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• W2022903577 abstract "MAPKKK dual leucine zipper-bearing kinases (DLKs) are regulators of synaptic development and axon regeneration. The mechanisms underlying their activation are not fully understood. Here, we show that C. elegans DLK-1 is activated by a Ca2+-dependent switch from inactive heteromeric to active homomeric protein complexes. We identify a DLK-1 isoform, DLK-1S, that shares identical kinase and leucine zipper domains with the previously described long isoform DLK-1L but acts to inhibit DLK-1 function by binding to DLK-1L. The switch between homo- or heteromeric DLK-1 complexes is influenced by Ca2+ concentration. A conserved hexapeptide in the DLK-1L C terminus is essential for DLK-1 activity and is required for Ca2+ regulation. The mammalian DLK-1 homolog MAP3K13 contains an identical C-terminal hexapeptide and can functionally complement dlk-1 mutants, suggesting that the DLK activation mechanism is conserved. The DLK activation mechanism is ideally suited for rapid and spatially controlled signal transduction in response to axonal injury and synaptic activity. MAPKKK dual leucine zipper-bearing kinases (DLKs) are regulators of synaptic development and axon regeneration. The mechanisms underlying their activation are not fully understood. Here, we show that C. elegans DLK-1 is activated by a Ca2+-dependent switch from inactive heteromeric to active homomeric protein complexes. We identify a DLK-1 isoform, DLK-1S, that shares identical kinase and leucine zipper domains with the previously described long isoform DLK-1L but acts to inhibit DLK-1 function by binding to DLK-1L. The switch between homo- or heteromeric DLK-1 complexes is influenced by Ca2+ concentration. A conserved hexapeptide in the DLK-1L C terminus is essential for DLK-1 activity and is required for Ca2+ regulation. The mammalian DLK-1 homolog MAP3K13 contains an identical C-terminal hexapeptide and can functionally complement dlk-1 mutants, suggesting that the DLK activation mechanism is conserved. The DLK activation mechanism is ideally suited for rapid and spatially controlled signal transduction in response to axonal injury and synaptic activity. DLK-1 short isoform acts as an endogenous inhibitor to DLK-1 functions A novel conserved hexapeptide is required for DLK-1 activation Ca2+ regulates DLK-1 isoform-specific interactions Human MAP3K13 contains the same hexapeptide and complements DLK-1 function in vivo MAP kinase-mediated signal transduction pathways have been implicated in many aspects of neuronal development and function (Huang and Reichardt, 2001Huang E.J. Reichardt L.F. Neurotrophins: roles in neuronal development and function.Annu. Rev. Neurosci. 2001; 24: 677-736Crossref PubMed Scopus (3333) Google Scholar; Ji et al., 2009Ji R.R. Gereau 4th, R.W. Malcangio M. Strichartz G.R. MAP kinase and pain.Brain Res. Brain Res. Rev. 2009; 60: 135-148Crossref PubMed Scopus (791) Google Scholar; Mielke and Herdegen, 2000Mielke K. Herdegen T. JNK and p38 stresskinases—degenerative effectors of signal-transduction-cascades in the nervous system.Prog. Neurobiol. 2000; 61: 45-60Crossref PubMed Scopus (437) Google Scholar; Samuels et al., 2009Samuels I.S. Saitta S.C. Landreth G.E. MAP’ing CNS development and cognition: an ERKsome process.Neuron. 2009; 61: 160-167Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar; Subramaniam and Unsicker, 2010Subramaniam S. Unsicker K. ERK and cell death: ERK1/2 in neuronal death.FEBS J. 2010; 277: 22-29Crossref PubMed Scopus (210) Google Scholar; Thomas and Huganir, 2004Thomas G.M. Huganir R.L. MAPK cascade signalling and synaptic plasticity.Nat. Rev. Neurosci. 2004; 5: 173-183Crossref PubMed Scopus (1139) Google Scholar). As neurons are highly polarized cells receiving spatially segregated information, a critical aspect of MAP kinases is their ability to be locally regulated within cells and with tight temporal control. For example, in developing axons, local activation of p38 and Erk MAP kinases by the MAPKK MEK1/MEK2 is differentially required for BDNF and netrin-1-induced growth cone turning (Ming et al., 2002Ming G.L. Wong S.T. Henley J. Yuan X.B. Song H.J. Spitzer N.C. Poo M.M. Adaptation in the chemotactic guidance of nerve growth cones.Nature. 