FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3100753829> ?p ?o ?g. }

• W3100753829 endingPage "100082" @default.
• W3100753829 startingPage "100082" @default.
• W3100753829 abstract "Proper brain development and function requires finely controlled mechanisms for protein turnover, and disruption of genes involved in proteostasis is a common cause of neurodevelopmental disorders. Kelch-like 15 (KLHL15) is a substrate adaptor for cullin3-containing E3 ubiquitin ligases, and KLHL15 gene mutations were recently described as a cause of severe X-linked intellectual disability. Here, we used a bioinformatics approach to identify a family of neuronal microtubule-associated proteins as KLHL15 substrates, which are themselves critical for early brain development. We biochemically validated doublecortin (DCX), also an X-linked disease protein, and doublecortin-like kinase 1 and 2 as bona fide KLHL15 interactors and mapped KLHL15 interaction regions to their tandem DCX domains. Shared with two previously identified KLHL15 substrates, a FRY tripeptide at the C-terminal edge of the second DCX domain is necessary for KLHL15-mediated ubiquitination of DCX and doublecortin-like kinase 1 and 2 and subsequent proteasomal degradation. Conversely, silencing endogenous KLHL15 markedly stabilizes these DCX domain-containing proteins and prolongs their half-life. Functionally, overexpression of KLHL15 in the presence of WT DCX reduces dendritic complexity of cultured hippocampal neurons, whereas neurons expressing FRY-mutant DCX are resistant to KLHL15. Collectively, our findings highlight the critical importance of the E3 ubiquitin ligase adaptor KLHL15 in proteostasis of neuronal microtubule-associated proteins and identify a regulatory network important for development of the mammalian nervous system. Proper brain development and function requires finely controlled mechanisms for protein turnover, and disruption of genes involved in proteostasis is a common cause of neurodevelopmental disorders. Kelch-like 15 (KLHL15) is a substrate adaptor for cullin3-containing E3 ubiquitin ligases, and KLHL15 gene mutations were recently described as a cause of severe X-linked intellectual disability. Here, we used a bioinformatics approach to identify a family of neuronal microtubule-associated proteins as KLHL15 substrates, which are themselves critical for early brain development. We biochemically validated doublecortin (DCX), also an X-linked disease protein, and doublecortin-like kinase 1 and 2 as bona fide KLHL15 interactors and mapped KLHL15 interaction regions to their tandem DCX domains. Shared with two previously identified KLHL15 substrates, a FRY tripeptide at the C-terminal edge of the second DCX domain is necessary for KLHL15-mediated ubiquitination of DCX and doublecortin-like kinase 1 and 2 and subsequent proteasomal degradation. Conversely, silencing endogenous KLHL15 markedly stabilizes these DCX domain-containing proteins and prolongs their half-life. Functionally, overexpression of KLHL15 in the presence of WT DCX reduces dendritic complexity of cultured hippocampal neurons, whereas neurons expressing FRY-mutant DCX are resistant to KLHL15. Collectively, our findings highlight the critical importance of the E3 ubiquitin ligase adaptor KLHL15 in proteostasis of neuronal microtubule-associated proteins and identify a regulatory network important for development of the mammalian nervous system. Ubiquitination is a highly coordinated multistep enzymatic cascade that requires the concerted action of three key factors: E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases (1Vucic D. Dixit V.M. Wertz I.E. Ubiquitylation in apoptosis: a post-translational modification at the edge of life and death.Nat. Rev. Mol. Cell Biol. 2011; 12: 439-452Crossref PubMed Scopus (295) Google Scholar), many of which are strongly implicated in the pathogenesis of neurodevelopmental disorders (2Cheon S. Dean M. Chahrour M. The ubiquitin proteasome pathway in neuropsychiatric disorders.Neurobiol. Learn. Mem. 2019; 165: 106791Crossref PubMed Scopus (10) Google Scholar, 3Hegde A.N. Upadhya S.C. The ubiquitin-proteasome pathway in health and disease of the nervous system.Trends Neurosci. 