMK KO mice. EDL muscle mass and cross-sectional area (CSA) were reduced in M-POMK KO mice compared to control mice (Figure 4A and B). Additionally, M-POMK KO EDL absolute isometric tetanic force production was significantly lower than that of controls (Figure 4C). However, when normalized to muscle CSA, force production was comparable to control values (Figure 4D). We also sought to determine if M-POMK KO muscle could withstand repeated eccentric contractions. EDL muscles of M-POMK KO mice demonstrated greater force deficits after five and eight lengthening contractions (LC) and recovered to a lower level after 45 min compared to Control EDL (Figure 4E). Together, the isometric and eccentric contractile studies suggest that the M-POMK KO EDL muscles display a specific force similar to controls (Figure 4D); however, muscle integrity is compromised following the stress of repeated eccentric contractions, as displayed by the slow, but progressive decline in force production and hampered recovery (Figure 4E). Thus, the current results demonstrate that the short matriglycan in POMK-deficient skeletal muscle can maintain specific force but cannot prevent eccentric contraction-induced force loss or muscle pathology. Figure 4 Download asset Open asset MckCre; Pax7Cre; PomkLoxP/LoxP Extensor Digitorum Longus (EDL) Muscle Demonstrates Eccentric Contraction-Induced Force Loss. (A) Mass (milligrams) of PomkLoxP/LoxP (Control) and MckCre; Pax7Cre; PomkLoxP/LoxP (M-POMK KO) EDL muscles tested for force production. ***Statistical significance with Student’s unpaired t-test with p-value<0.05 (p=0.0031). (B) Cross-sectional area (CSA) of EDL muscles. ***Statistical significance using Student’s unpaired t-test with p-value<0.05 (p=0.0463). (C) Maximum Absolute Tetanic Force production by Control and M-POMK KO EDL muscles. ***Statistical significance using Student’s unpaired t-test with a p-value<0.05 (p=0.0395). (D) Specific Force production in Control and M-POMK KO EDL muscles (p=0.921). (E) Force deficit and force recovery in Control (n=3) and M-POMK KO (n=4) mice after eccentric contractions. EDL muscles from 18- to 20-week-old male mice were tested and are represented by open (Control) or closed (M-POMK KO) circles. ***Statistical significance using Student’s unpaired t-test (p-value<0.0001) compared to Control EDL at given LC cycle. **Statistical significance using Student’s unpaired t-test (p-value=0.0027) compared to Control EDL at given LC cycle. Error bars represent SD. Biochemical analysis of control and M-POMK KO muscle showed a typical, lower molecular weight (MW) α-DG with anti-core DG antibody (Figure 5A), however, on laminin overlay, we observed laminin binding at 90–100 kDa (Figure 5B), similar to POMK D204N skeletal muscle (Figure 2D). IIH6 also showed binding at 90–100 kDa (Figure 5C). Solid-phase binding analyses of M-POMK KO and MckCre; PomkLoxP/LoxP skeletal muscle demonstrated a reduced binding capacity (relative Bmax) for laminin-111 compared to control muscle (Figure 5—figure supplement 1A), but higher than that of Largemyd muscle, which lacks matriglycan due to a deletion in Large. Figure 5 with 2 supplements see all Download asset Open asset Mice with a Muscle-Specific Loss of Pomk Express Matriglycan. (A) Biochemical analysis of Control and M-POMK KO skeletal muscle. Glycoproteins were enriched from quadriceps skeletal muscles of mice using wheat-germ agglutinin (WGA)-agarose. Immunoblotting was performed with antibody AF6868, which recognizes core α-DG and β-DG (three replicates). (B) Laminin overlay of quadriceps muscles of Control and M-POMK KO mice (three replicates). (C) IIH6 immunoblotting of Control and M-POMK KO quadriceps muscle. (D, E) Laminin overlay (D) and solid-phase analysis (E) of skeletal muscles of M-POMK KO mice treated in combination with two exoglycosidases, α-xylosidase (Xylsa) and β-glucuronidase (Bgus) for 17 hr (three replicates). To determine if matriglycan is responsible for the laminin binding at 90–100 kDa in POMK-null muscle, we treated glycoproteins