FK506-binding protein-like and FK506-binding protein 8 regulate dual leucine zipper kinase degradation and neuronal responses to axon injury

Abstract

The dual leucine zipper kinase (DLK) is a key regulator of axon regeneration and degeneration in response to neuronal injury; however, regulatory mechanisms of the DLK function via its interacting proteins are largely unknown. To better understand the molecular mechanism of DLK function, we performed yeast two-hybrid screening analysis and identified FK506-binding protein-like (FKBPL, also known as WAF-1/CIP1 stabilizing protein 39) as a DLK-binding protein. FKBPL binds to the kinase domain of DLK and inhibits its kinase activity. In addition, FKBPL induces DLK protein degradation through ubiquitin-dependent pathways. We further assessed other members in the FKBP protein family and found that FK506-binding protein 8 (FKBP8) also induced DLK degradation. We identified the lysine 271 residue in the kinase domain as a major site of DLK ubiquitination and SUMO3 conjugation and was thus responsible for regulating FKBP8-mediated proteasomal degradation that was inhibited by the substitution of the lysine 271 to arginine. FKBP8-mediated degradation of DLK is mediated by autophagy pathway because knockdown of Atg5 inhibited DLK destabilization. We show that in vivo overexpression of FKBP8 delayed the progression of axon degeneration and suppressed neuronal death after axotomy in sciatic and optic nerves. Taken together, this study identified FKBPL and FKBP8 as novel DLK-interacting proteins that regulate DLK stability via the ubiquitin-proteasome and lysosomal protein degradation pathways. The dual leucine zipper kinase (DLK) is a key regulator of axon regeneration and degeneration in response to neuronal injury; however, regulatory mechanisms of the DLK function via its interacting proteins are largely unknown. To better understand the molecular mechanism of DLK function, we performed yeast two-hybrid screening analysis and identified FK506-binding protein-like (FKBPL, also known as WAF-1/CIP1 stabilizing protein 39) as a DLK-binding protein. FKBPL binds to the kinase domain of DLK and inhibits its kinase activity. In addition, FKBPL induces DLK protein degradation through ubiquitin-dependent pathways. We further assessed other members in the FKBP protein family and found that FK506-binding protein 8 (FKBP8) also induced DLK degradation. We identified the lysine 271 residue in the kinase domain as a major site of DLK ubiquitination and SUMO3 conjugation and was thus responsible for regulating FKBP8-mediated proteasomal degradation that was inhibited by the substitution of the lysine 271 to arginine. FKBP8-mediated degradation of DLK is mediated by autophagy pathway because knockdown of Atg5 inhibited DLK destabilization. We show that in vivo overexpression of FKBP8 delayed the progression of axon degeneration and suppressed neuronal death after axotomy in sciatic and optic nerves. Taken together, this study identified FKBPL and FKBP8 as novel DLK-interacting proteins that regulate DLK stability via the ubiquitin-proteasome and lysosomal protein degradation pathways. Research in diverse model organisms has yielded critical insights into the molecular mechanisms of neuronal functions under various stress conditions. However, the mechanisms of axon regeneration and degeneration are not fully understood yet. Identifying the molecular mechanisms of regenerative response in injured neurons is required for developing methods promoting functional recovery in the adult mammalian nervous systems. In addition, understanding the mechanisms of axon degeneration is important for identifying therapeutic targets of neurodegeneration. Therefore, it is essential to identify the key molecules regulating axon regeneration and degeneration. Dual leucine zipper kinase (DLK), a mitogen-activated protein triple kinase 12, is a key regulator of neuronal signal transduction for axon regeneration and degeneration (1Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 763-768Google Scholar, 2Welsbie D.S. Schirok H. Mitchell K. Koch M. Kim B.