2007; 30: 587-595Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 4Groen E.J.N. Gillingwater T.H. UBA1: at the crossroads of ubiquitin homeostasis and neurodegeneration.Trends Mol. Med. 2015; 21: 622-632Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). E3 ubiquitin ligases have received particular attention, as they confer substrate specificity of the ubiquitination reaction by catalyzing the transfer of ubiquitin to proteins destined for degradation or other ubiquitin-dependent fates (5Upadhyay A. Joshi V. Amanullah A. Mishra R. Arora N. Prasad A. Mishra A. E3 ubiquitin ligases neurobiological mechanisms: development to degeneration.Front. Mol. Neurosci. 2017; 10: 151Crossref PubMed Scopus (36) Google Scholar, 6Louros S.R. Osterweil E.K. Perturbed proteostasis in autism spectrum disorders.J. Neurochem. 2016; 139: 1081-1092Crossref PubMed Scopus (33) Google Scholar, 7Upadhyay A. Amanullah A. Chhangani D. Mishra R. Mishra A. Selective multifaceted E3 ubiquitin ligases barricade extreme defense: potential therapeutic targets for neurodegeneration and ageing.Ageing Res. Rev. 2015; 24: 138-159Crossref PubMed Scopus (17) Google Scholar). Recent studies have identified KLHL15 as an X-linked intellectual disability (XLID) gene. Inactivating KLHL15 mutations are associated with various brain abnormalities as well as developmental and behavioral disorders in male patients (8Hu H. Haas S.A. Chelly J. Van Esch H. Raynaud M. de Brouwer A.P. Weinert S. Froyen G. Frints S.G. Laumonnier F. Zemojtel T. Love M.I. Richard H. Emde A.K. Bienek M. et al.X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.Mol. Psychiatry. 2016; 21: 133-148Crossref PubMed Scopus (154) Google Scholar, 9Mignon-Ravix C. Cacciagli P. Choucair N. Popovici C. Missirian C. Milh M. Megarbane A. Busa T. Julia S. Girard N. Badens C. Sigaudy S. Philip N. Villard L. Intragenic rearrangements in X-linked intellectual deficiency: results of a-CGH in a series of 54 patients and identification of TRPC5 and KLHL15 as potential XLID genes.Am. J. Med. Genet. A. 2014; 164A: 1991-1997Crossref PubMed Scopus (13) Google Scholar, 10Schumann M. Hofmann A. Krutzke S.K. Hilger A.C. Marsch F. Stienen D. Gembruch U. Ludwig M. Merz W.M. Reutter H. Array-based molecular karyotyping in fetuses with isolated brain malformations identifies disease-causing CNVs.J. Neurodev. Disord. 2016; 8: 11Crossref PubMed Scopus (13) Google Scholar, 11Karaca E. Harel T. Pehlivan D. Jhangiani S.N. Gambin T. Coban Akdemir Z. Gonzaga-Jauregui C. Erdin S. Bayram Y. Campbell I.M. Hunter J.V. Atik M.M. Van Esch H. Yuan B. Wiszniewski W. et al.Genes that affect brain structure and function identified by rare variant analyses of mendelian neurologic disease.Neuron. 2015; 88: 499-513Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Localized on chromosome Xp22.11, KLHL15 encodes a member of the Kelch-like (KLHL) family of proteins that function as adaptors for cullin3 (Cul3)-based E3 ubiquitin ligases to target specific substrates to the ubiquitin-proteasome system (12Shi X. Xiang S. Cao J. Zhu H. Yang B. He Q. Ying M. Kelch-like proteins: physiological functions and relationships with diseases.Pharmacol. Res. 2019; 148: 104404Crossref PubMed Scopus (14) Google Scholar). Numbering more than 40 in humans, KLHL proteins exert a wide range of biological functions, whereas genetic mutations and abnormal expression of KLHL genes have been linked to diverse diseases, ranging from cardiovascular disorders to cancer (12Shi X. Xiang S. Cao J. Zhu H. Yang B. He Q. Ying M. Kelch-like proteins: physiological functions and relationships with diseases.Pharmacol. Res. 2019; 148: 104404Crossref PubMed Scopus (14) Google Scholar, 13Dhanoa B.S. Cogliati T. Satish A.G. Bruford E.A. Friedman J.S. Update on the Kelch-like (KLHL) gene family.Hum. Genomics. 2013; 7: 13Crossref PubMed Scopus (116) Google Scholar, 14Gupta V.A. Beggs A.H. Kelch proteins: emerging roles in skeletal muscle development and diseases.Skelet. Muscle. 