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CDK5 Regulatory Subunit-associated Protein 1-Like 1 (CDKAL1) Is a Tail-anchored Protein in the Endoplasmic Reticulum (ER) of Insulinoma Cells*

  • Silvia Brambillasca
    Footnotes
    Affiliations
    Molecular Diabetology, Paul Langerhans Institute Dresden, Uniklinikum Carl Gustav Carus, Dresden University of Technology, Fetscherstrasse 74, 01307 Dresden, Germany
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  • Anke Altkrueger
    Affiliations
    Molecular Diabetology, Paul Langerhans Institute Dresden, Uniklinikum Carl Gustav Carus, Dresden University of Technology, Fetscherstrasse 74, 01307 Dresden, Germany
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  • Sara Francesca Colombo
    Affiliations
    Consiglio Nazionale delle Ricerche Institute of Neuroscience and Biometra Department, Università degli Studi di Milano, 20129 Milan, Italy
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  • Anne Friederich
    Affiliations
    Molecular Diabetology, Paul Langerhans Institute Dresden, Uniklinikum Carl Gustav Carus, Dresden University of Technology, Fetscherstrasse 74, 01307 Dresden, Germany
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  • Peter Eickelmann
    Affiliations
    Department of CardioMetabolic Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG., 88397 Biberach, Germany
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  • Michael Mark
    Affiliations
    Department of CardioMetabolic Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG., 88397 Biberach, Germany
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  • Nica Borgese
    Affiliations
    Consiglio Nazionale delle Ricerche Institute of Neuroscience and Biometra Department, Università degli Studi di Milano, 20129 Milan, Italy

    Department of Health Science, University of Catanzaro ”Magna Graecia,”, 88100 Catanzaro, Italy
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  • Michele Solimena
    Correspondence
    To whom correspondence should be addressed: Molecular Diabetology, Paul Langerhans Institute Dresden, Uniklinikum Carl Gustav Carus, Dresden University of Technology, Fetscherstrasse 74, 01307 Dresden, Germany. Tel.: 49-351-7963-6611; Fax: 49-351-7963-6698
    Affiliations
    Molecular Diabetology, Paul Langerhans Institute Dresden, Uniklinikum Carl Gustav Carus, Dresden University of Technology, Fetscherstrasse 74, 01307 Dresden, Germany

    Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
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  • Author Footnotes
    * This work was supported in part by funds from the German Ministry for Education and Research to the German Centre for Diabetes Research, a research grant from Boehringer Ingelheim (to M. S.), and by Italian Association for Cancer Research Grant IG9040 (to the N. B. laboratory).
    This article contains supplemental Experimental Procedures, Figs. S1–S5, Tables S1 and S2, and additional references.
    1 Recipient of a MedDrive grant from the Dresden University of Technology Medical School.
Open AccessPublished:October 09, 2012DOI:https://doi.org/10.1074/jbc.M112.376558
      Genome-wide association studies have led to the identification of numerous susceptibility genes for type 2 diabetes. Among them is Cdkal1, which is associated with reduced β-cell function and insulin release. Recently, CDKAL1 has been shown to be a methylthiotransferase that modifies tRNALys to enhance translational fidelity of transcripts, including the one encoding proinsulin. Here, we report that out of several CDKAL1 isoforms deposited in public databases, only isoform 1, which migrates as a 61-kDa protein by SDS-PAGE, is expressed in human islets and pancreatic insulinoma INS-1 and MIN6 cells. We show that CDKAL1 is a novel member of the tail-anchored protein family and exploits the TCR40/Get3-assisted pathway for insertion of its C-terminal transmembrane domain into the endoplasmic reticulum. Using endo-β-N-acetylglucosaminidase H and peptide:N-glycosidase F sensitivity assays on CDKAL1 constructs carrying an N-glycosylation site within the luminal domain, we further established that CDKAL1 is an endoplasmic reticulum-resident protein. Moreover, we observed that silencing CDKAL1 in INS-1 cells reduces the expression of secretory granule proteins prochromogranin A and proICA512/ICA512-TMF, in addition to proinsulin and insulin. This correlated with reduced glucose-stimulated insulin secretion. Taken together, our findings provide new insight into the role of CDKAL1 in insulin-producing cells and help to understand its involvement in the pathogenesis of diabetes.