enriched from skeletal muscles of M-POMK KO and MckCre; PomkLoxP/LoxP mice with two exoglycosidases, α-Xylosidase and β-Glucuronidase, which in combination digest matriglycan (Figure 5—figure supplements 1B, 2A and B; Briggs et al., 2016). Laminin overlay and solid-phase analysis showed a reduction in laminin binding from these muscles after dual exoglycosidase digestion (Figure 5D and E; Figure 5—figure supplement 2A and B). To study the role of POMK further, we used human POMK KO HAP1 cells, which have undetectable levels of POMK activity and expression (Figure 6A; Figure 6—figure supplement 1A; Zhu et al., 2016). A mass spectrometry (MS)-based glycomic analysis of O-glycans carried by recombinantly-expressed DG mucin-like domain indicated the near complete absence of an MS peak at m/z 873.5 corresponding to phosphorylated core M3 O-glycan (Figure 6D and E; Figure 6—figure supplement 2A and B), consistent with an undetectable level of POMK activity in POMK KO HAP1 cells. Compared to WT HAP1 cells, immunoblots of POMK KO HAP1 cells showed a reduction in IIH6 immunoreactivity, a decrease in MW of core α-DG, and the presence of laminin binding at ~90 kDa on laminin overlay (Figure 6C; Figure 6—figure supplement 1B and C). Laminin binding on overlay was rescued only after adenoviral transduction with wild-type (WT) POMK (POMK WT), but not with POMK containing D204N (POMK D204N) or D204A (POMK D204A) mutations (Figure 6C). POMK D204N also lacked POMK activity in vitro but showed normal B4GAT1, B3GALNT2, and LARGE1 activity, thus confirming the pathogenicity of the D204N mutation (Figure 6A and B; Figure 6—figure supplement 1D and E). Figure 6 with 2 supplements see all Download asset Open asset POMK D204N lacks Catalytic Activity. (A) POMK or (B) LARGE1 activity in POMK KO HAP1 cells transduced with adenoviruses encoding POMK D204N, POMK D204A, or POMK WT. Triple asterisks: statistical significance (p-value<0.0001) compared to POMK KO alone using one-way ANOVA with Dunnett’s test for multiple comparisons (three replicates, 95% Confidence intervals for POMK KO vs. WT HAP1: −106.7 to −81.0, POMK KO vs. POMK KO + POMK WT: −84.25 to −58.54). (C) Laminin overlay of POMK KO HAP1 cells expressing the indicated POMK mutants. (D, E) Mass Spectrometry (MS)-based O-glycomic analyses of DG mucin-like domain (DG390TevHis) expressed in Fukutin (FKTN) (D) or POMK (E) KO HAP1 cells. O-glycans were released from the protein backbone and permethylated prior to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analyses. MS peaks at m/z 779.5 (779.6) correspond to a mixture of core 2 and core M3 O-glycan, and at 873.5, phosphorylated core M3 O-glycan (red). MALDI-TOF is unable to determine anomeric or epimeric configurations of annotated O-glycans. To directly test if LARGE1 is required for synthesis of the 90 kDa laminin-binding glycoprotein in POMK KO HAP1 cells, we studied POMK/LARGE1 KO HAP1 cells, which bear a CRISPR/Cas9-mediated deletion in LARGE1 as well as POMK. POMK/LARGE1 KO HAP1 cells demonstrated the absence of the laminin binding at 90 kDa (Figure 7A; Figure 7—figure supplement 1A and B), indicating that LARGE1 is required for the synthesis of the matriglycan responsible for laminin binding at 90 kDa. Moreover, POMK/DAG1 KO HAP1 cells demonstrated a complete absence of laminin binding (Figure 7A) and IIH6 immunoreactivity at 90 kDa (Figure 7—figure supplement 1C), demonstrating that α-DG is the glycoprotein that binds laminin in the absence of POMK. We, therefore, refer to this glycoprotein as POMK-null α-DG (α-DG(POMK)). Since the length of matriglycan correlates with its binding capacity for ECM ligands (Goddeeris et al., 2013), we hypothesized that, given the MW of α-DG(POMK) at 90 kDa, the glycan must be shorter than full-length matriglycan, and therefore, have a lower Bmax for laminin. We measured the binding capacity of HAP1 α-DG using solid-phase binding assays. Bmax of α-DG(POMK) for laminin-111 was reduced compared to wild-type α-DG (α-DG(WT)) but was greater than that of α-DG