-J. Lobell M. Patel A.K. Holton S. Hristodorov D. Esteve-Rudd J. Berlinicke C. Terjung C. Hansen B.S. Werbeck N. Schubert W. et al.Identification of a retinal neuroprotective kinase inhibitor with preferential activity against DLK compared to LZK.Invest. Ophthalmol. Vis. Sci. 2018; 59: 2493Google Scholar). Dual leucine zipper kinase regulates the c-Jun N-terminal kinase (JNK) signaling pathway in neurons under stress conditions and is essential for injury-induced retrograde signaling that is responsible for differential gene expression (3Shin J.E. Ha H. Kim Y.K. Cho Y. DiAntonio A. DLK regulates a distinctive transcriptional regeneration program after peripheral nerve injury.Neurobiol. Dis. 2019; 127: 178-192Google Scholar, 4Shin J.E. Cho Y. Beirowski B. Milbrandt J. Cavalli V. DiAntonio A. Dual leucine zipper kinase is required for retrograde injury signaling and axonal regeneration.Neuron. 2012; 74: 1015-1022Google Scholar). In addition, DLK is required for axon degeneration as its depletion impairs the process (5Miller B.R. Press C. Daniels R.W. Sasaki Y. Milbrandt J. Diantonio A. A dual leucine kinase-dependent axon self-destruction program promotes Wallerian degeneration.Nat. Neurosci. 2009; 12: 387-389Google Scholar). Therefore, identifying DLK regulators is of major relevance for obtaining a better understanding of axon regeneration and degeneration. Protein degradation pathways are core signaling axes, regulating neuronal responses to a diverse range of stresses (6Nakata K. Abrams B. Grill B. Goncharov A. Huang X. Chisholm A.D. Jin Y. Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development.Cell. 2005; 120: 407-420Google Scholar, 7Collins C.A. Wairkar Y.P. Johnson S.L. DiAntonio A. Highwire restrains synaptic growth by attenuating a MAP kinase signal.Neuron. 2006; 51: 57-69Google Scholar). Because DLK is a key regulator of neurodegenerative signal transduction, the posttranslational modification of DLK has been studied to understand the mechanisms determining DLK activity, localization, and protein levels (1Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 763-768Google Scholar, 8Huntwork-Rodriguez S. Wang B. Watkins T. Ghosh A.S. Pozniak C.D. Bustos D. Newton K. Kirkpatrick D.S. Lewcock J.W. JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis.J. Cell Biol. 2013; 202: 747-763Google Scholar, 9Watkins T.A. Wang B. Huntwork-Rodriguez S. Yang J. Jiang Z. Eastham-Anderson J. Modrusan Z. Kaminker J.S. Tessier-Lavigne M. Lewcock J.W. DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 4039-4044Google Scholar, 10Larhammar M. Huntwork-Rodriguez S. Jiang Z. Solanoy H. Ghosh A.S. Wang B. Kaminker J.S. Huang K. Eastham-Anderson J. Siu M. Modrusan Z. Farley M.M. Tessier-Lavigne M. Lewcock J.W. Watkins T.A. Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult.Elife. 2017; 6e20725Google Scholar). For example, phosphorylation and palmitoylation of DLK regulate its function (11Montersino A. Thomas G.M. Slippery signaling: Palmitoylation-dependent control of neuronal kinase localization and activity.Mol. Membr. Biol. 2015; 32: 179-188Google Scholar, 12Niu J. Sanders S.S. Jeong H.K. Holland S.M. Sun Y. Collura K.M. Hernandez L.M. Huang H. Hayden M.R. Smith G.M. Hu Y. Jin Y. Thomas G.M. Coupled control of distal axon integrity and somal responses to axonal damage by the palmitoyl acyltransferase ZDHHC17.Cell Rep. 2020; 33: 108365Google Scholar, 13Martin D.D.O. Kanuparthi P.S. Holland S.M. Sanders S.S. Jeong H.K. Einarson M.B. Jacobson M.A. Thomas G.M. Identification of novel inhibitors of DLK palmitoylation and signaling by high content screening.Sci. Rep. 2019; 9: 1-12Google Scholar). In addition, DLK protein stability is modulated by the PHR1 E3 ubiquitin ligase and deubiquitinating enzyme USP9X in a key pathway that determines neuronal fate after injury (8Huntwork-Rodriguez S. Wang B. Watkins T. Ghosh A.S. Pozniak C.D. Bustos D. Newton K. Kirkpatrick D.S. Lewcock J.W. JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis.J. Cell Biol. 2013; 202: 747-763Google Scholar, 9Watkins T.A. Wang B. Huntwork-Rodriguez S. Yang J. Jiang Z. Eastham-Anderson J. Modrusan Z. Kaminker J.S. Tessier-Lavigne M. Lewcock J.W. DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 4039-4044Google Scholar, 10Larhammar M. Huntwork-Rodriguez S. Jiang Z. Solanoy H. Ghosh A.S. Wang B. Kaminker J.S. Huang K. Eastham-Anderson J. Siu M. Modrusan Z. Farley M.M. Tessier-Lavigne M. Lewcock J.W. Watkins T.A. Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult.Elife. 2017; 6e20725Google Scholar, 14Babetto E. Beirowski B. Russler E.V. Milbrandt J. DiAntonio A. The Phr1 ubiquitin ligase promotes injury-induced axon self-destruction.Cell Rep. 2013; 3: 1422-1429Google Scholar). Here, we discovered a molecule that interacts with DLK, regulating neuronal responses for axon degeneration. In the present study, we identified FK506-binding protein-like (FKBPL) and FK506-binding protein 8 (FKBP8; FKBP38) as DLK-binding proteins. FK506-binding proteins (FKBPs) belong to the immunophilin family, a group of receptors for immunosuppressive drugs like FK506, rapamycin, and cyclosporin A. FKBPs have a biological function that can regulate or stabilize the components of multiprotein complexes essential to cell function. FKBPL is a divergent immunophilin with distinct functions in disease states, especially appears to have neuroprotective properties. Furthermore, FKBP8 is known to regulate parkin-independent mitophagy, which involves the removal of mitochondria via autophagy and lysosomal degradation (15Yoo S.-M. Yamashita S. Kim H. Na D. Lee H. Kim S.J. Cho D.-H. Kanki T. Jung Y.-K. FKBP8 LIRL-dependent mitochondrial fragmentation facilitates mitophagy under stress conditions.FASEB J. 2020; 34: 2944-2957Google Scholar, 16Misaka T. Murakawa T. Nishida K. Omori Y. Taneike M. Omiya S. Molenaar C. Uno Y. Yamaguchi O. Takeda J. Shah A.M. Otsu K. FKBP8 protects the heart from hemodynamic stress by preventing the accumulation of misfolded proteins and endoplasmic reticulum-associated apoptosis in mice.J. Mol. Cell. Cardiol. 2018; 114: 93-104Google Scholar, 17Lim G.G. Lim K.-L. Parkin-independent mitophagy-FKBP8 takes the stage.EMBO Rep. 2017; 18: 864-865Google Scholar, 18Bhujabal Z. Birgisdottir Å.B. Sjøttem E. Brenne H.B. Øvervatn A. Habisov S. Kirkin V. Lamark T. Johansen T. FKBP8 recruits LC3A to mediate Parkin-independent mitophagy.EMBO Rep. 2017; 18: Scholar). We found that FKBPL is a of the FKBP family of a group of proteins to immunosuppressive as FK506, rapamycin, and cyclosporin A. FKBPL and FKBP8 to DLK to regulate its kinase activity and degradation in and in thus neuronal responses in sciatic and optic after To identify DLK-interacting we performed yeast two-hybrid screening analysis and found and as and the gene was as the of DLK A and FKBPL is a of the FKBP family, members binds to and has activity M. A protein for the has activity is distinct from Scholar). the screening as DLK and FKBPL in To the responsible for to of the N-terminal the domain of FKBPL and the DLK and FKBPL In addition, the N-terminal which the kinase domain of DLK, was to mediate the as of DLK with its N-terminal and to FKBPL that the N-terminal of proteins responsible for with the FKBPL domain and the DLK kinase domain into a to as the Taken together, yeast two-hybrid screening and that FKBPL was a of FKBPL to kinase we performed in kinase to DLK kinase activity was modulated via its with FKBPL (1Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 763-768Google was with or in the and with the protein kinase kinase in A and analysis that was after with as (1Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 763-768Google Scholar). However, phosphorylation was with the DLK that the of FKBPL inhibited DLK kinase activity and FKBPL was to the kinase this from interacting with the DLK kinase Dual leucine zipper kinase activity was required for its and the kinase of DLK the protein (8Huntwork-Rodriguez S. Wang B. Watkins T. Ghosh A.S. Pozniak C.D. Bustos D. Newton K. Kirkpatrick D.S. Lewcock J.W. JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis.J. Cell Biol. 2013; 202: 747-763Google Scholar). FKBPL to DLK and inhibited its kinase activity, we the protein of DLK with FKBPL and found that DLK protein levels FKBPL was in and However, DLK was not by a inhibitor of and In addition, inhibitor DLK Therefore, the by not responsible for the of DLK protein that FKBPL inhibits DLK kinase