      Introduction

      Diabetes mellitus is a chronic disorder characterized by high blood glucose levels due to insufficient secretion of insulin by pancreatic β-cells in relation to metabolic needs. Its most common form, type 2 diabetes (T2D),
      The abbreviations used are: T2D
      type 2 diabetes
      Cdkal1
      Cdk5 regulatory subunit-associated protein 1-like 1
      TA
      tail-anchored
      UPR
      unfolded protein response
      RM
      rough microsomes
      IAA
      iodoacetamide
      Ab
      antibody
      TMD
      transmembrane domain
      ER
      endoplasmic reticulum
      PF
      protected fragment
      CGA
      chromogranin A
      ICA512
      islet cells autoantigen 512
      Endo-H
      endoglycosidase H
      PNGaseF
      peptide:N-glycosidase F
      oligo
      oligonucleotide
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
      Nglyc
      N-glycosylated
      HSP
      high speed pellet.
      results from peripheral insulin resistance and β-cell dysfunction due to the unfavorable interaction of genes with environmental factors. Recent genome-wide association studies have led to the identification of >60 T2D susceptibility genes, including Cdkal1 (Cdk5 regulatory subunit-associated protein 1-like 1) (
      • Bonnefond A.
      • Froguel P.
      • Vaxillaire M.
      The emerging genetics of type 2 diabetes.
      ). Carriers of the Cdkal1 rs7754840 allele, in particular, display diminished glucose-stimulated insulin secretion and impaired proinsulin to insulin conversion (
      • Kirchhoff K.
      • Machicao F.
      • Haupt A.
      • Schäfer S.A.
      • Tschritter O.
      • Staiger H.
      • Stefan N.
      • Häring H.U.
      • Fritsche A.
      Polymorphisms in the TCF7L2CDKAL1SLC30A8 genes are associated with impaired proinsulin conversion.
      ,
      • Groenewoud M.J.
      • Dekker J.M.
      • Fritsche A.
      • Reiling E.
      • Nijpels G.
      • Heine R.J.
      • Maassen J.A.
      • Machicao F.
      • Schäfer S.A.
      • Häring H.U.
      • ‘t Hart L.M.
      • van Haeften T.W.
      Variants of CDKAL1 and IGF2BP2 affect first-phase insulin secretion during hyperglycaemic clamps.
      ). This gene was named after its close paralogue CDK5RAP1 and, similarly to it, was speculated to inhibit Cdk5 (
      • Ching Y.P.
      • Pang A.S.
      • Lam W.H.
      • Qi R.Z.
      • Wang J.H.
      Identification of a neuronal Cdk5 activator-binding protein as Cdk5 inhibitor.
      ) and thereby affect exocytosis of synaptic vesicles (
      • Dhavan R.
      • Tsai L.H.
      A decade of CDK5.
      ) and insulin granules (
      • Lilja L.
      • Yang S.N.
      • Webb D.L.
      • Juntti-Berggren L.
      • Berggren P.O.
      • Bark C.
      Cyclin-dependent kinase 5 promotes insulin exocytosis.
      ,
      • Lilja L.
      • Johansson J.U.
      • Gromada J.
      • Mandic S.A.
      • Fried G.
      • Berggren P.O.
      • Bark C.
      Cyclin-dependent kinase 5 associated with p39 promotes munc18-1 phosphorylation and Ca2+-dependent exocytosis.
      ,
      • Wei F.Y.
      • Nagashima K.
      • Ohshima T.
      • Saheki Y.
      • Lu Y.F.
      • Matsushita M.
      • Yamada Y.
      • Mikoshiba K.
      • Seino Y.
      • Matsui H.
      • Tomizawa K.
      Cdk5-dependent regulation of glucose-stimulated insulin secretion.
      ,
      • Schubert S.
      • Knoch K.P.
      • Ouwendijk J.
      • Mohammed S.
      • Bodrov Y.
      • Jäger M.
      • Altkrüger A.
      • Wegbrod C.
      • Adams M.E.
      • Kim Y.
      • Froehner S.C.
      • Jensen O.N.
      • Kalaidzidis Y.
      • Solimena M.
      β2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules.
      ). This hypothesis was confuted, however, as CDKAL1 was repeatedly shown neither to interact with Cdk5 nor inhibit its activity (
      • Ohara-Imaizumi M.
      • Yoshida M.
      • Aoyagi K.
      • Saito T.
      • Okamura T.
      • Takenaka H.
      • Akimoto Y.
      • Nakamichi Y.
      • Takanashi-Yanobu R.
      • Nishiwaki C.
      • Kawakami H.
      • Kato N.
      • Hisanaga S.
      • Kakei M.
      • Nagamatsu S.
      Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis.
      ,
      • Wei F.Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ). Meanwhile, independent work led to the discovery that Bacillus subtilis yqeV is a methylthiotransferase responsible for the modification of tRNAs (
      • Arragain S.
      • Handelman S.K.
      • Forouhar F.
      • Wei F.Y.
      • Tomizawa K.
      • Hunt J.F.
      • Douki T.
      • Fontecave M.
      • Mulliez E.
      • Atta M.
      Identification of eukaryotic and prokaryotic methylthiotransferase for biosynthesis of 2-methylthio-N6-threonylcarbamoyladenosine in tRNA.
      ). This modification was shown to improve codon recognition and accuracy of reading frame maintenance (
      • Gustilo E.M.
      • Vendeix F.A.
      • Agris P.F.
      tRNA's modifications bring order to gene expression.
      ), thus ensuring fidelity during protein synthesis. Remarkably, CDKAL1, which displays sequence similarity with YqeV, was able to rescue this enzymatic activity in a yqeV strain. Wei et al. (
      • Wei F.Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ) elegantly demonstrated that CDKAL1 is indeed a methylthiotransferase that specifically modifies tRNALys in mammals. The lack of CDKAL1 caused misreading of the Lys codon, and amino acids were erroneously incorporated during translation, resulting in aberrant proteolytic processing of proinsulin and a probable increase in its degradation.
      Here, we show that down-regulation of CDKAL1 in insulinoma cells perturbs levels of not only insulin but also chromogranin A and islet cell autoantigen 512 (ICA512/IA-2), two other components of insulin secretory granules. We additionally show that CDKAL1 is a new tail-anchored (TA) protein that exploits the TRC40/Get3 pathway for its insertion in the ER membrane. Finally, we report that CDKAL1 mRNA is up-regulated upon induction of the unfolded protein response, further suggesting a role for CDKAL1 in the regulation of the secretory pathway.