from LARGE1 KO HAP1 cells (Figure 7B). POMK/DAG1 KO HAP1 cells showed a reduction in Bmax compared to POMK KO HAP1 cells, but similar to the low levels observed in LARGE1 KO HAP1 cells (Figure 7B). These data indicate that a short, non-extended form of matriglycan is synthesized on α-DG(POMK), and this short form has a lower binding capacity for laminin-111, thus exhibiting a reduced level of α-DG receptor function. Figure 7 with 6 supplements see all Download asset Open asset LARGE1 requires POMK to Elongate Matriglycan. (A) WT, POMK KO, and POMK/LARGE1 KO HAP1 cells (left) or POMK/DAG1 KO HAP1 cells (right) (three replicates). (B) Solid-phase analysis of WT, POMK KO, POMK/DAG1 KO, and LARGE1 KO HAP1 cells (three replicates). (C, D, E) Laminin overlays of the following KO HAP1 cells (three replicates): POMK/ISPD expressing Ad-ISPD (C); POMK expressing Ad-LARGE1 (D); POMK/LARGE1 expressing Ad-LARGE1 with or without Ad-POMK (E). After POMK phosphorylates core M3, Fukutin (FKTN) modifies GalNAc with ribitol-phosphate for synthesis of full-length matriglycan (Figure 1; Yoshida-Moriguchi and Campbell, 2015; Hohenester, 2019; Kanagawa et al., 2016). Overexpression in POMK KO HAP1 cells of ISPD, which synthesizes the substrate (CDP-ribitol) of FKTN (Figure 1), increases the amount of matriglycan (without changing its migration on SDS-PAGE) responsible for laminin binding at 90 kDa (Figure 7—figure supplement 2A, B and C; Willer et al., 2012; Gerin et al., 2016; Riemersma et al., 2015). HAP1 cells lacking both POMK and ISPD do not express matriglycan, and adenoviral transduction of these cells with ISPD restores the 90 kDa laminin binding (Figure 7C; Figure 7—figure supplement 2D and E). FKTN overexpression in POMK KO HAP1 cells also increased the 90 kDa laminin binding (Figure 7—figure supplement 3A, B and C). These experiments collectively support a requirement for CDP-ribitol for synthesis of the non-extended form of matriglycan. This synthesis also requires the N-terminal domain of α-DG (DGN) (Hara et al., 2011a; Kanagawa et al., 2004), as a DG mutant lacking the DGN (DGE) expressed in POMK/DAG1 KO HAP1 cells did not show laminin binding at 90 kDa (Figure 7—figure supplement 4A, B and C). Similar experiments also indicated that synthesis of the non-extended matriglycan in HAP1 cells requires threonine-317 of the mucin-like domain of α-DG (Figure 7—figure supplement 4A, B and C). Overexpression of LARGE1 can rescue the defect in matriglycan synthesis in distinct forms of CMD as well as in LARGE1 KO HAP1 cells by generating very molecular weight matriglycan (Figure 7—figure supplement et al., However, overexpression of LARGE1 in POMK or POMK/LARGE1 KO HAP1 cells did not very molecular weight matriglycan (Figure and E; Figure 7—figure supplement and the rescue of POMK/LARGE1 KO HAP1 cells with POMK LARGE1 to molecular weight matriglycan (Figure Figure 7—figure supplement These indicate that LARGE1 requires phosphorylated core M3 to matriglycan on α-DG to its and molecular weight To understand phosphorylated core M3 is for LARGE1 to elongate matriglycan, we measured the binding of as well as for the phosphorylated core M3 using solution We showed that the core M3 binds to POMK with et al., 2016). The mannose anomeric is well and its intensity only with POMK protein (Figure supplement the intensity of the peak as a function of POMK we a of (Figure Figure supplement 1A and B). These results indicate that, compared to the core M3 of the phosphorylated core M3 of binds to POMK with a we measured the binding of LARGE1 for and in a similar results showed that LARGE1 binds with greater to compared to = for compared to for (Figure B and This that the core M3 increases the binding of LARGE1 for core M3 and could the of LARGE1 to elongate matriglycan in the presence of POMK. Figure with 3 supplements see all Download asset Open asset NMR of POMK and LARGE1 to and 1D NMR of the anomeric of (A) and (B) were for the glycan of in the presence of of LARGE1 as The peak is from the mannose anomeric indicate