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• W2973183998 abstract "•CDK8 is a negative regulator of insulin secretion•CDK8 regulates insulin secretion via OSBPL3 phosphorylation•CDK8 is required for postnatal silencing of NPY expression in β cells•CDK8 represses proapoptotic neuropeptides expression during metabolic stress Cyclin-dependent kinases (CDKs) contribute to vital cellular processes including cell cycle regulation. Loss of CDKs is associated with impaired insulin secretion and β cell survival; however, the function of CDK8 in β cells remains elusive. Here, we report that genetic ablation of Cdk8 improves glucose tolerance by increasing insulin secretion. We identify OSBPL3 as a CDK8-dependent phosphoprotein, which acts as a negative regulator of insulin secretion in response to glucose. We also show that embryonic gene silencing of neuropeptide Y in β cells is compromised in Cdk8-null mice, leading to continued expression into adulthood. Cdk8 ablation in β cells aggravates apoptosis and induces de novo expression of neuropeptides upon oxidative stress. Moreover, pancreatic islets exposed to stress display augmented apoptosis in the presence of these same neuropeptides. Our results reveal critical roles for CDK8 in β cell function and survival during metabolic stress that are in part mediated through de novo expression of neuropeptides. Cyclin-dependent kinases (CDKs) contribute to vital cellular processes including cell cycle regulation. Loss of CDKs is associated with impaired insulin secretion and β cell survival; however, the function of CDK8 in β cells remains elusive. Here, we report that genetic ablation of Cdk8 improves glucose tolerance by increasing insulin secretion. We identify OSBPL3 as a CDK8-dependent phosphoprotein, which acts as a negative regulator of insulin secretion in response to glucose. We also show that embryonic gene silencing of neuropeptide Y in β cells is compromised in Cdk8-null mice, leading to continued expression into adulthood. Cdk8 ablation in β cells aggravates apoptosis and induces de novo expression of neuropeptides upon oxidative stress. Moreover, pancreatic islets exposed to stress display augmented apoptosis in the presence of these same neuropeptides. Our results reveal critical roles for CDK8 in β cell function and survival during metabolic stress that are in part mediated through de novo expression of neuropeptides. Type 2 diabetes is a metabolic disease reaching epidemic proportions that primarily occurs as a result of obesity and lack of exercise. It is characterized by hyperglycemia and relative insulin deficiency (Alberti and Zimmet, 1998Alberti K.G. Zimmet P.Z. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation.Diabet. Med. 1998; 15: 539-553Google Scholar) due to impaired β cell function (Ashcroft and Rorsman, 2012Ashcroft F.M. Rorsman P. Diabetes mellitus and the β cell: the last ten years.Cell. 2012; 148: 1160-1171Google Scholar), β cell dedifferentiation (Talchai et al., 2012Talchai C. Xuan S. Lin H.V. Sussel L. Accili D. Pancreatic β cell dedifferentiation as a mechanism of diabetic β cell failure.Cell. 2012; 150: 1223-1234Google Scholar), and an imbalance of β cell apoptosis and proliferation (Bouwens and Rooman, 2005Bouwens L. Rooman I. Regulation of pancreatic beta-cell mass.Physiol. Rev. 2005; 85: 1255-1270Google Scholar). Thus, a better understanding of the molecular mechanisms underlying β cell function, differentiation, and survival may lead to new approaches to prevent or delay diabetes onset as well as improve therapies. Cyclin-dependent kinases (CDKs) are a family of multifunctional kinases that phosphorylate various protein substrates involved in critical cellular processes, most importantly the timing and coordination of cell cycle progression (Malumbres et al., 2009Malumbres M. Harlow E. Hunt T. Hunter T. Lahti J.M. Manning G. Morgan D.O. Tsai L.