2014; 4: 11Crossref PubMed Scopus (78) Google Scholar). We have previously demonstrated that KLHL15 mediates the ubiquitination and subsequent proteasomal degradation of the B'β (B56β, PR61β, PPP2R5B) regulatory subunit of the protein Ser/Thr phosphatase 2A (PP2A). KLHL15-mediated B'β degradation facilitates the formation of alternative PP2A holoenzymes by promoting the exchange with more than a dozen other regulatory subunits (15Oberg E.A. Nifoussi S.K. Gingras A.C. Strack S. Selective proteasomal degradation of the B'beta subunit of protein phosphatase 2A by the E3 ubiquitin ligase adaptor Kelch-like 15.J. Biol. Chem. 2012; 287: 43378-43389Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). PP2A/B'β is highly enriched in the mammalian nervous system (16Saraf A. Virshup D.M. Strack S. Differential expression of the B'beta regulatory subunit of protein phosphatase 2A modulates tyrosine hydroxylase phosphorylation and catecholamine synthesis.J. Biol. Chem. 2007; 282: 573-580Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 17McCright B. Virshup D.M. Identification of a new family of protein phosphatase 2A regulatory subunits.J. Biol. Chem. 1995; 270: 26123-26128Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 18Martens E. Stevens I. Janssens V. Vermeesch J. Gotz J. Goris J. Van Hoof C. Genomic organisation, chromosomal localisation tissue distribution and developmental regulation of the PR61/B' regulatory subunits of protein phosphatase 2A in mice.J. Mol. Biol. 2004; 336: 971-986Crossref PubMed Scopus (45) Google Scholar) and plays key roles in striatal dopamine biosynthesis (16Saraf A. Virshup D.M. Strack S. Differential expression of the B'beta regulatory subunit of protein phosphatase 2A modulates tyrosine hydroxylase phosphorylation and catecholamine synthesis.J. Biol. Chem. 2007; 282: 573-580Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), hippocampal long-term potentiation (19Fukunaga K. Muller D. Ohmitsu M. Bako E. DePaoli-Roach A.A. Miyamoto E. Decreased protein phosphatase 2A activity in hippocampal long-term potentiation.J. Neurochem. 2000; 74: 807-817Crossref PubMed Scopus (75) Google Scholar), and multiple intracellular signaling pathways in response to growth factor stimulation (20Bidinosti M. Botta P. Kruttner S. Proenca C.C. Stoehr N. Bernhard M. Fruh I. Mueller M. Bonenfant D. Voshol H. Carbone W. Neal S.J. McTighe S.M. Roma G. Dolmetsch R.E. et al.CLK2 inhibition ameliorates autistic features associated with SHANK3 deficiency.Science. 2016; 351: 1199-1203Crossref PubMed Scopus (81) Google Scholar, 21Brandt N. Franke K. Johannes S. Buck F. Harder S. Hassel B. Nitsch R. Schumacher S. B56beta, a regulatory subunit of protein phosphatase 2A, interacts with CALEB/NGC and inhibits CALEB/NGC-mediated dendritic branching.FASEB J. 2008; 22: 2521-2533Crossref PubMed Scopus (10) Google Scholar, 22Van Kanegan M.J. Strack S. The protein phosphatase 2A regulatory subunits B'beta and B'delta mediate sustained TrkA neurotrophin receptor autophosphorylation and neuronal differentiation.Mol. Cell Biol. 2009; 29: 662-674Crossref PubMed Scopus (31) Google Scholar). We also identified a tyrosine residue (Y52) within a phylogenetically well-conserved tripeptide motif (FRY) in vertebrate B'β as being necessary for KLHL15 binding and KLHL15-mediated B'β downregulation (15Oberg E.A. Nifoussi S.K. Gingras A.C. Strack S. Selective proteasomal degradation of the B'beta subunit of protein phosphatase 2A by the E3 ubiquitin ligase adaptor Kelch-like 15.J. Biol. Chem. 2012; 287: 43378-43389Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Since then, work by others identified a critical FRY tripeptide in the degron of a second KLHL15 substrate, the DNA repair protein CtIP/RBBP8 (23Ferretti L.P. Himmels S.F. Trenner A. Walker C. von Aesch C. Eggenschwiler A. Murina O. Enchev R.I. Peter M. Freire R. Porro A. Sartori A.A. Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection.Nat. Commun. 2016; 7: 12628Crossref PubMed Scopus (28) Google Scholar). Because of KLHL15’s link to severe intellectual disability and its ranking as one of the most clinically significant X-linked disease genes (8Hu H. Haas S.A. Chelly J. Van Esch H. Raynaud M. de Brouwer A.P. Weinert S. Froyen G. Frints S.G. Laumonnier F. Zemojtel T. Love M.I. Richard H. Emde A.K. Bienek M. et al.X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.Mol. Psychiatry. 2016; 21: 133-148Crossref PubMed Scopus (154) Google Scholar, 24Ge X. Kwok P.Y. Shieh J.T. Prioritizing genes for X-linked diseases using population exome data.Hum. Mol. Genet. 