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The X-Linked Intellectual Disability Gene Product and E3 Ubiquitin Ligase KLHL15 Degrades Doublecortin Proteins to Constrain Neuronal Dendritogenesis

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The X-Linked Intellectual Disability Gene Product and E3 Ubiquitin Ligase KLHL15 Degrades Doublecortin Proteins to Constrain Neuronal Dendritogenesis bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.324285; this version posted October 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. KLHL15 degrades doublecortin proteins The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis Jianing Song1,2, Ronald A. Merrill1,2, Andrew Y. Usachev1, and Stefan Strack1* From the 1Department of Neuroscience and Pharmacology and the Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa 52242 2 These authors contributed equally to the work. *To whom correspondence should be addressed: Stefan Strack: Dept. of Neuroscience and Pharmacology, The University of Iowa, Iowa City, IA 52242; [email protected]; Tel. (319) 384-4439; Fax. (319) 335-8930 Running Title: KLHL15 degrades doublecortin proteins Keywords: Kelch-like 15, KLHL15, E3 ubiquitin ligase, protein phosphatase 2A, signal transduction, doublecortin, doublecortin-like kinases, microtubule-associated protein, ubiquitination, proteasomal degradation, protein turnover, neurite outgrowth, dendritic complexity, pulse-chase, HaloTag ______________________________________________________________________________ ABSTRACT doublecortin (DCX), also an X-linked disease gene, and doublecortin-like kinases 1 and 2 Proper brain development and function (DCLK1/2) as bona fide KLHL15 interactors requires finely controlled mechanisms for and mapped KLHL15 interaction regions to protein turnover and disruption of genes their tandem DCX domains. Shared with two involved in proteostasis is a common cause of previously identified KLHL15 substrates, a neurodevelopmental disorders. Kelch-like 15 FRY tripeptide at the C-terminal edge of the (KLHL15) is a substrate adaptor for cullin3 second DCX domain is necessary for (Cul3)-containing E3 ubiquitin ligases and KLHL15-mediated ubiquitination of DCX KLHL15 gene mutations were recently and DCLK1/2 and subsequent proteasomal described as a cause of severe X-linked degradation. Conversely, silencing intellectual disability. Here, we used a endogenous KLHL15 markedly stabilizes bioinformatics approach to identify a family these DCX domain-containing proteins and of neuronal microtubule-associated proteins prolongs their half-life. Functionally, (MAPs) as KLHL15 substrates, which are overexpression of KLHL15 in the presence of themselves critical for early brain wild-type DCX reduces dendritic complexity development. We biochemically validated 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.324285; this version posted October 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. KLHL15 degrades doublecortin proteins of cultured hippocampal neurons, whereas genetic mutations and abnormal expression of neurons expressing FRY-mutant DCX are KLHL genes have been linked to diverse resistant to KLHL15. Collectively, our diseases, ranging from cardiovascular findings highlight the critical importance of disorders to cancer (12-14). the E3 ubiquitin ligase adaptor KLHL15 in We have previously demonstrated that proteostasis of neuronal MAPs and identify a KLHL15 mediates the ubiquitination and regulatory network important for development subsequent proteasomal degradation of the of the mammalian nervous system. B'β (B56β, PR61β, PPP2R5B) regulatory subunit of the protein Ser/Thr phosphatase 2A INTRODUCTION (PP2A). KLHL15-mediated B'β degradation facilitates the formation of alternative PP2A Ubiquitination is a highly coordinated holoenzymes by promoting the exchange with multi-step enzymatic cascade that requires the more than a dozen other regulatory subunits concerted action of three key factors: E1 (15). PP2A/B'β is highly enriched in the ubiquitin-activating enzymes, E2 ubiquitin- mammalian nervous system (16-18) and plays conjugating enzymes, and E3 ubiquitin ligases key roles in striatal dopamine biosynthesis (1), many of which are strongly implicated the (16), hippocampal long-term potentiation (19), pathogenesis of neurodevelopmental and multiple intracellular signaling pathways disorders (2-4). E3 ubiquitin ligases have in response to growth factor stimulation (20- received particular attention, as they confer 22). We also identified a tyrosine residue (Y52) substrate specificity of the ubiquitination within a phylogenetically well-conserved reaction by catalyzing the transfer of ubiquitin tripeptide motif (FRY) in vertebrate B'β as to