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. 2014 Feb 13;30(3):497–504. doi: 10.1007/s12264-013-1403-2

Protein kinase D: a new player among the signaling proteins that regulate functions in the nervous system

Gang Li 1, Yun Wang 1,2,✉
PMCID: PMC5562605  PMID: 24526660

Abstract

Protein kinase D (PKD) is an evolutionarily-conserved family of protein kinases. It has structural, regulatory, and enzymatic properties quite different from the PKC family. Many stimuli induce PKD signaling, including G-protein-coupled receptor agonists and growth factors. PKD1 is the most studied member of the family. It functions during cell proliferation, differentiation, secretion, cardiac hypertrophy, immune regulation, angiogenesis, and cancer. Previously, we found that PKD1 is also critically involved in pain modulation. Since then, a series of studies performed in our lab and by other groups have shown that PKDs also participate in other processes in the nervous system including neuronal polarity establishment, neuroprotection, and learning. Here, we discuss the connections between PKD structure, enzyme function, and localization, and summarize the recent findings on the roles of PKD-mediated signaling in the nervous system.

Keywords: PKD, neuronal polarity, pain modulation, neuroprotection, learning

References

  • [1].Rozengurt E, Rey O, Waldron RT. Protein kinase D signaling. J Biol Chem. 2005;280:13205–13208. doi: 10.1074/jbc.R500002200. [DOI] [PubMed] [Google Scholar]
  • [2].Hayashi A, Seki N, Hattori A, Kozuma S, Saito T. PKC, a new member of the protein kinase C family, composes a fourth subfamily with PKCμ. Biochim Biophys Acta. 1999;1450:99–106. doi: 10.1016/S0167-4889(99)00040-3. [DOI] [PubMed] [Google Scholar]
  • [3].Nishikawa K, Toker A, Johannes FJ, Songyang Z, Cantley LC. Determination of the specific substrate sequence motifs of protein kinase C isozymes. J Biol Chem. 1997;272:952–960. doi: 10.1074/jbc.272.2.952. [DOI] [PubMed] [Google Scholar]
  • [4].Valverde AM, Sinnett-Smith J, Van Lint J, Rozengurt E. Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain. Proc Natl Acad Sci U S A. 1994;91:8572–8576. doi: 10.1073/pnas.91.18.8572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Liljedahl M, Maeda Y, Colanzi A, Ayala I, Van Lint J, Malhotra V. Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network. Cell. 2001;104:409–420. doi: 10.1016/S0092-8674(01)00228-8. [DOI] [PubMed] [Google Scholar]
  • [6].Yeaman C, Ayala MI, Wright JR, Bard F, Bossard C, Ang A, et al. Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network. Nat Cell Biol. 2004;6:106–112. doi: 10.1038/ncb1090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Johannes FJ, Prestle J, Eis S, Oberhagemann P, Pfizenmaier K. PKCμ is a novel, atypical member of the protein kinase C family. J Biol Chem. 1994;269:6140–6148. [PubMed] [Google Scholar]
  • [8].Newton AC. Regulation of protein kinase C. Curr Opin Cell Biol. 1997;9:161–167. doi: 10.1016/S0955-0674(97)80058-0. [DOI] [PubMed] [Google Scholar]
  • [9].Mellor H, Parker PJ. The extended protein kinase C superfamily. Biochem J. 1998;332:281. doi: 10.1042/bj3320281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Sturany S, Van Lint J, Müller F, Wilda M, Hameister H, Höcker M, et al. Molecular cloning and characterization of the human protein kinase D2 a novel member of the protein kinase D family of serine threonine kinases. J Biol Chem. 2001;276:3310–3318. doi: 10.1074/jbc.M008719200. [DOI] [PubMed] [Google Scholar]
  • [11].Hanks SK. Genomic analysis of the eukaryotic protein kinase superfamily: a perspective. Genome Biol. 2003;4:111. doi: 10.1186/gb-2003-4-5-111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Johnson LN, Lowe ED, Noble ME, Owen DJ. The structural basis for substrate recognition and control by protein kinases. FEBS Lett. 1998;430:1–11. doi: 10.1016/S0014-5793(98)00606-1. [DOI] [PubMed] [Google Scholar]