2015; 24: 599-608Crossref PubMed Scopus (12) Google Scholar), we set out to uncover novel neuronal substrates of the E3 ubiquitin ligase by primary and secondary structure searches. This screen and subsequent biochemical validation identified doublecortin (DCX) and two doublecortin-like kinases (DCLK1, DCLK2), members of a family of neuronal microtubule-associated proteins (MAPs) that share a characteristic domain structure of N-terminal tandem DCX domains. These tandem DCX domains stabilize microtubule by enhancing tubulin polymerization, inhibiting tubulin catastrophe, and promoting microtubule bundling (25Reiner O. Coquelle F.M. Peter B. Levy T. Kaplan A. Sapir T. Orr I. Barkai N. Eichele G. Bergmann S. The evolving doublecortin (DCX) superfamily.BMC Genomics. 2006; 7: 188Crossref PubMed Scopus (80) Google Scholar, 26Fourniol F. Perderiset M. Houdusse A. Moores C. Structural studies of the doublecortin family of MAPs.Methods Cell Biol. 2013; 115: 27-48Crossref PubMed Scopus (15) Google Scholar, 27Coquelle F.M. Levy T. Bergmann S. Wolf S.G. Bar-El D. Sapir T. Brody Y. Orr I. Barkai N. Eichele G. Reiner O. Common and divergent roles for members of the mouse DCX superfamily.Cell Cycle. 2006; 5: 976-983Crossref PubMed Scopus (48) Google Scholar, 28Dijkmans T.F. van Hooijdonk L.W. Fitzsimons C.P. Vreugdenhil E. The doublecortin gene family and disorders of neuronal structure.Cent. Nerv Syst. Agents Med. Chem. 2010; 10: 32-46Crossref PubMed Scopus (48) Google Scholar). DCX plays essential roles in neurogenesis, neuronal migration, axonal/dendritic wiring, and cargo transport therefore ensuring normal brain development (28Dijkmans T.F. van Hooijdonk L.W. Fitzsimons C.P. Vreugdenhil E. The doublecortin gene family and disorders of neuronal structure.Cent. Nerv Syst. Agents Med. Chem. 2010; 10: 32-46Crossref PubMed Scopus (48) Google Scholar, 29Reiner O. LIS1 and DCX: implications for brain development and human disease in relation to microtubules.Scientifica (Cairo). 2013; 2013: 393975PubMed Google Scholar). The DCX gene is located on the X chromosome, and DCX mutations cause classic lissencephaly (agyria) in males and subcortical band heterotopia (also called double cortex syndrome) primarily in females as a result of neuronal migration defects (30Hehr U. Uyanik G. Aigner L. Couillard-Despres S. Winkler J. DCX-related disorders.in: Adam M.P. Ardinger H.H. Pagon R.A. Wallace S.E. Bean L.J.H. Stephens K. Amemiya A. GeneReviews((R)). University of Washington, 1993Google Scholar). Both disorders typically manifest with intellectual disability, epilepsy, language impairment, and global developmental delay (30Hehr U. Uyanik G. Aigner L. Couillard-Despres S. Winkler J. DCX-related disorders.in: Adam M.P. Ardinger H.H. Pagon R.A. Wallace S.E. Bean L.J.H. Stephens K. Amemiya A. GeneReviews((R)). University of Washington, 1993Google Scholar). DCLK1 and DCLK2 feature a C-terminal Ser/Thr kinase domain and play both common and distinct roles in neurodevelopment. Both kinases associate with microtubules and stimulate microtubule polymerization and dendritic development independently of their kinase activity (31Lin P.T. Gleeson J.G. Corbo J.C. Flanagan L. Walsh C.A. DCAMKL1 encodes a protein kinase with homology to doublecortin that regulates microtubule polymerization.J. Neurosci. 2000; 20: 9152-9161Crossref PubMed Google Scholar, 32Edelman A.M. Kim W.Y. Higgins D. Goldstein E.G. Oberdoerster M. Sigurdson W. Doublecortin kinase-2, a novel doublecortin-related protein kinase associated with terminal segments of axons and dendrites.J. Biol. Chem. 2005; 280: 8531-8543Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 33Shin E. Kashiwagi Y. Kuriu T. Iwasaki H. Tanaka T. Koizumi H. Gleeson J.G. Okabe S. Doublecortin-like kinase enhances dendritic remodelling and negatively regulates synapse maturation.Nat. Commun. 2013; 4: 1440Crossref PubMed Scopus (57) Google Scholar). DCLK1 and DCX synergistically regulate neuronal migration, axonal outgrowth, and hippocampal development (34Deuel T.A. Liu J.S. Corbo J.C. Yoo S.Y. Rorke-Adams L.B. Walsh C.A. Genetic interactions between doublecortin and doublecortin-like kinase in neuronal migration and axon outgrowth.Neuron. 