2002; 417: 411-418Crossref PubMed Scopus (357) Google Scholar) and slit-2-induced growth cone collapse (Piper et al., 2006Piper M. Anderson R. Dwivedy A. Weinl C. van Horck F. Leung K.M. Cogill E. Holt C. Signaling mechanisms underlying Slit2-induced collapse of Xenopus retinal growth cones.Neuron. 2006; 49: 215-228Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Local activation of MAP kinases by neuronal excitation plays important roles in dendritic spine dynamics (Wu et al., 2001Wu G.Y. Deisseroth K. Tsien R.W. Spaced stimuli stabilize MAPK pathway activation and its effects on dendritic morphology.Nat. Neurosci. 2001; 4: 151-158Crossref PubMed Scopus (328) Google Scholar). Axonal injury can trigger activation of Erk at the injury site to regulate signal transduction via retrograde transport (Perlson et al., 2005Perlson E. Hanz S. Ben-Yaakov K. Segal-Ruder Y. Seger R. Fainzilber M. Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve.Neuron. 2005; 45: 715-726Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). In a typical MAP kinase cascade, activation of the upstream MAPKKK is a critical control point for signal specificity and amplification (Chang and Karin, 2001Chang L. Karin M. Mammalian MAP kinase signalling cascades.Nature. 2001; 410: 37-40Crossref PubMed Scopus (4381) Google Scholar). However, our knowledge of how MAPKKKs are activated in vivo by local neuronal signals remains limited. The dual leucine zipper-bearing kinases (DLKs) are key regulators of synapse formation, axon regeneration, and axon degeneration from C. elegans to mammals (Collins et al., 2006Collins C.A. Wairkar Y.P. Johnson S.L. DiAntonio A. Highwire restrains synaptic growth by attenuating a MAP kinase signal.Neuron. 2006; 51: 57-69Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar; Hammarlund et al., 2009Hammarlund M. Nix P. Hauth L. Jorgensen E.M. Bastiani M. Axon regeneration requires a conserved MAP kinase pathway.Science. 2009; 323: 802-806Crossref PubMed Scopus (319) Google Scholar; Itoh et al., 2009Itoh A. Horiuchi M. Bannerman P. Pleasure D. Itoh T. Impaired regenerative response of primary sensory neurons in ZPK/DLK gene-trap mice.Biochem. Biophys. Res. Commun. 2009; 383: 258-262Crossref PubMed Scopus (69) Google Scholar; Miller et al., 2009Miller 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: 387-389Crossref PubMed Scopus (219) Google Scholar; Nakata et al., 2005Nakata 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: 407-420Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar; Xiong and Collins, 2012Xiong X. Collins C.A. A conditioning lesion protects axons from degeneration via the Wallenda/DLK MAP kinase signaling cascade.J. Neurosci. 2012; 32: 610-615Crossref PubMed Scopus (64) Google Scholar; Xiong et al., 2010Xiong X. Wang X. Ewanek R. Bhat P. Diantonio A. Collins C.A. Protein turnover of the Wallenda/DLK kinase regulates a retrograde response to axonal injury.J. Cell Biol. 2010; 191: 211-223Crossref PubMed Scopus (202) Google Scholar; Yan et al., 2009Yan D. Wu Z. Chisholm A.D. Jin Y. The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration.Cell. 2009; 138: 1005-1018Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar; Shin et al., 2012Shin 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: 1015-1022Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). The DLK kinases belong to the mixed-lineage family of MAPKKKs (Holzman et al., 1994Holzman L.B. Merritt S.E. Fan G. Identification, molecular cloning, and characterization of dual leucine zipper bearing kinase. A novel serine/threonine protein kinase that defines a second subfamily of mixed lineage kinases.J. Biol. Chem. 1994; 269: 30808-30817Abstract Full Text PDF PubMed Google Scholar). The hallmark of these kinases is a leucine zipper domain, which can mediate protein