      DISCUSSION

      Here, we have characterized several novel aspects of CDKAL1 biology in insulin-producing cells. We show that human islets and rat insulinoma INS-1 cells express only the transcript for isoform 1, which migrates as a 61-kDa protein by SDS-PAGE. In all tissues investigated, isoform 1 has been the only form detected (
      • Quaranta M.
      • Burden A.D.
      • Griffiths C.E.
      • Worthington J.
      • Barker J.N.
      • Trembath R.C.
      • Capon F.
      Differential contribution of CDKAL1 variants to psoriasis, Crohn's disease, and type II diabetes.
      ). It would be interesting to determine whether this is also the case in subjects with single nucleotide polymorphisms in CDKAL1, which increase the risk for T2D and could affect its splicing, possibly in a tissue-specific manner. Notably, all these single nucleotide polymorphisms map to intron 5 along with other single nucleotide polymorphisms found to confer susceptibility to psoriasis and Crohn disease (
      • Quaranta M.
      • Burden A.D.
      • Griffiths C.E.
      • Worthington J.
      • Barker J.N.
      • Trembath R.C.
      • Capon F.
      Differential contribution of CDKAL1 variants to psoriasis, Crohn's disease, and type II diabetes.
      ,
      • Wolf N.
      • Quaranta M.
      • Prescott N.J.
      • Allen M.
      • Smith R.
      • Burden A.D.
      • Worthington J.
      • Griffiths C.E.
      • Mathew C.G.
      • Barker J.N.
      • Capon F.
      • Trembath R.C.
      Psoriasis is associated with pleiotropic susceptibility loci identified in type II diabetes and Crohn disease.
      ,
      • Li Y.
      • Liao W.
      • Chang M.
      • Schrodi S.J.
      • Bui N.
      • Catanese J.J.
      • Poon A.
      • Matsunami N.
      • Callis-Duffin K.P.
      • Leppert M.F.
      • Bowcock A.M.
      • Kwok P.Y.
      • Krueger G.G.
      • Begovich A.B.
      Further genetic evidence for three psoriasis-risk genes. ADAM33CDKAL1PTPN22.
      ).
      Our results corroborate previous findings localizing CDKAL1 to the ER (
      • Ohara-Imaizumi M.
      • Yoshida M.
      • Aoyagi K.
      • Saito T.
      • Okamura T.
      • Takenaka H.
      • Akimoto Y.
      • Nakamichi Y.
      • Takanashi-Yanobu R.
      • Nishiwaki C.
      • Kawakami H.
      • Kato N.
      • Hisanaga S.
      • Kakei M.
      • Nagamatsu S.
      Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis.
      ,
      • Wei F.Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ). Although these studies indicated that CDKAL1 is associated with intracellular membranes, it was unclear if it is an integral membrane protein or is peripherally associated with the lipid bilayer (
      • Ohara-Imaizumi M.
      • Yoshida M.
      • Aoyagi K.
      • Saito T.
      • Okamura T.
      • Takenaka H.
      • Akimoto Y.
      • Nakamichi Y.
      • Takanashi-Yanobu R.
      • Nishiwaki C.
      • Kawakami H.
      • Kato N.
      • Hisanaga S.
      • Kakei M.
      • Nagamatsu S.
      Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis.
      ,
      • Wei F.Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ). We now show that CDKAL1 is a true transmembrane protein belonging to the TA protein family. This family includes several key enzymes and regulatory proteins, whose activities are inextricably linked to their localization. Among them are SNARE proteins, members of the Bcl-2 family and components of the translocation machinery in the ER, mitochondria, and peroxisomes (
      • Borgese N.
      • Colombo S.
      • Pedrazzini E.
      The tale of tail-anchored proteins. Coming from the cytosol and looking for a membrane.
      ). The majority of these transmembrane proteins rely on the TRC40/Get3 pathway for their ER membrane insertion. Notably, the Caenorhabditis elegans TRC40 homologue, Asna1, was reported to positively modulate insulin secretion (
      • Kao G.
      • Nordenson C.
      • Still M.
      • Rönnlund A.
      • Tuck S.
      • Naredi P.
      ASNA-1 positively regulates insulin secretion in C. elegans and mammalian cells.
      ). Our data may explain this observation given the broad role of TRC40 in the correct targeting of CDKAL1 and other SNARE proteins (