-H. Wolgemuth D.J. Cyclin-dependent kinases: a family portrait.Nat. Cell Biol. 2009; 11: 1275-1276Google Scholar). In the mouse and humans, a total of 20 CDKs and CDK-like proteins have been identified, all of which contain a conserved catalytic serine/threonine kinase domain regardless of molecular weight (Cao et al., 2014Cao L. Chen F. Yang X. Xu W. Xie J. Yu L. Phylogenetic analysis of CDK and cyclin proteins in premetazoan lineages.BMC Evol. Biol. 2014; 14: 10Google Scholar, Malumbres, 2014Malumbres M. Cyclin-dependent kinases.Genome Biol. 2014; 15: 122Google Scholar, Malumbres et al., 2009Malumbres M. Harlow E. Hunt T. Hunter T. Lahti J.M. Manning G. Morgan D.O. Tsai L.-H. Wolgemuth D.J. Cyclin-dependent kinases: a family portrait.Nat. Cell Biol. 2009; 11: 1275-1276Google Scholar). CDKs require the binding of their regulatory subunits (cyclins) to achieve full catalytic activity in controlling the cell cycle (Malumbres and Barbacid, 2005Malumbres M. Barbacid M. Mammalian cyclin-dependent kinases.Trends Biochem. Sci. 2005; 30: 630-641Google Scholar). Several different CDK complexes have been shown to control the progression of G1/S to G2/M, which is also associated with differential expression of CDKs (Bhaduri and Pryciak, 2011Bhaduri S. Pryciak P.M. Cyclin-specific docking motifs promote phosphorylation of yeast signaling proteins by G1/S Cdk complexes.Curr. Biol. 2011; 21: 1615-1623Google Scholar, Kõivomägi et al., 2011Kõivomägi M. Valk E. Venta R. Iofik A. Lepiku M. Morgan D.O. Loog M. Dynamics of Cdk1 substrate specificity during the cell cycle.Mol. Cell. 2011; 42: 610-623Google Scholar, Loog and Morgan, 2005Loog M. Morgan D.O. Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates.Nature. 2005; 434: 104-108Google Scholar, Pagliuca et al., 2011Pagliuca F.W. Collins M.O. Lichawska A. Zegerman P. Choudhary J.S. Pines J. Quantitative proteomics reveals the basis for the biochemical specificity of the cell-cycle machinery.Mol. Cell. 2011; 43: 406-417Google Scholar). Moreover, there is a considerable interest in developing CDK inhibitors as an approach to cancer therapy, as dysregulation of CDK activity has been linked to the development and progression of many tumor types (Malumbres and Barbacid, 2009Malumbres M. Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm.Nat. Rev. Cancer. 2009; 9: 153-166Google Scholar). For example, CDK4/6 inhibitor Palbociclib (PD-0332991) has been used for breast cancer treatment (Finn et al., 2015Finn R.S. Crown J.P. Lang I. Boer K. Bondarenko I.M. Kulyk S.O. Ettl J. Patel R. Pinter T. Schmidt M. et al.The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first-line treatment of oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 study.Lancet Oncol. 2015; 16: 25-35Google Scholar); Dinaciclib (SCH 727965), an inhibitor of multiple CDKs, shows antiproliferative activity in a broad spectrum of human tumor cell lines (Parry et al., 2010Parry D. Guzi T. Shanahan F. Davis N. Prabhavalkar D. Wiswell D. Seghezzi W. Paruch K. Dwyer M.P. Doll R. et al.Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinase inhibitor.Mol. Cancer Ther. 2010; 9: 2344-2353Google Scholar); CDKI-73, a CDK9 inhibitor, induces human leukemia cell apoptosis (Walsby et al., 2014Walsby E. Pratt G. Shao H. Abbas A.Y. Fischer P.M. Bradshaw T.D. Brennan P. Fegan C. Wang S. Pepper C. A novel Cdk9 inhibitor preferentially targets tumor cells and synergizes with fludarabine.Oncotarget. 2014; 5: 375-385Google Scholar); and the novel CDK8 inhibitor Senexin A increases the efficacy of chemotherapy against xenograft of tumors formed by cell/fibroblast mixtures (Porter et al., 2012Porter D.C. Farmaki E. Altilia S. Schools G.P. West D.K. Chen M. Chang B.