  • [13].Vertommen D, Rider M, Ni Y, Waelkens E, Merlevede W, Vandenheede JR, et al. Regulation of protein kinase D by multisite phosphorylation identification of phosphorylation sites by mass spectrometry and characterization by sitedirected mutagenesis. J Biol Chem. 2000;275:19567–19576. doi: 10.1074/jbc.M001357200. [DOI] [PubMed] [Google Scholar]
  • [14].Iglesias T, Waldron RT, Rozengurt E. Identification of in vivo phosphorylation sites required for protein kinase D activation. J Biol Chem. 1998;273:27662–27667. doi: 10.1074/jbc.273.42.27662. [DOI] [PubMed] [Google Scholar]
  • [15].Matthews SA, Rozengurt E, Cantrell D. Characterization of serine 916 as an in vivo autophosphorylation site for protein kinase D/protein kinase Cμ. J Biol Chem. 1999;274:26543–26549. doi: 10.1074/jbc.274.37.26543. [DOI] [PubMed] [Google Scholar]
  • [16].Waldron RT, Rey O, Iglesias T, Tugal T, Cantrell D, Rozengurt E. Activation loop Ser744 and Ser748 in protein kinase D are transphosphorylated in vivo. J Biol Chem. 2001;276:32606–32615. doi: 10.1074/jbc.M101648200. [DOI] [PubMed] [Google Scholar]
  • [17].Waldron RT, Rozengurt E. Protein kinase C phosphorylates protein kinase D activation loop Ser744 and Ser748 and releases autoinhibition by the pleckstrin homology domain. J Biol Chem. 2003;278:154–163. doi: 10.1074/jbc.M208075200. [DOI] [PubMed] [Google Scholar]
  • [18].Brändlin I, Hübner S, Eiseler T, Martinez-Moya M, Horschinek A, Hausser A, et al. Protein kinase C (PKC) η-mediated PKCμ activation modulates ERK and JNK signal pathways. J Biol Chem. 2002;277:6490–6496. doi: 10.1074/jbc.M106083200. [DOI] [PubMed] [Google Scholar]
  • [19].Waldron RT, Iglesias T, Rozengurt E. The pleckstrin homology domain of protein kinase D interacts preferentially with the η isoform of protein kinase C. J Biol Chem. 1999;274:9224–9230. doi: 10.1074/jbc.274.14.9224. [DOI] [PubMed] [Google Scholar]
  • [20].Jamora C, Yamanouye N, Van Lint J, Laudenslager J, Vandenheede JR, Faulkner DJ, et al. Gβγ-mediated regulation of Golgi organization is through the direct activation of protein kinase D. Cell. 1999;98:59–68. doi: 10.1016/S0092-8674(00)80606-6. [DOI] [PubMed] [Google Scholar]
  • [21].Añel AMD, Malhotra V. PKCη is required for β1γ2/β3γ2- and PKD-mediated transport to the cell surface and the organization of the Golgi apparatus. J Cell Biol. 2005;169:83–91. doi: 10.1083/jcb.200412089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Rey O, Sinnett-Smith J, Zhukova E, Rozengurt E. Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation. J Biol Chem. 2001;276:49228–49235. doi: 10.1074/jbc.M109395200. [DOI] [PubMed] [Google Scholar]
  • [23].Endo K, Oki E, Biedermann V, Kojima H, Yoshida K, Johannes FJ, et al. Proteolytic cleavage and activation of protein kinase C μ by caspase-3 in the apoptotic response of cells to 1-β-d-arabinofuranosylcytosine and other genotoxic agents. J Biol Chem. 2000;275:18476–18481. doi: 10.1074/jbc.M002266200. [DOI] [PubMed] [Google Scholar]
  • [24].Iglesias T, Rozengurt E. Protein kinase D activation by mutations within its pleckstrin homology domain. J Biol Chem. 1998;273:410–416. doi: 10.1074/jbc.273.1.410. [DOI] [PubMed] [Google Scholar]
  • [25].Storz P, Döppler H, Johannes FJ, Toker A. Tyrosine phosphorylation of protein kinase D in the pleckstrin homology domain leads to activation. J Biol Chem. 2003;278:17969–17976. doi: 10.1074/jbc.M213224200. [DOI] [PubMed] [Google Scholar]
  • [26].Iglesias T, Rozengurt E. Protein kinase D activation by deletion of its cysteine-rich motifs. FEBS Lett. 1999;454:53–56. doi: 10.1016/S0014-5793(99)00772-3. [DOI] [PubMed] [Google Scholar]
  • [27].Iglesias T, Matthews S, Rozengurt E. Dissimilar phorbol ester binding properties of the individual cysteine-rich motifs of protein kinase D. FEBS Lett. 1998;437:19–23. doi: 10.1016/S0014-5793(98)01189-2. [DOI] [PubMed] [Google Scholar]