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method.
      ), which are essentials for vesicle fusion.
      The ER localization of CDKAL1 implies the coupling of this methylthiotransferase with polysomes engaged in the translation of proteins destined only for the secretory pathway. Consistent with this conclusion, CDKAL1 knockdown decreases the levels of at least two other secretory granule components, pro-CGA and proICA512/ICA512-TMF, in addition to proinsulin/insulin (
      • Wei F.Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ). However, not all transcripts translated at the ER seem susceptible to regulation by CDKAL1, as its silencing did not alter the expression of other secretory proteins, e.g. the SNAREs syntaxin1a and VAMP2/synaptobrevin, as well as Kir6.2, SUR1 (
      • Ohara-Imaizumi M.
      • Yoshida M.
      • Aoyagi K.
      • Saito T.
      • Okamura T.
      • Takenaka H.
      • Akimoto Y.
      • Nakamichi Y.
      • Takanashi-Yanobu R.
      • Nishiwaki C.
      • Kawakami H.
      • Kato N.
      • Hisanaga S.
      • Kakei M.
      • Nagamatsu S.
      Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis.
      ), PC1/3, PC2, and Phogrin/IA2-β (this study). Notably, these last six proteins are all found on insulin secretory granules (
      • Varadi A.
      • Grant A.
      • McCormack M.
      • Nicolson T.
      • Magistri M.
      • Mitchell K.J.
      • Halestrap A.P.
      • Yuan H.
      • Schwappach B.
      • Rutter G.A.
      Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells. Against a role in organelle cation homeostasis.
      ,
      • Steiner D.F.
      • Rouillé Y.
      • Gong Q.
      • Martin S.
      • Carroll R.
      • Chan S.J.
      The role of prohormone convertases in insulin biosynthesis: evidence for inherited defects in their action in men and experimental animals.
      ,
      • Bailyes E.M.
      • Bennett D.L.
      • Hutton J.C.
      Proprotein-processing endopeptidases of the insulin secretory granule.
      ). Thus efficient translation of granule proteins does not always depend on CDKAL1 activity. Indeed, the number of insulin granules in CDKAL1−/− mice was not reduced (
      • Ohara-Imaizumi M.
      • Yoshida M.
      • Aoyagi K.
      • Saito T.
      • Okamura T.
      • Takenaka H.
      • Akimoto Y.
      • Nakamichi Y.
      • Takanashi-Yanobu R.
      • Nishiwaki C.
      • Kawakami H.
      • Kato N.
      • Hisanaga S.
      • Kakei M.
      • Nagamatsu S.
      Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis.
      ).
      Bioinformatics analysis revealed no significant differences in the frequency of Lys residues among proteins translocated through the ER versus those that are cytosolic (data not shown). Lys is the target of many post-translational modifications and is also crucial for the correct sorting and maturation of proteins along the secretory pathway (
      • Zhou A.
      • Webb G.
      • Zhu X.
      • Steiner D.F.
      Proteolytic processing in the secretory pathway.
      ,
      • Teasdale R.D.
      • Jackson M.R.
      Signal-mediated sorting of membrane proteins between the endoplasmic reticulum and the Golgi apparatus.
      ). In professional secretory cells, such as insulin-producing β-cells, proteins destined for secretion can account for up to 50% of the total protein content (
      • Schuit F.C.
      • In't Veld P.A.
      • Pipeleers D.G.
      Glucose stimulates proinsulin biosynthesis by a dose-dependent recruitment of pancreatic beta cells.
      ). Lower Lys incorporation into proinsulin and ER stress of β-cells upon deletion of CDKAL1 could result in the misfolding of proinsulin (
      • Wei F.Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ). Proteins misfolded in the ER are retrotranslocated to the cytosol for proteasomal degradation (
      • Vembar S.S.
      • Brodsky J.L.
      One step at a time. Endoplasmic reticulum-associated degradation.