-D. Puzyrev A.T. Lim C.U. Rokow-Kittell R. et al.Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities.Proc. Natl. Acad. Sci. USA. 2012; 109: 13799-13804Google Scholar). CDK4, CDK8, and CDK9 are among the highest expressed CDK in human islets (Taneera et al., 2013Taneera J. Fadista J. Ahlqvist E. Zhang M. Wierup N. Renström E. Groop L. Expression profiling of cell cycle genes in human pancreatic islets with and without type 2 diabetes.Mol. Cell. Endocrinol. 2013; 375: 35-42Google Scholar) and isolated β cells from human donors (Blodgett et al., 2015Blodgett D.M. Nowosielska A. Afik S. Pechhold S. Cura A.J. Kennedy N.J. Kim S. Kucukural A. Davis R.J. Kent S.C. et al.Novel observations from next-generation RNA sequencing of highly purified human adult and fetal islet cell subsets.Diabetes. 2015; 64: 3172-3181Google Scholar, Nica et al., 2013Nica A.C. Ongen H. Irminger J.C. Bosco D. Berney T. Antonarakis S.E. Halban P.A. Dermitzakis E.T. Cell-type, allelic, and genetic signatures in the human pancreatic beta cell transcriptome.Genome Res. 2013; 23: 1554-1562Google Scholar) and mice (Adriaenssens et al., 2016Adriaenssens A.E. Svendsen B. Lam B.Y. Yeo G.S. Holst J.J. Reimann F. Gribble F.M. Transcriptomic profiling of pancreatic alpha, beta and delta cell populations identifies delta cells as a principal target for ghrelin in mouse islets.Diabetologia. 2016; 59: 2156-2165Google Scholar, DiGruccio et al., 2016DiGruccio M.R. Mawla A.M. Donaldson C.J. Noguchi G.M. Vaughan J. Cowing-Zitron C. van der Meulen T. Huising M.O. Comprehensive alpha, beta and delta cell transcriptomes reveal that ghrelin selectively activates delta cells and promotes somatostatin release from pancreatic islets.Mol. Metab. 2016; 5: 449-458Google Scholar). Over the past decades, several studies have reported the involvement of CDKs in various metabolic processes of pancreatic β cells. In vivo, CDKs regulate both β cell proliferation and insulin secretion. For example, CDK4 knockout mice fail to undergo postnatal β cell expansion, leading to β cell hypoplasia (Rane et al., 1999Rane S.G. Dubus P. Mettus R.V. Galbreath E.J. Boden G. Reddy E.P. Barbacid M. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.Nat. Genet. 1999; 22: 44-52Google Scholar). Furthermore, inhibition of CDK4 results in decreased expression of Kir6.2, impaired insulin secretion, and glucose intolerance in mice (Annicotte et al., 2009Annicotte J.-S. Blanchet E. Chavey C. Iankova I. Costes S. Assou S. Teyssier J. Dalle S. Sardet C. Fajas L. The CDK4-pRB-E2F1 pathway controls insulin secretion.Nat. Cell Biol. 2009; 11: 1017-1023Google Scholar). Mice with β cell-specific genetic deletion of CDK2 display glucose intolerance primarily due to defects in mitochondrial function and glucose-stimulated insulin secretion (Kim et al., 2017Kim S.Y. Lee J.-H. Merrins M.J. Gavrilova O. Bisteau X. Kaldis P. Satin L.S. Rane S.G. Loss of cyclin-dependent kinase 2 in the pancreas links primary β-cell dysfunction to progressive depletion of β-cell mass and diabetes.J. Biol. Chem. 2017; 292: 3841-3853Google Scholar). Moreover, CDK5 inhibition has been shown to increase apoptosis in INS 832/13 cells (Daval et al., 2011Daval M. Gurlo T. Costes S. Huang C.J. Butler P.C. Cyclin-dependent kinase 5 promotes pancreatic β-cell survival via Fak-Akt signaling pathways.Diabetes. 2011; 60: 1186-1197Google Scholar), and overexpression of CDK6 in human β cells promotes β cell replication (Fiaschi-Taesch et al., 2010Fiaschi-Taesch N.M. Salim F. Kleinberger J. Troxell R. Cozar-Castellano I. Selk K. Cherok E. Takane K.K. Scott D.K. Stewart A.F. Induction of human β-cell proliferation and engraftment using a single G1/S regulatory molecule, cdk6.Diabetes. 