DCX, a new mediator of the JNK pathway
Mutations in the X‐linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here w...
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| Published in | The EMBO journal Vol. 23; no. 4; pp. 823 - 832 |
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| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Chichester, UK
John Wiley & Sons, Ltd
25.02.2004
Blackwell Publishing Ltd Nature Publishing Group |
| Subjects | |
| Online Access | Get full text |
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| Summary: | Mutations in the X‐linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c‐Jun N‐terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus‐end directed molecular motor) versus dynein (a minus‐end directed molecular motor). |
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| Bibliography: | istex:6662AE2276D3D45EDB74925A909245540840BF8E Supplementary Figure S1 a) The phosphorylation of GST-DCX is by JNK and not by MKK7. Cells were transfected with HA-JNK and prior to the immunoprecipitations JNK was activated by arsenate. GST-DCX was phosphorylated by the activated kinase (indicated). In this autoradiogram, the presence of phosphorylated JNK is marked as well. b) In vitro on phosphorylation of GST-DCX and GST-DCX mutated in single or double putative phosphorylation sites. GST-DCX wild type or mutated in the designated amino acids, and c-Jun (positive control) were phosphorylated in vitro by JNK. Note that the double mutant T331, S334A protein was not phosphorylated. c) DCX is phosphorylated in vitro on threonine 321 and on threonine 331 and serine 334 (right panel). GST-DCX wild type or mutated in the designated amino acids were phosphorylated in vitro by JNK, and the proteins were blotted and reacted with anti-p-DCX antibodies. The unphosphorylated protein was not recognized by the p-specific antibodies. Reduced reactivity was observed with DCX mutated in threonine 321, or mutated in threonine 321 and serine 327. Enhanced recognition was observed in case of DCX mutated in serine 327. Therefore, we conclude that antibodies recognize DCX phosphorylated on threonine 321. d) DCX is phosphorylated in vitro on threonine 331 and serine 334. GST-DCX wild type or mutated in the designated amino acids were phosphorylated in vitro by JNK, and the proteins were blotted and reacted with the indicated anti-p-DCX antibodies. The unphosphorylated protein was barely recognized by the p-specific antibodies. Reduced reactivity was observed with DCX mutated in threonine 321, or mutated in threonine 331 and serine 334. Our conclusion is that the antibodies recognize both p-threonine 321and p-serine 334. e) Phospho-specific DCX threonine 321 antibodies recognized the phospho-protein. HEK 293 cells were co-transfected with FLAG-DCX and constitutively active JNK kinase or the kinase-dead version (KD). Cell extract was analyzed by Western blot using p-DCX antibodies recognizing p-threonine 321. Note the reduced recognition in case of co-expression with the kinase-dead expression construct. f) Phospho-specific DCX threonine 331 and serine 334 antibodies recognized the phospho-protein. HEK 293 cells were co-transfected with FLAG-DCX and constitutively active JNK kinase or the kinase-dead version (KD). Cell extract was analyzed by Western blot using p-DCX antibodies recognizing p-threonine 331 and serine 334. Note the reduced recognition in case of co-expression with the kinase-dead expression construct. g) HEK 293 cells were transfected with FLAG-DCX and constitutively active JNK kinase. The pattern of immunostaining using phospho-specific DCX threonine 321 antibodies was similar to that observed by immunostaining with anti-FLAG antibodies. Similar results were obtained with phospho-specific DCX threonine 331 and serine 334 antibodies (data not shown).Supplementary Figure S2 DCX interacts with JIP. a,b) Cells were transfected with myc-JIP-1 and with either FLAG-tagged constructs of DCX S47R, 247X, pep1, pep2. Myc-tagged JIP-1 was immunoprecipitated using anti-FLAG antibodies (a), or the FLAG-tagged constructs were immunoprecipitated with anti-myc antibodies (b). The expression of each of the plasmids was verified by Western blot analysis (lower panels, extract). c) Yeast expression constructs with DCX, JIP-1 (SH3+PID), JIP-1 (SH3+PID, 687 mutation), and JNK2 were tested for interaction in the two-hybrid system. DCX and JIP-1 strongly interacted and the PID mutation reduced the interaction in both directions tested. No interaction was observed with JNK2.Supplementary Figure S3 Typical transfected DCX PC12 cells (24 hrs after transfection). The mutations are indicated on the panels. Three of the four mutations were on the backbone of DCX-DsRed contruct. T321A was on the backbone of FLAG-DCX, to visulalize transfected cells, cells were co-transfected in a 1:8 ratio with EGFP. In each transfection the length of neurons per cell was measured, and the number of neurites per cell was counted. The number of analyzed cells for the 24 hr time point were for T321A; 87 cells, T331, S334A; 85 cells, wild-type DCX; 130 cells, and T331, S334E; 34 cells. The results of the averages are shown in Figure 8.Supplementary Figure S4 Typical transfected DCX PC12 cells (48 hrs after transfection). The mutations are indicated on the panels. Three of the four mutations were on the backbone of DCX-DsRed contruct. T321A was on the backbone of FLAG-DCX, to visulalize transfected cells, cells were co-transfected in a 1:8 ratio with EGFP. In each transfection the length of neurons per cell was measured, and the number of neurites per cell was counted, The number of analyzed cells for the 48 hr time point were for T321A; 87 cells, T331, S334A; 116 cells, wild-type DCX; 170 cells, and T331, S334E; 40 cells. The results of the averages are shown in Figure 8.Supplementary Figure S5 Typical transfected DCX N2A cells. The mutations are indicated on the panels. Three of the four mutations were on the backbone of DCX-DsRed contruct. T321A was on the backbone of FLAG-DCX, to visulalize transfected cells, cells were co-transfected in a 1:8 ratio with EGFP. In each transfection the length of neurons per cell was measured, and the number of neurites per cell was counted, the number of analyzed cells for each transfection were for T321A; 125 cells, T331, S334A; 92 cells, wild-type DCX; 149 cells, and T331, S334E; 138 cells. The results of the averages are shown in Figure 8.Supplementary Figure S6 Typical transfected DCX-DsRed primary cerebellar cells. Primary cerebellar neurons were transfected with either unphospho-mimicry form T331, S334A, (top panel), wild-type DCX (middle panel) and the phospho-mimicry form T331, S334E (bottom panel). In each transfection the length of neurons was measured, the number of analyzed cells for each transfection were for T331, S334A; 68 cells, wild-type DCX; 56 cells, and for T331, S334E; 61 cells. The results of the averages are shown in Figure 8.Supplementary Table S1 ark:/67375/WNG-7GV5ZWFK-K ArticleID:EMBJ7600079 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 These authors contributed equally to this work |
| ISSN: | 0261-4189 1460-2075 |
| DOI: | 10.1038/sj.emboj.7600079 |