activity and its protein FKBPs are a group of proteins FK506-binding domain and a domain J. the of protein signal transduction, and Biol. Scholar, L.M. M. in protein Protein Scholar, Y. S. FKBP family with biological Scholar, G. Z. Wang X. M.M. O. Y. S. J. J. X. analysis of FKBP family and function in and Biol. 2018; 18: Scholar, M. Jiang Y. FK506-binding proteins and diverse Mol. 2015; 9: we other FKBP proteins to the of DLK protein we the expression levels of in and sciatic from (3Shin J.E. Ha H. Kim Y.K. Cho Y. DiAntonio A. DLK regulates a distinctive transcriptional regeneration program after peripheral nerve injury.Neurobiol. Dis. 2019; 127: 178-192Google Scholar, J.E. Ha H. Cho Kim Y.K. Cho Y. analysis of the of injured nerve responses to damage in mice.J. 2018; Scholar, B. Lee J. Y. Kim H. M. J.E. Cho Y. axon regeneration by the function of the gene Scholar). In addition, the from neurons was as with the levels of neuronal expression Y. K.M. Cavalli V. is essential for axon 2013; Scholar). and the in adult and sciatic In addition, the neuronal expression of that and compared to other family which was in neurons Y. K.M. Cavalli V. is essential for axon 2013; Scholar). analysis that and FKBPs in we DLK under of the FKBPL in DLK protein levels to compared to control levels analysis that FKBP8 DLK protein to and a DLK protein levels and In addition, analysis that and FKBP8 with DLK, not with it FKBP8 regulates parkin-independent mitophagy, which facilitates the removal of mitochondria via autophagy and lysosomal degradation (15Yoo S.-M. Yamashita S. Kim H. Na D. Lee H. Kim S.J. Cho D.-H. Kanki T. Jung Y.-K. FKBP8 LIRL-dependent mitochondrial fragmentation facilitates mitophagy under stress conditions.FASEB J. 2020; 34: 2944-2957Google Scholar, 16Misaka T. Murakawa T. Nishida K. Omori Y. Taneike M. Omiya S. Molenaar C. Uno Y. Yamaguchi O. Takeda J. Shah A.M. Otsu K. FKBP8 protects the heart from hemodynamic stress by preventing the accumulation of misfolded proteins and endoplasmic reticulum-associated apoptosis in mice.J. Mol. Cell. Cardiol. 2018; 114: 93-104Google Scholar, 17Lim G.G. Lim K.-L. Parkin-independent mitophagy-FKBP8 takes the stage.EMBO Rep. 2017; 18: 864-865Google Scholar, 18Bhujabal Z. Birgisdottir Å.B. Sjøttem E. Brenne H.B. Øvervatn A. Habisov S. Kirkin V. Lamark T. Johansen T. FKBP8 recruits LC3A to mediate Parkin-independent mitophagy.EMBO Rep. 2017; 18: Scholar, Y. S. FKBP family with biological Scholar). FKBP8 to DLK and its protein we or DLK was mediated via lysosomal degradation. with and DLK degradation was that DLK was via the the protein levels of DLK FKBP8 or FKBPL under further lysosomal degradation as a pathway DLK protein To the in FKBP8 was by into DLK proteins from neurons by that FKBP8 DLK protein stability in and Taken together, we that FKBPL and FKBP8 are DLK-interacting proteins that its degradation via the Dual leucine zipper kinase protein degradation is by the PHR1 E3 ligase (6Nakata K. Abrams B. Grill B. Goncharov A. Huang X. Chisholm A.D. Jin Y. Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development.Cell. 2005; 120: 407-420Google Scholar, 7Collins C.A. Wairkar Y.P. Johnson S.L. DiAntonio A. Highwire restrains synaptic growth by attenuating a MAP kinase signal.Neuron. 2006; 51: 57-69Google Scholar). or DLK protein required lysosomal function, we DLK protein was mediated via ubiquitin-dependent degradation. DLK was to ubiquitination by ubiquitin in that was with DLK in FKBP8 was with DLK in DLK proteins which was by with inhibitor of ubiquitin-dependent protein degradation that FKBP8 induced DLK degradation via the ubiquitin-dependent protein degradation To identify lysine responsible for we assessed lysine the kinase domain of DLK as the site of FKBPL We that was by a residue known to by and essential for DLK kinase activity, as the (8Huntwork-Rodriguez S. Wang B. Watkins T. Ghosh A.S. Pozniak C.D. Bustos D. Newton K. Kirkpatrick D.S. Lewcock J.W. JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis.J. Cell Biol. 2013; 202: 747-763Google Scholar). The residue was the for DLK phosphorylation and the residue this was Therefore, we that it as for DLK by its kinase activity its