dimerization or oligomerization and has been implicated in kinase activation (Nihalani et al., 2000Nihalani D. Merritt S. Holzman L.B. Identification of structural and functional domains in mixed lineage kinase dual leucine zipper-bearing kinase required for complex formation and stress-activated protein kinase activation.J. Biol. Chem. 2000; 275: 7273-7279Crossref PubMed Scopus (59) Google Scholar). Although C. elegans and Drosophila each has only one gene encoding DLK kinase (Nakata et al., 2005Nakata 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: 407-420Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar; Collins et al., 2006Collins C.A. Wairkar Y.P. Johnson S.L. DiAntonio A. Highwire restrains synaptic growth by attenuating a MAP kinase signal.Neuron. 2006; 51: 57-69Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), mammalian genomes encode two closely related DLK family kinases known as MAP3K12/DLK/MUK/ZPK (Blouin et al., 1996Blouin R. Beaudoin J. Bergeron P. Nadeau A. Grondin G. Cell-specific expression of the ZPK gene in adult mouse tissues.DNA Cell Biol. 1996; 15: 631-642Crossref PubMed Scopus (22) Google Scholar; Hirai et al., 1996Hirai S. Izawa M. Osada S. Spyrou G. Ohno S. Activation of the JNK pathway by distantly related protein kinases, MEKK and MUK.Oncogene. 1996; 12: 641-650PubMed Google Scholar; Holzman et al., 1994Holzman L.B. Merritt S.E. Fan G. Identification, molecular cloning, and characterization of dual leucine zipper bearing kinase. A novel serine/threonine protein kinase that defines a second subfamily of mixed lineage kinases.J. Biol. Chem. 1994; 269: 30808-30817Abstract Full Text PDF PubMed Google Scholar) and MAP3K13/LZK (Sakuma et al., 1997Sakuma H. Ikeda A. Oka S. Kozutsumi Y. Zanetta J.P. Kawasaki T. Molecular cloning and functional expression of a cDNA encoding a new member of mixed lineage protein kinase from human brain.J. Biol. Chem. 1997; 272: 28622-28629Crossref PubMed Scopus (72) Google Scholar). Both kinases are widely expressed in the nervous system, and DLK/MAP3K12 was identified as a synapse-associated MAPKKK (Mata et al., 1996Mata M. Merritt S.E. Fan G. Yu G.G. Holzman L.B. Characterization of dual leucine zipper-bearing kinase, a mixed lineage kinase present in synaptic terminals whose phosphorylation state is regulated by membrane depolarization via calcineurin.J. Biol. Chem. 1996; 271: 16888-16896Crossref PubMed Scopus (62) Google Scholar). The in vivo functions of these kinases were discovered through genetic studies of the synaptic E3 ubiquitin ligases known as PHR proteins, including C. elegans RPM-1, Drosophila Highwire, mouse Phr1, and human Pam (Collins et al., 2006Collins C.A. Wairkar Y.P. Johnson S.L. DiAntonio A. Highwire restrains synaptic growth by attenuating a MAP kinase signal.Neuron. 2006; 51: 57-69Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar; Lewcock et al., 2007Lewcock J.W. Genoud N. Lettieri K. Pfaff S.L. The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics.Neuron. 2007; 56: 604-620Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar; Nakata et al., 2005Nakata 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: 407-420Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Activated DLK kinases are targeted for degradation by these E3 ligases, resulting in a tight control of duration of signal transduction. In C. elegans, loss-of-function mutations in dlk-1 genetically suppress the neuronal defects of rpm-1 mutants, but dlk-1 mutants themselves are viable and grossly normal (Nakata et al., 2005Nakata 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: 407-420Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Constitutive activation of the DLK-1 pathway induces developmental defects mimicking rpm-1(lf) (Nakata et al., 2005Nakata 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: 407-420Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Moreover, expression of a constitutively active MAK-2, a downstream kinase of DLK-1, at synapses can disrupt synapse morphology and decrease synapse number (Yan et al., 2009Yan D. Wu Z. Chisholm A.D. Jin Y. The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration.Cell. 2009; 138: 1005-1018Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), suggesting that local activation of the DLK-1 pathway plays important roles in synapse formation. In adult neurons, DLK-1 is essential for injured axons to regenerate, and its activity is required within a limited time window after injury (Hammarlund et al., 2009Hammarlund M. Nix P. Hauth L. Jorgensen E.M. Bastiani M. Axon regeneration requires a conserved MAP kinase pathway.Science. 