proteins destined for degradation or other being necessary for KLHL15 binding and ubiquitin-dependent fates (5-7). KLHL15-mediated B'β downregulation (15). Recent studies have identified Since then, work by others identified a critical KLHL15 as a X-linked intellectual disability FRY tripeptide in the degron of a second (XLID) gene. Inactivating KLHL15 mutations KLHL15 substrate, the DNA repair protein are associated with various brain CtIP/RBBP8 (23). abnormalities as well as developmental and Because of KLHL15’s link to severe behavioral disorders in male patients (8-11). intellectual disability and its ranking as one of Localized on chromosome Xp22.11, KLHL15 the most clinically significant X-linked encodes a member of the Kelch-like (KLHL) disease genes (8,24), we set out to uncover family of proteins that function as adaptors for novel neuronal substrates of the E3 ubiquitin Cullin3 (Cul3)-based E3 ubiquitin ligases to ligase by primary and secondary structure target specific substrates to the ubiquitin- searches. This screen and subsequent proteasome system (12). Numbering more biochemical validation identified doublecortin than 40 in humans, KLHL proteins exert a (DCX) and two doublecortin-like kinases wide range of biological functions, while (DCLK1, DCLK2), members of a family of 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.324285; this version posted October 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. KLHL15 degrades doublecortin proteins neuronal microtubule-associated proteins degradation requires a degron containing both (MAPs) that share a characteristic domain N-terminal DCX domains and a conserved structure of N-terminal tandem DCX domains. FRY tripeptide. Functionally, we show that These tandem DCX domains stabilize KLHL15 expression reduces dendritic microtubule by enhancing tubulin arborization in primary hippocampal cultures polymerization, inhibiting tubulin catastrophe, unless neurons also express DCX with a and promoting microtubule bundling (25-28). stabilizing FRY mutation. Our study uncovers DCX plays essential roles in neurogenesis, a novel role of the E3 ubiquitin ligase adaptor neuronal migration, axonal/dendritic wiring, KLHL15 as a negative regulator of DCX and cargo transport therefore ensuring normal protein abundance and dendritogenesis and brain development (28,29). The DCX gene is suggests possible mechanistic connections in located on the X chromosome and DCX X-linked neurodevelopmental disorders. mutations cause classic lissencephaly (agyria) RESULTS in males and subcortical band heterotopia (also called double cortex syndrome) KLHL15 regulates signal transduction in primarily in females as a result of neuronal hippocampal neurons by PP2A-dependent migration defects (30). Both disorders and independent mechanisms typically manifest with intellectual disability, To determine whether KLHL15 epilepsy, language impairment, and global possesses functions in neurons independent of developmental delay (30). DCLK1 and B'β, a neuron-enriched regulatory subunit of DCLK2 feature a C-terminal Ser/Thr kinase PP2A (16-18), we investigated the effect of domain and play both common and distinct KLHL15 on activation of extracellular signal- roles in neurodevelopment. Both kinases regulated kinases (ERKs). We previously associate with microtubules and stimulate reported that the B'β-containing PP2A microtubule polymerization and dendritic holoenzyme potentiates nerve growth factor development independent of their kinase (NGF) signaling at the tropomyosin-related activity (31-33). DCLK1 and DCX kinase A (TrkA) receptor level to sustain ERK synergistically regulate neuronal migration, signaling and neuronal differentiation of axonal outgrowth, and hippocampal neuroendocrine PC12 cells (22). To address development (34-36), whereas DCLK2 works the hypothesis that PP2A/B'β may similarly in concert with DCX to organize hippocampal potentiate TrkB receptor activity in central lamination and prevent spontaneous seizures neurons, we measured ERK activity in (37). transiently transfected primary hippocampal This report documents that three neurons from E18 rat embryos using a dual members of the DCX family of neuronal luciferase reporter assay (PathDetect Elk1 MAPs, are subject to KLHL15-mediated reporter, Fig. 1A) (22). In GFP control- downregulation. KLHL15-dependent transfected neurons, a 3 h incubation with a polyubiquitination and proteasomal sub-saturating concentration of brain-derived neurotrophic factor (BDNF) resulted in a ~17- 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.324285; this version posted October 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed
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