with posttranslational To ubiquitination the that this lysine with the of ubiquitin conjugation to DLK in that the lysine 271 was a major site of DLK Furthermore, also as the site of as DLK was with SUMO3 was in the was not To DLK in sciatic and after The in conditions and to analysis that DLK protein levels the injured and Taken together, that was the major site of ubiquitination and SUMO3 the residue was responsible for DLK we this site was required for DLK degradation. analysis that the was to degradation a substitution inhibited FKBP8-mediated DLK degradation that the ubiquitination of DLK was required for its FKBP8-mediated that DLK protein to the protein degradation In addition, the a with that DLK ubiquitination DLK and FKBP8 To the in DLK protein levels by analysis of neurons with or overexpression DLK protein was FKBP8 was in However, the DLK against degradation in that FKBP8 induced DLK degradation via the pathway in In addition, DLK degradation was Atg5 was in neurons and that autophagy was in FKBP8-mediated DLK degradation. To this in with as in and to sciatic nerve injury analysis that sciatic of DLK protein. sciatic for DLK protein was the injury site in and However, DLK accumulation was under FKBP8 that FKBP8 neuronal DLK protein levels via ubiquitin and autophagy pathways. FKBP8 with DLK to regulate its we neuronal injury responses under FKBP8 overexpression in In vivo gene FKBP8 overexpression in To axon degeneration in sciatic from or and the distal that was from the cell was after axotomy analysis that FKBP8 overexpression delayed axon degeneration in sciatic because sciatic of the distal nerve in and the of axonal with the we that control sciatic of with a of sciatic from of a DLK is responsible for retinal cell apoptosis after optic nerve injury (8Huntwork-Rodriguez S. Wang B. Watkins T. Ghosh A.S. Pozniak C.D. Bustos D. Newton K. Kirkpatrick D.S. Lewcock J.W. JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis.J. Cell Biol. 2013; 202: 747-763Google Scholar, 10Larhammar M. Huntwork-Rodriguez S. Jiang Z. Solanoy H. Ghosh A.S. Wang B. Kaminker J.S. Huang K. Eastham-Anderson J. Siu M. Modrusan Z. Farley M.M. Tessier-Lavigne M. Lewcock J.W. Watkins T.A. Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult.Elife. 2017; 6e20725Google we FKBP8 overexpression death after of after injury and for as a of We a in in the with injury compared to control of injury the to However, the of after optic nerve injury that injury-induced after was under FKBP8 that FKBP8 is a in the molecular mechanism regulating axon degeneration and neuronal DLK is a core protein responsible for the of neuronal responses to DLK a in axon regeneration and it has been to as a in A. The DLK pathway a in and Rep. 2013; Scholar). Therefore, it is essential to the molecular mechanisms regulating DLK function to a better understanding of neuronal responses to In this study, we identified DLK-interacting proteins FKBPL and FKBP8 as the regulators of DLK degradation and kinase activity. FKBPL and FKBP8 to the kinase domain of DLK to its activity. In addition, FKBP8 induced the degradation of DLK through the lysosomal degradation In vivo gene of FKBP8 delayed axon degeneration in sciatic after axotomy and a against death after Dual leucine zipper kinase protein levels are neurons are to as axotomy and signaling is important for somal not axonal degeneration of retinal following axonal injury.Neurobiol. Dis. Scholar, E. Milbrandt J. DiAntonio A. DLK activation with mitochondrial to axon and promote axon 2020; Scholar, V. E. E. DiAntonio A. the which promotes axonal regeneration and a injury.Neurobiol. Dis. 2015; Scholar, Y. B. Dual leucine in neuronal and stress Cell Biol. 2019; Scholar, M. distinct 2016; Scholar, A. insights to therapeutic for the and of peripheral 2019; Scholar). DLK protein levels in neuronal death in optic nerve injury (8Huntwork-Rodriguez S. Wang B. Watkins T. Ghosh A.S. Pozniak C.D. Bustos D. Newton K. Kirkpatrick D.S. Lewcock J.W. JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis.J. Cell Biol. 2013; 202: 747-763Google Scholar, 10Larhammar M. Huntwork-Rodriguez S. Jiang Z. Solanoy H. Ghosh A.S. Wang B. Kaminker J.S. Huang K. Eastham-Anderson J. Siu M. Modrusan Z. Farley M.M. Tessier-Lavigne M. Lewcock J.W. Watkins T.A. Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult.Elife. 2017; 6e20725Google Scholar). Therefore, identifying the molecular mechanism DLK protein is important for understanding neuronal fate is in response to PHR1 E3 ligase and the deubiquitinating enzyme USP9X are the key regulators of DLK protein levels (8Huntwork-Rodriguez S. Wang B. Watkins T. Ghosh A.S. Pozniak C.D. Bustos D. Newton K. Kirkpatrick D.S. Lewcock J.W. JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis.J. Cell Biol. 2013; 202: 747-763Google Scholar, 14Babetto E. Beirowski B. Russler E.V. Milbrandt J. DiAntonio A. The Phr1 ubiquitin ligase promotes injury-induced axon self-destruction.Cell Rep. 2013; 3: 1422-1429Google Scholar). we the DLK protein degradation through the of lysine 271 as the residue responsible for the DLK ubiquitination and the kinase that ubiquitination and this In addition, the lysine 271 is required for ubiquitin-dependent DLK lysosomal degradation. In the and FKBP8-mediated DLK protein degradation via the a for the of DLK protein levels in vivo for the study of and neurodegenerative conditions. and in the present and by the was performed under following regulatory nerve injury performed as Y. V. D. N. S. Cavalli V. expression is by mammalian of in injured neurons to promote axon Biol. Scholar). to of the sciatic nerve and a injury was with for J. J.E. Lee B. Kim H. Y. Cho Y. The cell promotes axon regeneration by via signaling.Proc. Natl. Acad. Sci. U. S. A. 2020; Scholar). gene was to knockdown in was with as Y. Cavalli V. is a novel axon J. 2012; Scholar). For in gene was to To or Atg5 in identified by the or and into a with was with a and a as Y. J.E. M. Cho E. Cavalli V. Cho Y. In vivo gene of promotes axon regeneration in sciatic Scholar). was To in of and was into via a with a The expression of and the gene in sciatic and was via and The following for for for and for and We in for which was in with as or in the of We inhibitor and inhibitor and and expression for and from expression for and from The into two-hybrid analysis was performed a with The was from the and into the of a with the and DLK to from To study and into following the Cell in and and for protein in the was via protein with as of protein into for and a The with in with for with and with The with for and with Protein expression levels via and in a inhibitor was with to Protein A from for The and to for and or in a inhibitor Dual leucine zipper kinase was with to Protein A from for For and in and for a inhibitor was with to Protein A from neurons was performed the as The was for and the for expression levels via the with as Dual leucine zipper kinase activity was assessed as (1Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 763-768Google Scholar). with or Cell in a inhibitor was with Protein The was following a (1Holland S.M. Collura K.M. Ketschek A. Noma K. Ferguson T.A. Jin Y. Gallo G. Thomas G.M. Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 763-768Google Scholar). for in of kinase and and of or The by the of and to analysis with and sciatic nerve in for after and in in a of and as the in and in for and with in The with in with for with and or The as under a and For in vivo degeneration the distal of the sciatic nerve was from the injury site after axotomy in control and in sciatic a of and with a The into and with a of We the in the distal nerve and compared and To of for from biological with a We in and compared and the To the optic the from the of the was and the optic nerve was for a not to damage the was and after the injury to the from after the and via in a for in the to for The optic with the in a for and or is in the The that have of with the of this was by a B. A. V. J. E. and Y. C. B. E. J. E. H. W. and Y. C. B. Y. E. J. E. H. W. and Y. C. B. Y. E. J. E. H. W. and Y. C. J. E. S. and Y. C. J. E. S. and Y. C. A. V. J. E. and Y. C. A. V. J. E. and Y. C. B. H. W. and Y. C. B. H. W. and Y. C. B. and Y. C. B. A. V. J. E. H. W. and Y. C. and was by a Research of by the to Y. and to J. E.

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