  • [28].Storz P, Hausser A, Link G, Dedio J, Ghebrehiwet B, Pfizenmaier K, et al. Protein kinase C μ is regulated by the multifunctional chaperon protein p32. J Biol Chem. 2000;275:24601–24607. doi: 10.1074/jbc.M002964200. [DOI] [PubMed] [Google Scholar]
  • [29].Matthews SA, Iglesias T, Rozengurt E, Cantrell D. Spatial and temporal regulation of protein kinase D (PKD) EMBO J. 2000;19:2935–2945. doi: 10.1093/emboj/19.12.2935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Hausser A, Link G, Bamberg L, Burzlaff A, Lutz S, Pfizenmaier K, et al. Structural requirements for localization and activation of protein kinase C μ (PKCμ) at the Golgi compartment. J Cell Biol. 2002;156:65–74. doi: 10.1083/jcb.200110047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Rey O, Young SH, Cantrell D, Rozengurt E. Rapid protein kinase D translocation in response to G protein-coupled receptor activation dependence on protein kinase C. J Biol Chem. 2001;276:32616–32626. doi: 10.1074/jbc.M101649200. [DOI] [PubMed] [Google Scholar]
  • [32].Matthews S, Iglesias T, Cantrell D, Rozengurt E. Dynamic redistribution of protein kinase D (PKD) as revealed by a GFPPKD fusion protein: dissociation from PKD activation. FEBS Lett. 1999;457:515–521. doi: 10.1016/S0014-5793(99)01090-X. [DOI] [PubMed] [Google Scholar]
  • [33].Maeda Y, Beznoussenko GV, Van Lint J, Mironov AA, Malhotra V. Recruitment of protein kinase D to the trans-Golgi network via the first cysteine-rich domain. EMBO J. 2001;20:5982–5990. doi: 10.1093/emboj/20.21.5982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Ghanekar Y, Lowe M. Protein kinase D: activation for Golgi carrier formation. Trends Cell Biol. 2005;15:511–514. doi: 10.1016/j.tcb.2005.08.001. [DOI] [PubMed] [Google Scholar]
  • [35].Baron CL, Malhotra V. Role of diacylglycerol in PKD recruitment to the TGN and protein transport to the plasma membrane. Science. 2002;295:325–328. doi: 10.1126/science.1066759. [DOI] [PubMed] [Google Scholar]
  • [36].Pfeffer S. Membrane domains in the secretory and endocytic pathways. Cell. 2003;112:507–517. doi: 10.1016/S0092-8674(03)00118-1. [DOI] [PubMed] [Google Scholar]
  • [37].Wang QJ. PKD at the crossroads of DAG and PKC signaling. Trends Pharmacol Sci. 2006;27:317. doi: 10.1016/j.tips.2006.04.003. [DOI] [PubMed] [Google Scholar]
  • [38].Bossard C, Bresson D, Polishchuk RS, Malhotra V. Dimeric PKD regulates membrane fission to form transport carriers at the TGN. J Cell Biol. 2007;179:1123–1131. doi: 10.1083/jcb.200703166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Oancea E, Bezzerides VJ, Greka A, Clapham DE. Mechanism of persistent protein kinase D1 translocation and activation. Dev Cell. 2003;4:561–574. doi: 10.1016/S1534-5807(03)00087-X. [DOI] [PubMed] [Google Scholar]
  • [40].Auer A, von Blume J, Sturany S, von Wichert G, Van Lint J, Vandenheede J, et al. Role of the regulatory domain of protein kinase D2 in phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling. Mol Biol Cell. 2005;16:4375–4385. doi: 10.1091/mbc.E05-03-0251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Irie K, Nakahara A, Ohigashi H, Fukuda H, Wender PA, Konishi H, et al. Synthesis and phorbol ester-binding studies of the individual cysteine-rich motifs of protein kinase D. Bioorg Med Chem Lett. 1999;9:2487–2490. doi: 10.1016/S0960-894X(99)00413-8. [DOI] [PubMed] [Google Scholar]
  • [42].Van Lint J, Rykx A, Maeda Y, Vantus T, Sturany S, Malhotra V, et al. Protein kinase D: an intracellular traffic regulator on the move. Trends Cell Biol. 2002;12:193–200. doi: 10.1016/S0962-8924(02)02262-6. [DOI] [PubMed] [Google Scholar]
  • [43].Rey O, Rozengurt E. Protein kinase D interacts with Golgi via its cysteine-rich domain. Biochem Biophys Res Commun. 2001;287:21–26. doi: 10.1006/bbrc.2001.5530. [DOI] [PubMed] [Google Scholar]
  • [44].Szallasi A, Cortright DN, Blum CA, Eid SR. The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept. Nat Rev Drug Discov. 2007;6:357–372. doi: 10.1038/nrd2280. [DOI] [PubMed] [Google Scholar]