2010; 59: 1926-1936Google Scholar). CDK8 is a key member of the CDK family and is a component of the Mediator complex, which directly interacts with RNA polymerase II through its large subunit’s C-terminal domain (CTD) to regulate gene transcription (Cai et al., 2009Cai G. Imasaki T. Takagi Y. Asturias F.J. Mediator structural conservation and implications for the regulation mechanism.Structure. 2009; 17: 559-567Google Scholar, Nemet et al., 2014Nemet J. Jelicic B. Rubelj I. Sopta M. The two faces of Cdk8, a positive/negative regulator of transcription.Biochimie. 2014; 97: 22-27Google Scholar, Tsai et al., 2013Tsai K.-L. Sato S. Tomomori-Sato C. Conaway R.C. Conaway J.W. Asturias F.J. A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction.Nat. Struct. Mol. Biol. 2013; 20: 611-619Scopus (111) Google Scholar). Investigation of CDK8 has mainly focused on cancer development and progression, whereby it has been shown that CDK8 can regulate multiple oncogenic pathways, including β-catenin, TGF-β, and p53 signaling (Alarcón et al., 2009Alarcón C. Zaromytidou A.-I. Xi Q. Gao S. Yu J. Fujisawa S. Barlas A. Miller A.N. Manova-Todorova K. Macias M.J. et al.Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-β pathways.Cell. 2009; 139: 757-769Google Scholar, Donner et al., 2007Donner A.J. Szostek S. Hoover J.M. Espinosa J.M. CDK8 is a stimulus-specific positive coregulator of p53 target genes.Mol. Cell. 2007; 27: 121-133Google Scholar, Firestein et al., 2008Firestein R. Bass A.J. Kim S.Y. Dunn I.F. Silver S.J. Guney I. Freed E. Ligon A.H. Vena N. Ogino S. et al.CDK8 is a colorectal cancer oncogene that regulates β-catenin activity.Nature. 2008; 455: 547-551Google Scholar). Surprisingly, inducible-Cre-mediated ablation of Cdk8 in young mice revealed no gross or histopathological defects in many tissues expressing Cdk8 (McCleland et al., 2015McCleland M.L. Soukup T.M. Liu S.D. Esensten J.H. de Sousa e Melo F. Yaylaoglu M. Warming S. Roose-Girma M. Firestein R. Cdk8 deletion in the Apc(Min) murine tumour model represses EZH2 activity and accelerates tumourigenesis.J. Pathol. 2015; 237: 508-519Google Scholar). In the context of metabolism, Zhao et al., 2012Zhao X. Feng D. Wang Q. Abdulla A. Xie X.-J. Zhou J. Sun Y. Yang E.S. Liu L.-P. Vaitheesvaran B. et al.Regulation of lipogenesis by cyclin-dependent kinase 8-mediated control of SREBP-1.J. Clin. Invest. 2012; 122: 2417-2427Google Scholar revealed through transient CDK8 knockdown in mouse liver that CDK8 plays an essential role in lipogenesis; however, it remains unknown whether CDK8 is also involved in glucose homeostasis. In this study, we generated conditional knockout mice of Cdk8 in pancreatic β cells and investigated its effects on cell function and survival under physiological and metabolic stress conditions. We discovered that mice lacking Cdk8 exhibit improved glucose tolerance due to elevated insulin secretion, an outcome that is distinctive among all studied Cdks and that is likely mediated in part by its target Oxysterol-binding protein related protein-3 (OSBPL3). Furthermore, we demonstrate that CDK8-deficient β cells express a defined set of neuroendocrine secretory proteins that, under metabolic stress conditions, contribute to β cell demise. To explore the function of CDK8 in mouse pancreatic β cells, we generated a conditional Cdk8 allele in which exon 5 was flanked with loxP sites (Figures S1A and S1B). Cdk8fl/fl mice were intercrossed with Rip-Cre mice to generate mice with β cell-specific deletion of Cdk8 (called Rip-Cre Cdk8fl/fl). Mice lacking Cdk8 in the entire pancreas were generated by crossing Cdk8fl/fl mice with Pdx1-Cre mice (Pdx1-Cre Cdk8fl/fl). A profound reduction in CDK8 protein of ≈90% in Rip-Cre Cdk8fl/fl and Pdx1-Cre Cdk8fl/fl mice compared to Cdk8fl/fl control mice