2006; 49: 41-53Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 35Koizumi H. Tanaka T. Gleeson J.G. Doublecortin-like kinase functions with doublecortin to mediate fiber tract decussation and neuronal migration.Neuron. 2006; 49: 55-66Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 36Tanaka T. Koizumi H. Gleeson J.G. The doublecortin and doublecortin-like kinase 1 genes cooperate in murine hippocampal development.Cereb. Cortex. 2006; 16 Suppl 1: i69-i73Crossref PubMed Scopus (31) Google Scholar), whereas DCLK2 works in concert with DCX to organize hippocampal lamination and prevent spontaneous seizures (37Kerjan G. Koizumi H. Han E.B. Dube C.M. Djakovic S.N. Patrick G.N. Baram T.Z. Heinemann S.F. Gleeson J.G. Mice lacking doublecortin and doublecortin-like kinase 2 display altered hippocampal neuronal maturation and spontaneous seizures.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 6766-6771Crossref PubMed Scopus (62) Google Scholar). This report documents that three members of the DCX family of neuronal MAPs are subject to KLHL15-mediated downregulation. KLHL15-dependent polyubiquitination and proteasomal degradation requires a degron containing both N-terminal DCX domains and a conserved FRY tripeptide. Functionally, we show that KLHL15 expression reduces dendritic arborization in primary hippocampal cultures unless neurons also express DCX with a stabilizing FRY mutation. Our study uncovers a novel role of the E3 ubiquitin ligase adaptor KLHL15 as a negative regulator of DCX protein abundance and dendritogenesis and suggests possible mechanistic connections in X-linked neurodevelopmental disorders. Two KLHL15 substrates have been identified to date, the neuron-enriched PP2A regulatory subunit B'β (15Oberg E.A. Nifoussi S.K. Gingras A.C. Strack S. Selective proteasomal degradation of the B'beta subunit of protein phosphatase 2A by the E3 ubiquitin ligase adaptor Kelch-like 15.J. Biol. Chem. 2012; 287: 43378-43389Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and CtIP/RBBP8, a widely expressed endonuclease involved in DNA repair by homologous recombination (23Ferretti L.P. Himmels S.F. Trenner A. Walker C. von Aesch C. Eggenschwiler A. Murina O. Enchev R.I. Peter M. Freire R. Porro A. Sartori A.A. Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection.Nat. Commun. 2016; 7: 12628Crossref PubMed Scopus (28) Google Scholar). According to gene expression databases (e.g., https://www.ebi.ac.uk/gxa), B'β is more highly expressed in the adult than in the embryonic human brain. This raises the question whether this PP2A subunit is relevant for the neurodevelopmental consequences of KLHL15 mutations. To determine whether KLHL15 possesses functions in neurons independent of PP2A/B'β, we investigated the effect of KLHL15 on activation of extracellular signal-regulated kinases (ERKs, Fig. 1A). We previously reported that the B'β-containing PP2A holoenzyme potentiates nerve growth factor signaling at the tropomyosin-related kinase A (TrkA) receptor level to sustain ERK signaling and neuronal differentiation of neuroendocrine PC12 cells (22Van Kanegan M.J. Strack S. The protein phosphatase 2A regulatory subunits B'beta and B'delta mediate sustained TrkA neurotrophin receptor autophosphorylation and neuronal differentiation.Mol. Cell Biol. 2009; 29: 662-674Crossref PubMed Scopus (31) Google Scholar). To address the hypothesis that PP2A/B'β may similarly potentiate TrkB receptor activity in central neurons, we measured ERK activity in transiently transfected primary hippocampal neurons from E18 rat embryos using a dual luciferase reporter assay (PathDetect Elk1 reporter, Fig. 1A) (22Van Kanegan M.J. Strack S. The protein phosphatase 2A regulatory subunits B'beta and B'delta mediate sustained TrkA neurotrophin receptor autophosphorylation and neuronal differentiation.Mol. Cell Biol. 2009; 29: 662-674Crossref PubMed Scopus (31) Google Scholar). In GFP control-transfected neurons, a 3-h incubation with a subsaturating concentration of brain-derived neurotrophic factor (BDNF) resulted in a several-fold increase in Elk1 transcriptional activity (Fig. 1B). Compared with control, overexpression of GFP-KLHL15 attenuated ERK activation by 2.5-fold, whereas B'β overexpression amplified the BDNF response by 3-fold (Fig. 