2009; 323: 802-806Crossref PubMed Scopus (319) Google Scholar; Yan et al., 2009Yan D. Wu Z. Chisholm A.D. Jin Y. The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration.Cell. 2009; 138: 1005-1018Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). These results indicate that the activation of DLK-1 must be precisely controlled in time and space by neuronal activity or injury. Despite extensive studies of DLK kinase function and their negative regulation by PHR proteins, the mechanisms by which DLK kinases are activated have remained elusive. Here we identify a short isoform, DLK-1S, that shares identical kinase and leucine zipper domains with the previously reported long isoform DLK-1L but binds to and inhibits the activity of the active DLK-1L. We identify a unique hexapeptide at the DLK-1L C terminus that plays critical roles in DLK-1 isoform-specific interactions. We further show that mammalian MAP3K13 contains identical hexapeptide and can complement DLK-1 function. Our results reveal a mechanism for tight spatial and temporal control of MAPKKK activity in neurons. The previously reported C. elegans DLK-1 protein contains 928 amino acid (aa) residues, including a kinase domain (aa 133–382) and a leucine zipper (LZ, aa 459–480) (Figures 1A and 1B ). By our analysis of new dlk-1 cDNA clones, and subsequently by RT-PCR and northern blotting, we found that the dlk-1 locus generates a second shorter transcript by use of an alternative polyadenylation site in intron 7 (Figure 1A, Experimental Procedures, and see Figure S1A available online). This transcript encodes a DLK-1 isoform of 577 residues. We here name the two isoforms DLK-1L (long) and DLK-1S (short). Both isoforms contain identical N-terminal kinase and LZ domains. The C terminus of DLK-1S consists of 11 isoform-specific residues, whereas the DLK-1L-specific C terminus contains 361 residues. Neither C-terminal domain contains known protein motifs. Analysis of expressed sequence tags (ESTs) for human and rat DLK family members indicates that these genes can also encode long and short isoforms (Figure 1B). To gain clues about the functions of the two isoforms of DLK-1, we took advantage of our collection of genetic loss-of-function mutations in dlk-1, all of which were isolated as suppressors of rpm-1(lf) (Nakata et al., 2005Nakata 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: 407-420Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). A large number of missense mutations affect conserved residues in the kinase domain (Figures 1B, S1B, and S1C and Table S1); one mutation (ju591) changes the conserved Leu at residue 459 in the LZ domain (Figure 1B). The strong loss-of-function phenotypes induced by these mutations are consistent with the essential roles of the kinase and LZ domains (Figure S1C). Unexpectedly, another set of strong loss-of-function mutations affect the C terminus specific to DLK-1L and are not predicted to affect DLK-1S (Figures 1B and S1C and Table S1). RT-PCR analysis showed that DLK-1S transcripts were produced at normal levels in the C-terminal mutants (Figure S1D). These observations raised the possibility that DLK-1S does not have the same activity as DLK-1L. To more directly address the role of DLK-1S, we assayed its function in synaptogenesis and developmental axon outgrowth, using transgenic rescue of the phenotypes of dlk-1(lf); rpm-1(lf) double mutants. rpm-1 mutants exhibit defects in motor neuron synapse development and in touch neuron axon growth (Schaefer et al., 2000Schaefer A.M. Hadwiger G.D. Nonet M.L. rpm-1, a conserved neuronal gene that regulates targeting and synaptogenesis in C. elegans.Neuron. 