was measured by immunoblot analysis (Figures S1C and S1D). The Rip-Cre-dependent ablation was β cell-specific, with no evidence of Cre-mediated recombination in the hypothalamus (Figure S1B). To investigate the physiological consequences of CDK8 ablation in pancreatic β cells, we performed intraperitoneal glucose tolerance tests (IPGTTs). Surprisingly, Rip-Cre Cdk8fl/fl and Pdx1-Cre Cdk8fl/fl mice fed a normal diet displayed augmented glucose tolerance (Figures 1A and S1E). To further understand this improvement, we measured circulating insulin in Rip-Cre Cdk8fl/fl mice during the glucose challenge. Plasma insulin levels were increased in Rip-Cre Cdk8fl/fl mice at 2 and 5 min, but not at later time points (15 and 30 min) compared with Cdk8fl/fl control mice (Figures 1B and 1C). Similar results were obtained in Pdx1-Cre Cdk8fl/fl mice (Figure S1F). However, insulin sensitivity (Figure 1D) and insulin content (Figure 1E) remained unchanged. Moreover, β cell mass and islet morphology in Rip-Cre Cdk8fl/fl were indistinguishable from control mice (Figures 1F and 1G). Transgenic Rip-Cre mice exhibited normal blood glucose levels during IPGTT and intraperitoneal insulin tolerance test (IPITT), excluding the possibility that the phenotypic effects resulted from expression of the Cre-transgene (Figures S1G and S1H). We next stressed Rip-Cre Cdk8fl/fl mice with a high-fat diet (HFD) (20% protein, 36% lipids, and 36.7% carbohydrate) for 12 weeks and observed improved IPGTT and insulin secretion, and unchanged IPITT tests, consistent with the observations in chow-fed mice (Figures S1I–S1K). Furthermore, food intake, energy expenditure, random-fed blood glucose, and body weight of HFD fed mice were measured and found to be similar in Rip-Cre Cdk8fl/fl and control mice (Figures S1L–S1P). To study the effects of CDK8 overexpression in pancreatic β cells, we also generated transgenic mice (Tg/+) expressing Cdk8 under the control of the Rip-promoter (Figure S1Q). Although CDK8 levels in Ins-CDK8 mice were increased ≈5-fold (Figure S1R), this did not affect glucose tolerance (Figure S1S) or pancreatic insulin content (Figure S1T). Together, these data demonstrate that loss of Cdk8 in β cells improves glucose tolerance by increasing first-phase insulin secretion, consistent with the importance of early insulin release for the control of glucose tolerance (Del Prato and Tiengo, 2001Del Prato S. Tiengo A. The importance of first-phase insulin secretion: implications for the therapy of type 2 diabetes mellitus.Diabetes Metab. Res. Rev. 2001; 17: 164-174Google Scholar), and that Cdk8 expression is not limiting. Protein phosphorylation is recognized as the major mechanism by which intracellular events in mammalian tissues are controlled by external physiological stimuli. In islets, glucose-stimulated insulin secretion (GSIS) follows an oscillatory pattern with a period of 3–6 min, which is generated through the pulsatile release of insulin by pancreatic β cells (Rorsman et al., 2000Rorsman P. Eliasson L. Renström E. Gromada J. Barg S. Göpel S. The cell physiology of biphasic insulin secretion.News Physiol. Sci. 2000; 15: 72-77Google Scholar). The rapid timescale of these events implies regulation by fast post-translational modifications of proteins, acting through intracellular signal transduction pathways rather than by manipulation of gene expression (Chen et al., 2012Chen X.-Y. Gu X.-T. Saiyin H. Wan B. Zhang Y.-J. Li J. Wang Y.-L. Gao R. Wang Y.-F. Dong W.-P. et al.Brain-selective kinase 2 (BRSK2) phosphorylation on PCTAIRE1 negatively regulates glucose-stimulated insulin secretion in pancreatic β-cells.J. Biol. Chem. 2012; 287: 30368-30375Google Scholar, Nesher et al., 2002Nesher R. Anteby E. Yedovizky M. Warwar N. Kaiser N. Cerasi E. Beta-cell protein kinases and the dynamics of the insulin response to glucose.Diabetes. 