1B). Conversely, silencing of endogenous KLHL15 amplified BDNF signaling to ERK by 1.5-fold over control shRNA-transfected cells, whereas shRNA targeting endogenous B'β nearly eliminated the BDNF response (Fig. 1C). These results suggest that KLHL15 inhibits neurotrophin signaling in hippocampal neurons primarily by targeting PP2A/B'β for proteasomal degradation. The neuromodulator pituitary adenylate cyclase-activating polypeptide (PACAP) signals through the type 1 G protein–coupled receptor PAC1 to ERK via cAMP-dependent activation of Rap1 (38Vaudry D. Stork P.J. Lazarovici P. Eiden L.E. Signaling pathways for PC12 cell differentiation: making the right connections.Science. 2002; 296: 1648-1649Crossref PubMed Scopus (677) Google Scholar) (Fig. 1A). In control-transfected neurons, PACAP at subsaturating concentration induced Elk1 transcriptional activity by ∼5-fold (Fig. 1D). Overexpression of GFP-KLHL15 dampened this response by ∼2-fold, whereas KLHL15 silencing amplified PACAP signaling to ERK by ∼3-fold (Fig. 1, D–E). In contrast to BDNF stimulation, neither overexpression nor knockdown of PP2A/B'β significantly affected the reporter response to PACAP (Fig. 1, D–E), suggesting the involvement of a distinct KLHL15 substrate. To uncover additional neuronal KLHL15 substrates, we searched the human UniProt database for proteins that contain the FRY sequence, which is necessary for the interaction of KLHL15 with its two previously identified targets, PP2A/B'β (15Oberg E.A. Nifoussi S.K. Gingras A.C. Strack S. Selective proteasomal degradation of the B'beta subunit of protein phosphatase 2A by the E3 ubiquitin ligase adaptor Kelch-like 15.J. Biol. Chem. 2012; 287: 43378-43389Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and the DNA endonuclease CtIP/RBBP8 (23Ferretti L.P. Himmels S.F. Trenner A. Walker C. von Aesch C. Eggenschwiler A. Murina O. Enchev R.I. Peter M. Freire R. Porro A. Sartori A.A. Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection.Nat. Commun. 2016; 7: 12628Crossref PubMed Scopus (28) Google Scholar). This search identified a list of 616 proteins, which was further narrowed down according to secondary structure prediction (Fig. 2A). The FRY sequence in B'β and CtIP/RBBP8 is predicted to adopt a β-strand flanked by extended unstructured regions, which we reasoned provides flexibility for protein–protein interactions. Constraining the list of FRY-containing proteins to those in which the tripeptide is within a 2- to 4-residue-long β-strand surrounded by at least five unstructured residues on both sides yielded a list of 24 proteins (Fig. 2A), which includes signaling proteins, transcription factors, and proteins involved in proteostasis (Table 1). We focused on three proteins: DCX and two doublecortin-like kinases DCLK1 and DCLK2, which belong to a group of 11 neuronal MAPs characterized by microtubule binding, tandem DCX domains that function during brain development (25Reiner O. Coquelle F.M. Peter B. Levy T. Kaplan A. Sapir T. Orr I. Barkai N. Eichele G. Bergmann S. The evolving doublecortin (DCX) superfamily.BMC Genomics. 2006; 7: 188Crossref PubMed Scopus (80) Google Scholar, 33Shin E. Kashiwagi Y. Kuriu T. Iwasaki H. Tanaka T. Koizumi H. Gleeson J.G. Okabe S. Doublecortin-like kinase enhances dendritic remodelling and negatively regulates synapse maturation.Nat. Commun. 2013; 4: 1440Crossref PubMed Scopus (57) Google Scholar, 34Deuel T.A. Liu J.S. Corbo J.C. Yoo S.Y. Rorke-Adams L.B. Walsh C.A. Genetic interactions between doublecortin and doublecortin-like kinase in neuronal migration and axon outgrowth.Neuron. 2006; 49: 41-53Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 35Koizumi H. Tanaka T. Gleeson J.G. Doublecortin-like kinase functions with doublecortin to mediate fiber tract decussation and neuronal migration.Neuron. 2006; 49: 55-66Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 36Tanaka T. Koizumi H. Gleeson J.G. The doublecortin and doublecortin-like kinase 1 genes cooperate in murine hippocampal development.Cereb. Cortex. 2006; 16 Suppl 1: i69-i73Crossref PubMed Scopus (31) Google Scholar, 37Kerjan G. Koizumi H. Han E.B. Dube C.M. Djakovic S.N. Patrick G.N. Baram T.Z. Heinemann S.F. Gleeson J.G. Mice lacking doublecortin and doublecortin-like kinase 2 display altered hippocampal neuronal maturation and spontaneous seizures.