      ). By increasing the translation fidelity of mRNAs coding for proteins in transit through the ER, and thus minimizing misfolding and energy consumption involved in ER-associated protein degradation, CDKAL1 may be critical in professional secretory cells. Accordingly, lymphocytes also display high CDKAL1 expression levels (
      • Quaranta M.
      • Burden A.D.
      • Griffiths C.E.
      • Worthington J.
      • Barker J.N.
      • Trembath R.C.
      • Capon F.
      Differential contribution of CDKAL1 variants to psoriasis, Crohn's disease, and type II diabetes.
      ).
      The need for CDKAL1 may be more overt in conditions that place a high metabolic load on the ER and trigger a stress response. Indeed β-cell-restricted CDKAL1−/− mice showed UPR activation and profound glucose intolerance when fed a high fat diet (
      • Wei F.Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ). Here, we showed silencing CDKAL1 in INS-1 cells was sufficient to up-regulate the ER stress marker CHOP10. Intriguingly, we found that the CDKAL1 mRNA content was also enhanced by thapsigargin-induced ER stress, although the protein levels were unchanged. It is plausible that increased CDKAL1 transcription prompts the cell for more efficient translation, once the constraints imposed by the UPR are alleviated.
      Reduction of CDKAL1 expression in INS-1 cells further correlated with reduced expression of the granule precursor proteins proinsulin, pro-CGA, and pro-ICA512. As proposed for proinsulin (
      • Wei F.Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ), the lower levels of pro-CGA and pro-ICA512 could result from aberrant folding and processing consequent to incorrect Lys incorporation. Reduced expression of these granule proteins could account, at least in part, for the decreased insulin secretion of CDKAL1-depleted INS-1 (this study) and mouse β-cells (
      • Wei F.Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ). Alternatively, CDKAL1 may affect secretion by regulating β-cell processes other than insulin granule biogenesis. Islet cells of systemic (
      • Ohara-Imaizumi M.
      • Yoshida M.
      • Aoyagi K.
      • Saito T.
      • Okamura T.
      • Takenaka H.
      • Akimoto Y.
      • Nakamichi Y.
      • Takanashi-Yanobu R.
      • Nishiwaki C.
      • Kawakami H.
      • Kato N.
      • Hisanaga S.
      • Kakei M.
      • Nagamatsu S.
      Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis.
      ) and β-cell-restricted (
      • Wei F.Y.
      • Suzuki T.
      • Watanabe S.
      • Kimura S.
      • Kaitsuka T.
      • Fujimura A.
      • Matsui H.
      • Atta M.
      • Michiue H.
      • Fontecave M.
      • Yamagata K.
      • Suzuki T.
      • Tomizawa K.
      Deficit of tRNALys modification by Cdkal1 causes the development of type 2 diabetes in mice.
      ) CDKAL1−/− mice displayed lower ATP levels due to mitochondrial dysfunction. Reduced ATP generation could impair the function of KATP channels, which are key for depolarization of β-cells and insulin secretion. Thus, both β-cell ER stress and deficits in later stages of granule exocytosis could contribute to deficient insulin release in carriers of CDKAL1 alleles associated with an increased risk for T2D.
      In conclusion, elucidation of CDKAL1 regulation in normal and pathological conditions and identification of the protein subset that depends on CDKAL1 for efficient translation will help reveal how its genetic variants confer susceptibility to diabetes and other disorders, such as Crohn disease and psoriasis.

      Acknowledgments

      We are grateful to Barbara Ludwig, Anja Steffen, and Stefan Bornstein for providing human islets and Klaus Knoch for sharing RNA extracts. We thank Ramanujan S. Hegde (Medical Research Council, Cambridge, UK) and Stephen High (University of Manchester, UK) for the preprolactin and Sec61b-Nglyc constructs, respectively; Gert Kreibich (New York University School of Medicine), and Paul Hargrave (University of Florida, Gainsville), and Richard Zimmermann (Saarland University, Homburg, Germany) for the anti-ribophorin I, anti-opsin, and anti-Sec61a antibodies, respectively. We thank all members of the Solimena laboratory for advice and support.

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