2002; 51: S68-S73Google Scholar, Sacco et al., 2016Sacco F. Humphrey S.J. Cox J. Mischnik M. Schulte A. Klabunde T. Schäfer M. Mann M. Glucose-regulated and drug-perturbed phosphoproteome reveals molecular mechanisms controlling insulin secretion.Nat. Commun. 2016; 7: 13250Google Scholar). As a protein kinase that regulates insulin secretion in vivo, CDK8 is a candidate for mediating the phosphoproteomic response to glucose stimulation. We therefore applied an unbiased mass spectrometry (MS)-based phosphoproteomic approach to ex vivo cultured islets from Rip-Cre Cdk8fl/fl and control Cdk8fl/fl mice, enabling exploration of the global phosphoproteomic changes that are dependent on CDK8 and may influence in insulin secretion. Considering that insulin secretion in Rip-Cre Cdk8fl/fl mice was increased in the early phases following glucose stimulation, we stimulated isolated islets from Rip-Cre Cdk8fl/fl and control Cdk8fl/fl littermates for 5 and 10 min with high- and low-glucose concentrations prior to an unbiased determination of phosphoproteomic changes using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS). Indeed, we measured, both qualitatively and quantitatively, more phosphorylation-regulated peptides in response to short-term, high-glucose culture conditions, although prolonged (1-h) exposure of Rip-Cre Cdk8fl/fl islets with high or low glucose also revealed mild regulation of the phosphoproteome (p < 0.05; fold change ≥ 2) (Figure 2A; Table S1). The decrease in phosphorylation events in Rip-Cre Cdk8fl/fl islets is consistent with a loss of kinase activity and with the in vivo phenotype in which the strongest effect of CDK8 on insulin secretion occurred within minutes after glucose injection (Figure 1B). Proteins of Rip-Cre Cdk8fl/fl versus Cdk8fl/fl islets with downregulated phosphorylation events in both the 5- and 10-min datasets mostly localized to the cytoplasm (Figure 2B; Table S1), indicating their potential participation in the insulin secretion pathway. Among these proteins, PCLO, RPH3AL, CADPS, and NFATC2 have previously been shown to affect GSIS (Fujimoto et al., 2002Fujimoto K. Shibasaki T. Yokoi N. Kashima Y. Matsumoto M. Sasaki T. Tajima N. Iwanaga T. Seino S. Piccolo, a Ca2+ sensor in pancreatic β-cells. Involvement of cAMP-GEFII.Rim2. Piccolo complex in cAMP-dependent exocytosis.J. Biol. Chem. 2002; 277: 50497-50502Google Scholar, Keller et al., 2016Keller M.P. Paul P.K. Rabaglia M.E. Stapleton D.S. Schueler K.L. Broman A.T. Ye S.I. Leng N. Brandon C.J. Neto E.C. et al.The transcription factor Nfatc2 regulates β-cell proliferation and genes associated with type 2 diabetes in mouse and human islets.PLoS Genet. 2016; 12: e1006466Google Scholar, Matsumoto et al., 2004Matsumoto M. Miki T. Shibasaki T. Kawaguchi M. Shinozaki H. Nio J. Saraya A. Koseki H. Miyazaki M. Iwanaga T. Seino S. Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells.Proc. Natl. Acad. Sci. USA. 2004; 101: 8313-8318Google Scholar, Speidel et al., 2008Speidel D. Salehi A. Obermueller S. Lundquist I. Brose N. Renström E. Rorsman P. CAPS1 and CAPS2 regulate stability and recruitment of insulin granules in mouse pancreatic β cells.Cell Metab. 