2000; 26: 345-356Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar; Zhen et al., 2000Zhen M. Huang X. Bamber B. Jin Y. Regulation of presynaptic terminal organization by C. elegans RPM-1, a putative guanine nucleotide exchanger with a RING-H2 finger domain.Neuron. 2000; 26: 331-343Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Both synaptic and axonal rpm-1 defects are strongly suppressed by dlk-1(lf) (Nakata et al., 2005Nakata 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: 407-420Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) (Figures 1C, 1D, and S2A). Neuronal expression of a DLK-1L cDNA at low concentrations fully rescued the dlk-1(lf) suppression phenotype (Figures 1C, 1D, and S2A, juEx2789, juEx2519). Expression of a DLK-1 minigene that produces both DLK-1L and DLK-1S proteins at comparable levels (Figure S2B) also fully rescued dlk-1 suppression phenotype (Figure 1D, juEx3452). In contrast, expression of DLK-1S alone showed no rescuing activity (Figures 1C and 1D, juEx2791, juEx2523), consistent with the interpretation that DLK-1S cannot substitute for DLK-1L. Furthermore, the rescuing activity of DLK-1L was strongly attenuated by co-overexpression with DLK-1S (Figures 1C and 1D, juEx2802, juEx2813). This inhibitory effect of DLK-1S was eliminated when the LZ domain was deleted from DLK-1S (Figure S2C). However, expression of a kinase-dead mutant DLK-1S(K162A), in which the Lys162 at the ATP binding site of the kinase domain was mutated to Ala (Nakata et al., 2005Nakata 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: 407-420Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar), inhibited DLK-1L to a similar degree as did wild-type DLK-1S (Figure S2C). These data suggest that the ability of DLK-1S to inhibit DLK-1L requires its LZ domain but not its kinase activity. As a further test for the role of DLK-1S, we expressed various DLK-1 constructs in the wild-type background (Figure S2D). Overexpression of DLK-1L alone caused abnormal neuronal development, whereas overexpression of DLK-1(mini) gene had a much weaker effect. Removing intron 7 from DLK-1(mini), which would prevent production of DLK-1S, resulted in gain-of-function effects similar to DLK-1(L). Finally, to address whether transgenically expressed DLK-1S could interfere with endogenous DLK-1L, we overexpressed DLK-1S in rpm-1(lf) single mutants and observed significant suppression of rpm-1(lf) phenotypes (Figure S3A). Together, these analyses demonstrate that despite sharing identical kinase and LZ domains, DLK-1S is a potent inhibitor of DLK-1L function. If DLK-1S acts as an endogenous inhibitory isoform, how does DLK-1L become activated at all? Since DLK-1L and DLK-1S differ only in their C termini, we hypothesized that the C terminus of DLK-1L may contain elements important for its kinase activation and that DLK-1S may act by preventing the interactions between such elements and the kinase domain. To test this idea, we generated a series of DLK-1L variants in which the C terminus was either truncated or contained internal deletions (Supplemental Experimental Procedures) and assayed rescuing activity of these constructs in the dlk-1(lf); rpm-1(lf) double mutant strain (Figure 2, Table S2). We found that a region of 25 amino acids from residues 856 to 881 in the DLK-1L C terminus was necessary for DLK-1L activity (Figure 2, juEx3586). Remarkably, a construct lacking all of the DLK-1L C terminus except for aa 856–881 recapitulated the activity of the full-length DLK-1L (Figure 2, juEx3657), suggesting that this region is sufficient for DLK-1L regulation. Upon closer inspection of the amino acid sequences, we found a six residue motif SDGLSD (aa 874–879, hereafter referred to as the hexapeptide) that is completely conserved between C. elegans DLK-1 and vertebrate MAP3K13/LZK (Figure 3A); the remainder of