peaks from (C, of the NMR binding data of POMK (C) and LARGE1 (D) to core M3 of and The was from the NMR data by the in the peak intensity of the anomeric in the absence and presence of POMK or by the peak intensity of the form. Discussion POMK is a muscular dystrophy gene that phosphorylates mannose of the core M3 trisaccharide (GalNAc-β1,3-GlcNAc-β1,4-Man) on α-DG during synthesis of the O-mannose-linked polysaccharide ending in matriglycan. LARGE1 is responsible for the synthesis of matriglycan, and addition of matriglycan enables α-DG to serve as a predominant ECM receptor in many tissues, in skeletal muscle and Over genes are in matriglycan synthesis, and complete loss-of-function mutations in these genes abrogate synthesis of the modification and preclude the addition of matriglycan, thereby to dystroglycanopathies, congenital and limb-girdle muscular dystrophies with or without structural brain and eye Here, we have used a multidisciplinary approach to show that the absence of POMK activity not preclude addition of matriglycan. in the absence of core M3 phosphorylation by LARGE1 synthesizes a short, non-extended form of matriglycan on α-DG (~90 kDa). However, in to generate full-length matriglycan on α-DG (~150 LARGE1 requires phosphorylation of core M3 by POMK (Figure supplement 2A and B). study shows that the short form of matriglycan is to to laminin with and thus enables α-DG(POMK) to function as an ECM the very in MW in α-DG(POMK) compared to α-DG from cells and muscle lacking LARGE1 (Figure 5—figure supplement 2A; Figure 7—figure supplement 1A; Figure supplement the short, non-extended form of matriglycan contains However, can laminin only a single repeat is for laminin binding (Briggs et al., but cannot function as an ECM This short matriglycan muscular dystrophy in patient NH13-284 with a complete loss-of-function mutation in preventing the severe that is observed in the complete absence of the other known dystroglycanopathy POMK KO mice express the short, non-extended form of matriglycan on ~90 kDa α-DG and a mild muscular dystrophy Muscle studies demonstrate that the short matriglycan expressed in the absence of POMK can maintain specific force but cannot prevent eccentric contraction-induced force loss or skeletal muscle pathology. Interestingly, mutations in that cause also show reduced expression of matriglycan (Yoshida-Moriguchi and Campbell, and a muscular dystrophy. Thus, M-POMK KO mice are an of forms of dystroglycanopathy in which short matriglycan is expressed and be for studies of these forms of α-DG is of three the which at by a during α-DG post-translational a mucin-like and a et al., et al., The domain of in is to three sites of matriglycan synthesis within the mucin-like domain of α-DG. Biochemical studies using POMK KO HAP1 demonstrated that the synthesis of the short, non-extended form of matriglycan on threonine-317 of the mucin-like domain and, like full-length matriglycan, requires and experiments demonstrated that the DGN is necessary for synthesis of the short form of matriglycan. the binding of LARGE1 to the DGN is essential for the synthesis of full-length matriglycan on α-DG et al., et al., is required for synthesis of the short form of matriglycan as Solution NMR studies revealed that LARGE1 binds to core M3, and the binding increases in the presence of the mannose The phosphorylated core M3, therefore serve to LARGE1 to the proper during the of full-length matriglycan In the absence of the mannose the LARGE1 only the matriglycan to the to the Synthesis of full-length matriglycan therefore, a of and phosphorylated core M3. The phosphorylated core M3 also serve to LARGE1 to α-DG during matriglycan elongation. In the absence of the binding of LARGE1 to the DGN and the core M3 only be for synthesis of a short form of matriglycan. structural and biochemical studies be required to understand the between and the phosphorylated core M3. our results indicate that LARGE1 requires DGN to the short, non-extended form of matriglycan but both the DGN