2008; 7: 57-67Google Scholar). Together, our findings imply that CDK8 participates in the GSIS signaling pathways in pancreatic β cells by affecting downstream protein phosphorylation. We next reasoned that silencing of putative CDK8 targets may partially mimic the dephosphorylation status of these proteins. To characterize which differentially phosphorylated proteins impact GSIS, we knocked down the top-ranked 21 candidates (from Table S2) using small interfering RNAs (siRNAs) in low-passage INS1E cells and measured insulin secretion in response to a glucose or other insulin secretagogues. A ≈90% mRNA knockdown of Osbpl3 (the candidate with greatest fold change in both 5- and 10-min datasets) led to increased insulin secretion following stimulation with glucose but not arginine (Figures 2C, 2D, and S2A). Moreover, we also stimulated islets from Rip-Cre Cdk8fl/fl and control Cdk8fl/fl mice with arginine and other regulators of the insulin secretion pathway (diazoxide, tolbutamide) that affect depolarization of the membrane potential; however, neither of these compounds affected insulin secretion (Figure S2B). Furthermore, OSBPL3 was the only putative CDK8 target where downregulation stimulated insulin secretion (whereas silencing of other targets such Rab effector Noc2 [Rph3al] inhibited insulin secretion). No other proteins consistently affected GSIS following knockdown of the corresponding transcript (Figure S2C). Mass spectrometry analysis of OSBPL3 revealed one threonine (T248) and four serine residues (S250, S264, S271, and S272) to be dramatically dephosphorylated in islets of Rip-Cre Cdk8fl/fl versus control mice in response to 5 or 10 min of high-glucose stimulation, and moderately dephosphorylated after prolonged glucose stimulation (1 h) (Figure 2E). Interestingly, we found a complete CDK consensus sequence (S/T–P–X–R/K) (Songyang et al., 1994Songyang Z. Blechner S. Hoagland N. Hoekstra M.F. Piwnica-Worms H. Cantley L.C. Use of an oriented peptide library to determine the optimal substrates of protein kinases.Curr. Biol. 1994; 4: 973-982Google Scholar) at a conserved serine-264 residue of OSBPL3 (S264–P–K–K), thereby providing further support that OSBPL3 is a direct target of CDK8. We next generated an OSBPL3 phosphorylation-specific antibody against S272 (Osbpl3-S272) to validate the phosphorylation changes in OSBPL3 in the absence of CDK8. We used this affinity-purified antibody in western blots of isolated islets cultured in high glucose for 5 min, revealing decreased phospho-OSBPL3-specific signal in islets of Rip-Cre Cdk8fl/fl compared to Cdk8fl/fl control mice (Figure 2F). In order to investigate whether glucose per se can regulate the phosphorylation of OSBPL3, we treated islets from wild-type animals in low and high glucose. No change in the phosphorylation of OSBPL3 (S272) was observed, demonstrating that the regulation of OSBPL3 phosphorylation is CDK8-dependent but not influenced by glucose (Figure S2D). Furthermore, cell fractionation/immunoblot analysis and immunofluorescence staining demonstrated that CDK8 and OSBPL3 both localized predominantly to the cytoplasm (Figures 2G and 2H). The above findings suggest that OSBPL3 may act as a sensor of CDK8 kinase activity in β cells and that its phosphorylation status may regulate insulin secretion. We first showed that CDK8 is also a negative regulator of insulin secretion in human islets and INS1E cells by pharmacological CDK8 inhibition or silencing CDK8 transcripts by RNAi, respectively, leading to increased insulin secretion in human and rat cells (Figures 2I and S2E). Next, we generated expression plasmids harboring wild-type, “mimic” and “dead” mutations by substituting the phosphorylated amino acids in OSBPL3 with aspartic acid or alanine, respectively. These expression constructs were transfected in INS1E cells and analyzed following glucose stimulation. We found that under 16.7 mM glucose stimulation, wild-type Osbpl3 overexpression caused a 50% decrease in insulin secretion compared to the control (empty vector transfection). The unphosphorylated form of Osbpl3 (Dead), however, was unable to repress insulin secretion, despite equal protein overexpression (Figures 2J and 2K). Unexpectedly, the “mimic” mutant did not inhibit but rather exhibited similar