the C termini of these kinases show little sequence conservation. Moreover, dlk-1(ju620), a strong loss-of-function mutation, results in a missense alteration (G870E) adjacent to this hexapeptide. We expressed a DLK-1L(G870E) mutant cDNA and observed little rescuing activity (Figure 2, juEx3659). Deleting the conserved hexapeptide in DLK-1L also completely abolished rescuing activity (Figure 2, juEx4098). Together, these results identify the conserved C-terminal hexapeptide as critical for DLK-1L function.Figure 3DLK-1L Can Bind to Itself or to DLK-1S, and the Interactions Are Regulated by a C-Terminal Conserved HexapeptideShow full caption(A) Sequence conservation of the C-terminal peptide of DLK-1L (aa 868–881) with human MAP3K13/LZK (accession number EAW78225.1), mouse (accession number NM_172821.3), Bos taurus (accession number NM_001101853.1), and Xenopus laevis (accession number NM_001172189.1). Asterisk marks dlk-1(ju620) mutation. Sequence alignments used ClustW. Protein phosphorylation sites were predicted by the NetPhos program (http://www.cbs.dtu.dk/services/NetPhos).(B–F) Yeast cells were plated in a dilution series on Trp– Leu– media (left). Histidine (His) selection plates contained 10 mM 3-AT (right).(B) DLK-1S binds to DLK-1L. BD, GAL4 binding domain; AD, GAL4 activation domain; V, empty vector; L, DLK-1L; S, DLK-1S; ΔLZ, leucine zipper domain deletion.(C) The C-terminal hexapeptide regulates DLK-1 interactions. L(Δ), DLK-1L(Δ874-879); L(EE), DLK-1L(S874E, S878E); L(AA), DLK-1L(S874A, S878A).(D) aa 605–814 of DLK-1L are also required for the interactions between DLK-1L and DLK-1S.(E) aa 850–881 of DLK-1L bind to the kinase domain (KiD). The interaction is noticeably enhanced by phosphomimetic mutations (EE) and slightly reduced by nonphosphorylatable mutations (AA) of the hexapeptide.(F) The DLK-1L C-terminal aa 850–881 interacts with the kinase domain of human MAP3K13 (KiD(H)).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Sequence conservation of the C-terminal peptide of DLK-1L (aa 868–881) with human MAP3K13/LZK (accession number EAW78225.1), mouse (accession number NM_172821.3), Bos taurus (accession number NM_001101853.1), and Xenopus laevis (accession number NM_001172189.1). Asterisk marks dlk-1(ju620) mutation. Sequence alignments used ClustW. Protein phosphorylation sites were predicted by the NetPhos program (http://www.cbs.dtu.dk/services/NetPhos). (B–F) Yeast cells were plated in a dilution series on Trp– Leu– media (left). Histidine (His) selection plates contained 10 mM 3-AT (right). (B) DLK-1S binds to DLK-1L. BD, GAL4 binding domain; AD, GAL4 activation domain; V, empty vector; L, DLK-1L; S, DLK-1S; ΔLZ, leucine zipper domain deletion. (C) The C-terminal hexapeptide regulates DLK-1 interactions. L(Δ), DLK-1L(Δ874-879); L(EE), DLK-1L(S874E, S878E); L(AA), DLK-1L(S874A, S878A). (D) aa 605–814 of DLK-1L are also required for the interactions between DLK-1L and DLK-1S. (E) aa 850–881 of DLK-1L bind to the kinase domain (KiD). The interaction is noticeably enhanced by phosphomimetic mutations (EE) and slightly reduced by nonphosphorylatable mutations (AA) of the hexapeptide. (F) The DLK-1L C-terminal aa 850–881 interacts with the kinase domain of human MAP3K13 (KiD(H)). To determine how DLK-1S interacts with DLK-1L and how the C-terminal hexapeptide regulates their interaction, we next performed protein interaction studies using yeast two-hybrid assays. We found that full-length DLK-1L interacted with itself and also with DLK-1S (Figure 3B). Removal of the LZ domain in DLK-1L or DLK-1S eliminated these interactions. Unexpectedly, despite containing the LZ domain, DLK-1S did not show interaction with itself, suggesting that the LZ domain is not sufficient for DLK-1 dimerization or oligomerization. To test the role of the C-terminal hexapeptide SDGLSD in the interactions between DLK-1 isoforms, we deleted it from DLK-1L. We found that a DLK-1L construct lacking the hexapeptide failed to show any homomeric interaction and, instead, displayed an enhanced heteromeric