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 6766-6771Crossref PubMed Scopus (62) Google Scholar, 39Shu T. Tseng H.C. Sapir T. Stern P. Zhou Y. Sanada K. Fischer A. Coquelle F.M. Reiner O. Tsai L.H. Doublecortin-like kinase controls neurogenesis by regulating mitotic spindles and M phase progression.Neuron. 2006; 49: 25-39Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The FRY tripeptide is situated at the extreme C-terminal border of the second DCX domain (Fig. 2B). This region is highly conserved with the FRY sequence present in all vertebrate orthologs of DCX, DCLK1, and DCLK2. The region is not conserved in DCLK3, an atypical and largely uncharacterized member of the DCX family, whose homology to DCLK1 and DCLK2 is largely confined to the kinase domain (40Ohmae S. Takemoto-Kimura S. Okamura M. Adachi-Morishima A. Nonaka M. Fuse T. Kida S. Tanji M. Furuyashiki T. Arakawa Y. Narumiya S. Okuno H. Bito H. Molecular identification and characterization of a family of kinases with homology to Ca2+/calmodulin-dependent protein kinases I/IV.J. Biol. Chem. 2006; 281: 20427-20439Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar).Table 1List of 24 putative KLHL15 substrates identified by the two-step in silico screenProteinAnnotationATXN2RNA stability regulator Ataxin-2/SCA2AXIN1β-Catenin destruction complex component Axin-1CACNG8Voltage-dependent calcium channel γ8 subunitCADH3Cadherin-3DCLK1Serine/threonine-protein kinase DCLK1DCLK2Serine/threonine-protein kinase DCLK2DCXNeuronal migration protein doublecortinDIXDC1Wnt signaling pathway effector DixinDUSP2Dual specificity protein phosphatase 2EPB41L2Erythrocyte membrane protein band 4.1 like 2HERC4Probable E3 ubiquitin-protein ligase HERC4PDE8Cyclic nucleotide phosphodiesterase 8BPDZD8PDZ domain-containing protein 8PPP2R5BProtein phosphatase 2A regulatory subunit B'βPPIAL4CPeptidyl-prolyl cis-trans isomerase A-like 4CPSMG2Proteasome assembly chaperone 2PTPN4Tyrosine-protein phosphatase non-receptor type 4RABL6ARAB-like GTPase RABL6ARBBP8DNA endonuclease RBBP8/CtIPTCF20Transcription factor 20TMEM161ATransmembrane protein 161ATRPS1Zinc finger transcription factor Trps1USPL1SUMO-specific isopeptidase USPL1ZFP37Zinc finger protein 37 Open table in a new tab To test whether DCX, DCLK1, and DCLK2 are KLHL15 interactors, we coexpressed GFP-tagged KLHL15 and FLAG-tagged DCX or DCLKs in COS-1 cells and conducted reciprocal immunoprecipitation (IP) experiments. GFP-IP of KLHL15 selectively enriched DCX, DCLK1, and DCLK2, but not DCLK3 or another negative control (FLAG-RIβ). Similarly, only FLAG-IPs of DCX and DCLK1/2 isolated GFP-KLHL15 (Fig. 2C). No interactions were detected with dynamin-related protein 1 (GFP-Drp1), indicating that DCX and DCLK1/2 do not interact with GFP. Notably, compared with GFP-Drp1, coexpression of GFP-KLHL15 dramatically decreased steady-state levels of the three interacting proteins (Fig. 2C, input). To obtain evidence for direct interactions with endogenous proteins, we performed glutathione-S-transferase (GST) pulldown experiments with brain lysates from E18 rat embryos. A GST fusion of the substrate-binding Kelch domain of KLHL15 (amino acids 255–604, GST-KLHL15Kelch [15Oberg E.A. Nifoussi S.K. Gingras A.C. Strack S. Selective proteasomal degradation of the B'beta subunit of protein phosphatase 2A by the E3 ubiquitin ligase adaptor Kelch-like 15.J. Biol. Chem. 2012; 287: 43378-43389Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]) was able to capture the endogenous PP2A/B'β regulatory subunit, as well as DCX, DCLK1, and DCLK2 (Fig. 2D). Indicating specificity for KLHL15, none of the proteins were pulled down with the Kelch domain of KLHL9 (GST-KLHL9Kelch) or GST alone (EV, Fig. 2D). To map the KLHL15-binding domain of DCX proteins, we constructed chimeras between the interactor DCLK2 and the non-interactor DCLK3. Chimera DCLK3-2 is composed of the N terminus of DCLK3 (amino acids 1–130) an" @default.
• W3100753829 created "2020-11-23" @default.
• W3100753829 creator A5007533191 @default.
• W3100753829 creator A5040636021 @default.
• W3100753829 creator A5064866168 @default.
• W3100753829 creator A5070992833 @default.