GSIS to “WT.” However, aspartic acid phosphomimic mutants frequently do not mirror the phosphorylated functional state of a protein, which is likely due to differences in charge densities, distributions and pKas of the carboxyl group of aspartic acid, that are quite different from a phosphate group (Dissmeyer and Schnittger, 2011Dissmeyer N. Schnittger A. Use of phospho-site substitutions to analyze the biological relevance of phosphorylation events in regulatory networks.Methods Mol. Biol. 2011; 779: 93-138Google Scholar). Together, these results indicate that the ability of OSBPL3 to regulate GSIS depends on its phosphorylation status. In contrast, when endogenous CDK8 levels were reduced by siRNAs prior to Osbpl3 overexpression, we found that the wild-type and OSBPL3 dead mutant markedly stimulated GSIS and that wild-type OSBPL3 did not inhibit insulin secretion under these conditions (Figure 2L). These results are all consistent with a scenario where regulation of GSIS is in part mediated by OSBPL3 under the upstream influence of CDK8. In summary, our results support a model by which CDK8-dependent phosphorylation of OSBPL3 at multiple serine positions desensitizes β cells to glucose-stimulated insulin secretion. CDK8 can be associated with the Mediator complex that has been reported to play indispensable roles in transcriptional regulation from yeast to mammals (Donner et al., 2010Donner A.J. Ebmeier C.C. Taatjes D.J. Espinosa J.M. CDK8 is a positive regulator of transcriptional elongation within the serum response network.Nat. Struct. Mol. Biol. 2010; 17: 194-201Google Scholar, Hirst et al., 1999Hirst M. Kobor M.S. Kuriakose N. Greenblatt J. Sadowski I. GAL4 is regulated by the RNA polymerase II holoenzyme-associated cyclin-dependent protein kinase SRB10/CDK8.Mol. Cell. 1999; 3: 673-678Google Scholar, Nelson et al., 2003Nelson C. Goto S. Lund K. Hung W. Sadowski I. Srb10/Cdk8 regulates yeast filamentous growth by phosphorylating the transcription factor Ste12.Nature. 2003; 421: 187-190Google Scholar, Raithatha et al., 2012Raithatha S. Su T.-C. Lourenco P. Goto S. Sadowski I. Cdk8 regulates stability of the transcription factor Phd1 to control pseudohyphal differentiation of Saccharomyces cerevisiae.Mol. Cell. Biol. 2012; 32: 664-674Google Scholar, Zhao et al., 2012Zhao X. Feng D. Wang Q. Abdulla A. Xie X.-J. Zhou J. Sun Y. Yang E.S. Liu L.-P. Vaitheesvaran B. et al.Regulation of lipogenesis by cyclin-dependent kinase 8-mediated control of SREBP-1.J. Clin. Invest. 2012; 122: 2417-2427Google Scholar). To investigate the consequence of Cdk8 ablation on genome-wide transcript levels, we performed RNA sequencing on isolated islets. Surprisingly, analysis of islets lacking Cdk8 revealed only a small number of genes that exhibited altered expression levels, with 27 upregulated and 3 downregulated genes compared to islets of Cdk8fl/fl control mice (false-discovery rate [FDR] < 0.01; fold change [FC] ≥ 2) (Figure 3A). Specifically, expression of neuropeptide Y (NPY) was found t" @default.
• W2973183998 created "2019-09-19" @default.
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• W2973183998 creator A5020062473 @default.
• W2973183998 creator A5081825319 @default.
• W2973183998 date "2019-09-01" @default.
• W2973183998 modified "2023-10-14" @default.
• W2973183998 title "CDK8 Regulates Insulin Secretion and Mediates Postnatal and Stress-Induced Expression of Neuropeptides in Pancreatic β Cells" @default.
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• W2973183998 doi "https://doi.org/10.1016/j.celrep.2019.08.025" @default.
• W2973183998 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/31509750" @default.
• W2973183998 hasPublicationYear "2019" @default.
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