  PublisherDOI

• The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress

  Cell Death and Differentiation · 2020 · 37 citations

  • Cancer research
  • Biology
  • Cell biology

  To survive proteotoxic stress, cancer cells activate the proteotoxic-stress response pathway, which is controlled by the transcription factor heat shock factor 1 (HSF1). This pathway supports cancer initiation, cancer progression and chemoresistance and thus is an attractive therapeutic target. As developing inhibitors against transcriptional regulators, such as HSF1 is challenging, the identification and targeting of upstream regulators of HSF1 present a tractable alternative strategy. Here we demonstrate that in triple-negative breast cancer (TNBC) cells, the dual specificity tyrosine-regulated kinase 2 (DYRK2) phosphorylates HSF1, promoting its nuclear stability and transcriptional activity. DYRK2 depletion reduces HSF1 activity and sensitises TNBC cells to proteotoxic stress. Importantly, in tumours from TNBC patients, DYRK2 levels positively correlate with active HSF1 and associates with poor prognosis, suggesting that DYRK2 could be promoting TNBC. These findings identify DYRK2 as a key modulator of the HSF1 transcriptional programme and a potential therapeutic target.

  PublisherOA PDFDOI

• POMK regulates dystroglycan function via LARGE-mediated elongation of matriglycan

  bioRxiv (Cold Spring Harbor Laboratory) · 2020-04-07 · 1 citations

  preprintOpen access

  Abstract Matriglycan [-GlcA-β1,3-Xyl-α1,3-] n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetylglucosaminyltransferase-1 (LARGE) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK) , which phosphorylates mannose of core M3 (GalNac-β1,3-GlcNac-β1,4-Man) preceding matriglycan synthesis, is required for LARGE-mediated generation of full-length matriglycan on α-DG (∼150 kDa). In the absence of POMK , LARGE synthesizes a very short matriglycan resulting in a ∼90 kDa α-DG in mouse skeletal muscle which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.

  PublisherOA PDFDOI

• POMK regulates dystroglycan function via LARGE1-mediated elongation of matriglycan

  eLife · 2020-09-25 · 30 citations

  articleOpen access

  Matriglycan [-GlcA-β1,3-Xyl-α1,3-] n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetyl-glucosaminyltransferase 1 (LARGE1) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O -Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNAc-β1,3-GlcNAc-β1,4-Man) preceding matriglycan synthesis, is required for LARGE1-mediated generation of full-length matriglycan on α-DG (~150 kDa). In the absence of Pomk gene expression in mouse skeletal muscle, LARGE1 synthesizes a very short matriglycan resulting in a ~ 90 kDa α-DG which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE1 directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.

  PublisherDOI

Recent grants

• NIH Grant R01GM090328

  NIH · $1.3M · 2015

• NIH Grant R01AI060662

  NIH · $1.8M · 2010

• NIH Grant R01AM018849

  NIH · $134k · 1989

• NIH Grant R01AM018024

  NIH · $105k · 1988

• Novel Secreted Kinases

  NIH · $2.3M · 1991–2020

Frequent coauthors

• Carolyn A. Worby

  University of California, San Diego

  78 shared

• C D Minth

  University of Michigan–Ann Arbor

  46 shared

• Bernard A. Roos

  44 shared

• Philip Andrews

  University of Alaska Fairbanks

  40 shared

• Randy S. Haun

  Arkana Laboratories

  38 shared

• Sandra E. Wiley

  MEI Pharma (United States)

  36 shared

• Junyu Xiao

  Peking University

  35 shared

• Tomohiko Maehama

  Kobe University

  33 shared

Labs

• Dixon LabPI

  1-2 sentence research focus