• W3100753829 date "2021-01-01" @default.
• W3100753829 modified "2023-10-02" @default.
• W3100753829 title "The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis" @default.
• W3100753829 cites W1607410226 @default.
• W3100753829 cites W1659618367 @default.
• W3100753829 cites W1838932899 @default.
• W3100753829 cites W1926834468 @default.
• W3100753829 cites W1940044602 @default.
• W3100753829 cites W1965217223 @default.
• W3100753829 cites W1969557614 @default.
• W3100753829 cites W1978516110 @default.
• W3100753829 cites W1979666591 @default.
• W3100753829 cites W1982410382 @default.
• W3100753829 cites W1985425984 @default.
• W3100753829 cites W1986951078 @default.
• W3100753829 cites W1989171146 @default.
• W3100753829 cites W1990991867 @default.
• W3100753829 cites W2017707927 @default.
• W3100753829 cites W2021885593 @default.
• W3100753829 cites W2026404289 @default.
• W3100753829 cites W2027922031 @default.
• W3100753829 cites W2031266198 @default.
• W3100753829 cites W2032466814 @default.
• W3100753829 cites W2035106298 @default.
• W3100753829 cites W2046001747 @default.
• W3100753829 cites W2046499002 @default.
• W3100753829 cites W2049623473 @default.
• W3100753829 cites W2050723187 @default.
• W3100753829 cites W2052875624 @default.
• W3100753829 cites W2055861028 @default.
• W3100753829 cites W2062470404 @default.
• W3100753829 cites W2063377436 @default.
• W3100753829 cites W2063511140 @default.
• W3100753829 cites W2065231473 @default.
• W3100753829 cites W2077457398 @default.
• W3100753829 cites W2083933392 @default.
• W3100753829 cites W2084575956 @default.
• W3100753829 cites W2087787736 @default.
• W3100753829 cites W2088745158 @default.
• W3100753829 cites W2099133509 @default.
• W3100753829 cites W2101326883 @default.
• W3100753829 cites W2107941234 @default.
• W3100753829 cites W2110530948 @default.
• W3100753829 cites W2113602122 @default.
• W3100753829 cites W2121847900 @default.
• W3100753829 cites W2124671935 @default.
• W3100753829 cites W2133082302 @default.
• W3100753829 cites W2144114582 @default.
• W3100753829 cites W2147686902 @default.
• W3100753829 cites W2155376306 @default.
• W3100753829 cites W2156104877 @default.
• W3100753829 cites W2164707957 @default.
• W3100753829 cites W2170600158 @default.
• W3100753829 cites W2217482446 @default.
• W3100753829 cites W2286983239 @default.
• W3100753829 cites W2327235326 @default.
• W3100753829 cites W2468124248 @default.
• W3100753829 cites W2506551582 @default.
• W3100753829 cites W2515412931 @default.
• W3100753829 cites W2544355903 @default.
• W3100753829 cites W2553689505 @default.
• W3100753829 cites W2558331372 @default.
• W3100753829 cites W2614146854 @default.
• W3100753829 cites W2755866416 @default.
• W3100753829 cites W2789561966 @default.
• W3100753829 cites W2794270490 @default.
• W3100753829 cites W2969937509 @default.
• W3100753829 cites W2972775142 @default.
• W3100753829 cites W2977930626 @default.
• W3100753829 doi "https://doi.org/10.1074/jbc.ra120.016210" @default.
• W3100753829 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7948412" @default.
• W3100753829 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/33199366" @default.
• W3100753829 hasPublicationYear "2021" @default.
• W3100753829 type Work @default.
• W3100753829 sameAs 3100753829 @default.
• W3100753829 citedByCount "5" @default.
• W3100753829 countsByYear W31007538292022 @default.
• W3100753829 countsByYear W31007538292023 @default.
• W3100753829 crossrefType "journal-article" @default.
• W3100753829 hasAuthorship W3100753829A5007533191 @default.
• W3100753829 hasAuthorship W3100753829A5040636021 @default.
• W3100753829 hasAuthorship W3100753829A5064866168 @default.
• W3100753829 hasAuthorship W3100753829A5070992833 @default.
• W3100753829 hasBestOaLocation W31007538291 @default.
• W3100753829 hasConcept C104317684 @default.
• W3100753829 hasConcept C134459356 @default.
• W3100753829 hasConcept C150194340 @default.
• W3100753829 hasConcept C169760540 @default.
• W3100753829 hasConcept C185592680 @default.
• W3100753829 hasConcept C195286587 @default.
• W3100753829 hasConcept C2524010 @default.
• W3100753829 hasConcept C25602115 @default.

• next