interaction with DLK-1S (Figure 3C). These results suggest that the C-terminal hexapeptide plays a critical regulatory role in DLK-1 isoform-specific interactions. Since the C-terminal aa 856–881 region can endow a truncated DLK-1(kinase+LZ) with complete function (Figure 2, juEx3588), we tested whether this domain might interact with the kinase domain. In yeast two-hybrid assays, we observed that the aa 850–881 region interacted with the kinase domain of DLK-1 (Figure 3E). The hexapeptide SDGLSD contains two potential phosphorylation sites (Ser 874 and Ser878, Figure 3A). We addressed whether these serines were sites of regulation by generating phosphomimetic and nonphosphorylatable forms of the hexapeptide. We found that full-length DLK-1L containing phosphomimetic (S874E, S878E) hexapeptide showed stronger binding to itself (Figure 3C). Conversely, full-length DLK-1L containing nonphosphorylatable (S874A, S878A) hexapeptide showed an enhanced interaction with DLK-1S (Figure 3C). The phosphomimetic C-terminal aa 850–881 region also showed stronger binding to the kinase domain of DLK-1 (Figure 3E). The C-terminal domain alone did not interact with itself in yeast two-hybrid assays (data not shown), although a region of 209 amino acids between LZ domain and the hexapeptide was necessary for DLK-1L to interact with DLK-1S (Figure 3D). Taken together, the results from yeast two-hybrid interaction assays suggest that phosphorylation of the DLK-1L hexapeptide could regulate the balance between active DLK-1L homomers and inactive DLK-1L/S heteromers. To address the in vivo importance of DLK-1 C-terminal hexapeptide phosphorylation, we turned to transgenic expression. DLK-1L with a nonphosphorylatable hexapeptide (S874A, S878A) was expressed normally (Figure S4) but lacked rescuing activity (Figure 4A, juEx4708). However, coexpression of the phosphomimetic DLK-1L(S874E, S878E) overcame the inhibitory effects of DLK-1S (Figure 4A, juEx4694). Strikingly, coexpression of DLK-1S with the DLK-1L C-terminal 328 aa or the aa 850–881 fragment in dlk-1; rpm-1 mutants significantly rescued the suppression effects of dlk-1(lf) (Figure 4A, juEx3661, juEx3729). These results suggest that the C terminus of DLK-1L can activate DLK-1 in trans. Vertebrate MAP3K13/LZK proteins contain C-terminal hexapeptides identical to that of DLK-1L (Figure 3A). We therefore tested whether the function of DLK-1L was conserved with human MAP3K13. The kinase domain of MAP3K13 is 60% identical to that of DLK-1 (Figure S1B). We found that DLK-1L (aa 850–881) could bind to the kinase domain of human MAP3K13 in the yeast two-hybrid assay (Figure 3F). We then expressed the human MAP3K13 cDNA under a panneural promoter in dlk-1(lf); rpm-1(lf) animals (Supplemental Experimental Procedures) and observed a significant rescue of dlk-1(lf) phenotypes (Figures 4B and S3, juEx4748). In contrast, expression of a mutant MAP3K13 containing Ala mutations in the hexapeptide (S903A, S907A) did not rescue dlk-1(lf) (Figure 4B, juEx4995). The MAP3K12/DLK shares an almost identical kinase domain with MAP3K13/LZK but lacks the C-terminal hexapeptide. However, expression of MAP3K12/DLK alone failed to rescue dlk-1 phenotypes (Figure 4B, juEx4701). Interestingly, coexpression of MAP3K12/DLK with a fragment containing the DLK-1 C-terminal hexapeptide partially rescued dlk-1(lf) (Figure 4B, juEx5167). These results show that human MAP3K13 complements dlk-1 function and suggest that MAP3K13 can be activated by a similar mechanism involving the conserved hexapep" @default.
• W2022903577 created "2016-06-24" @default.
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• W2022903577 date "2012-11-01" @default.
• W2022903577 modified "2023-10-12" @default.
• W2022903577 title "Regulation of DLK-1 Kinase Activity by Calcium-Mediated Dissociation from an Inhibitory Isoform" @default.
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• W2022903577 doi "https://doi.org/10.1016/j.neuron.2012.08.043" @default.
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