  • [45].Immke DC, Gavva NR. The TRPV1 receptor and nociception. Semin Cell Dev Biol. 2006;17:582. doi: 10.1016/j.semcdb.2006.09.004. [DOI] [PubMed] [Google Scholar]
  • [46].Cortright DN, Szallasi A. Biochemical pharmacology of the vanilloid receptor TRPV1. Eur J Biochem. 2004;271:1814–1819. doi: 10.1111/j.1432-1033.2004.04082.x. [DOI] [PubMed] [Google Scholar]
  • [47].Wang Y, Kedei N, Wang M, Wang QJ, Huppler AR, Toth A, et al. Interaction between protein kinase Cμ and the vanilloid receptor type 1. J Biol Chem. 2004;279:53674–53682. doi: 10.1074/jbc.M410331200. [DOI] [PubMed] [Google Scholar]
  • [48].Zhu H, Yang Y, Zhang H, Han Y, Li Y, Zhang Y, et al. Interaction between protein kinase D1 and transient receptor potential V1 in primary sensory neurons is involved in heat hypersensitivity. Pain. 2008;137:574–588. doi: 10.1016/j.pain.2007.10.025. [DOI] [PubMed] [Google Scholar]
  • [49].Wang Y. The functional regulation of TRPV1 and its role in pain sensitization. Neurochem Res. 2008;33:2008–2012. doi: 10.1007/s11064-008-9750-5. [DOI] [PubMed] [Google Scholar]
  • [50].Craig AM, Banker G. Neuronal polarity. Annu Rev Neurosci. 1994;17:267–310. doi: 10.1146/annurev.ne.17.030194.001411. [DOI] [PubMed] [Google Scholar]
  • [51].Dotti CG, Sullivan CA, Banker GA. The establishment of polarity by hippocampal neurons in culture. J Neurosci. 1988;8:1454–1468. doi: 10.1523/JNEUROSCI.08-04-01454.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [52].Yin DM, Huang YH, Zhu YB, Wang Y. Both the establishment and maintenance of neuronal polarity require the activity of protein kinase D in the Golgi apparatus. J Neurosci. 2008;28:8832–8843. doi: 10.1523/JNEUROSCI.1291-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [53].Czondor K, Ellwanger K, Fuchs YF, Lutz S, Gulyas M, Mansuy IM, et al. Protein kinase D controls the integrity of Golgi apparatus and the maintenance of dendritic arborization in hippocampal neurons. Mol Biol Cell. 2009;20:2108–2120. doi: 10.1091/mbc.E08-09-0957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [54].Bisbal M, Conde C, Donoso M, Bollati F, Sesma J, Quiroga S, et al. Protein kinase d regulates trafficking of dendritic membrane proteins in developing neurons. J Neurosci. 2008;28:9297–9308. doi: 10.1523/JNEUROSCI.1879-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [55].Sánchez-Ruiloba L, Cabrera-Poch N, Rodríguez-Martínez M, López-Menéndez C, Jean-Mairet RM, Higuero AM, et al. Protein kinase D intracellular localization and activity control kinase D-interacting substrate of 220-kDa traffic through a postsynaptic density-95/discs large/zonula occludens-1-binding motif. J Biol Chem. 2006;281:18888–18900. doi: 10.1074/jbc.M603044200. [DOI] [PubMed] [Google Scholar]
  • [56].Bracale A, Cesca F, Neubrand VE, Newsome TP, Way M, Schiavo G. Kidins220/ARMS is transported by a kinesin-1-based mechanism likely to be involved in neuronal differentiation. Mol Biol Cell. 2007;18:142–152. doi: 10.1091/mbc.E06-05-0453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [57].Higuero AM, Sánchez-Ruiloba L, Doglio LE, Portillo F, Abad-Rodríguez J, Dotti CG, et al. Kidins220/ARMS modulates the activity of microtubule-regulating proteins and controls neuronal polarity and development. J Biol Chem. 2010;285:1343–1357. doi: 10.1074/jbc.M109.024703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [58].Cabrera-Poch N, Sánchez-Ruiloba L, Rodríguez-Martínez M, Iglesias T. Lipid raft disruption triggers protein kinase C and Src-dependent protein kinase D activation and Kidins220 phosphorylation in neuronal cells. J Biol Chem. 2004;279:28592–28602. doi: 10.1074/jbc.M312242200. [DOI] [PubMed] [Google Scholar]
  • [59].Benton R, Johnston DS. Drosophila PAR-1 and 14-3-3 inhibit Bazooka/PAR-3 to establish complementary cortical domains in polarized cells. Cell. 2003;115:691–704. doi: 10.1016/S0092-8674(03)00938-3. [DOI] [PubMed] [Google Scholar]
  • [60].Chen Y, Wang Q, Hu H, Yu P, Zhu J, Drewes G, et al. Microtubule affinity-regulating kinase 2 functions downstream of the PAR-3/PAR-6/atypical PKC complex in regulating hippocampal neuronal polarity. Proc Natl Acad Sci U S A. 2006;103:8534–8539. doi: 10.1073/pnas.0509955103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [61].Lin D, Edwards AS, Fawcett JP, Mbamalu G, Scott JD, Pawson T. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nat Cell Biol. 2000;2:540–547. doi: 10.1038/35019592. [DOI] [PubMed] [Google Scholar]
  • [62].Wu Q, DiBona VL, Bernard LP, Zhang H. The polarity protein partitioning-defective 1 (PAR-1) regulates dendritic spine morphogenesis through phosphorylating postsynaptic density protein 95 (PSD-95) J Biol Chem. 2012;287:30781–30788. doi: 10.1074/jbc.M112.351452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [63].Watkins JL, Lewandowski KT, Meek SE, Storz P, Toker A, Piwnica-Worms H. Phosphorylation of the Par-1 polarity kinase by protein kinase D regulates 14-3-3 binding and membrane association. Proc Natl Acad Sci U S A. 2008;105:18378–18383. doi: 10.1073/pnas.0809661105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [64].Asaithambi A, Kanthasamy A, Saminathan H, Anantharam V, Kanthasamy AG. Protein kinase D1 (PKD1) activation mediates a compensatory protective response during early stages of oxidative stress-induced neuronal degeneration. Mol Neurodegener. 2011;6:43. doi: 10.1186/1750-1326-6-43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [65].Stetler RA, Cao G, Gao Y, Zhang F, Wang S, Weng Z, et al. Hsp27 protects against ischemic brain injury via attenuation of a novel stress-response cascade upstream of mitochondrial cell death signaling. J Neurosci. 2008;28:13038–13055. doi: 10.1523/JNEUROSCI.4407-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [66].Doppler H, Storz P, Li J, Comb MJ, Toker A. A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D. J Biol Chem. 2005;280:15013–15019. doi: 10.1074/jbc.C400575200. [DOI] [PubMed] [Google Scholar]
  • [67].Stetler RA, Gao Y, Zhang L, Weng Z, Zhang F, Hu X, et al. Phosphorylation of HSP27 by protein kinase D is essential for mediating neuroprotection against ischemic neuronal injury. J Neurosci. 2012;32:2667–2682. doi: 10.1523/JNEUROSCI.5169-11.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [68].Feng H, Ren M, Chen L, Rubin CS. Properties, regulation, and in vivo functions of a novel protein kinase D Caenorhabditis elegans DKF-2 links diacylglycerol second messenger to the regulation of stress responses and life span. J Biol Chem. 2007;282:31273–31288. doi: 10.1074/jbc.M701532200. [DOI] [PubMed] [Google Scholar]
  • [69].Ren M, Feng H, Fu Y, Land M, Rubin CS. Protein kinase D (DKF-2), a diacylglycerol effector, is an essential regulator of C. elegans innate immunity. Immunity. 2009;30:521. doi: 10.1016/j.immuni.2009.03.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [70].Hukema RK, Rademakers S, Dekkers MP, Burghoorn J, Jansen G. Antagonistic sensory cues generate gustatory plasticity in Caenorhabditis elegans. EMBO J. 2006;25:312–322. doi: 10.1038/sj.emboj.7600940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [71].Jansen G, Weinkove D, Plasterk RH. The G-protein {gamma} subunit gpc-1 of the nematode C. elegans is involved in taste adaptation. Sci Signal. 2002;21:986. doi: 10.1093/emboj/21.5.986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [72].Saeki S, Yamamoto M, Iino Y. Plasticity of chemotaxis revealed by paired presentation of a chemoattractant and starvation in the nematode Caenorhabditis elegans. J Exp Biol. 2001;204:1757–1764. doi: 10.1242/jeb.204.10.1757. [DOI] [PubMed] [Google Scholar]
  • [73].Fu Y, Ren M, Feng H, Chen L, Altun ZF, Rubin CS. Neuronal and intestinal protein kinase d isoforms mediate Na+ (salt taste)-induced learning. Sci Signal 2009, 2: ra42. [DOI] [PubMed]

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