QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00312

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tootle, T. L. Articles by Rebay, I. Search for Related Content PubMed PubMed Citation Articles by Tootle, T. L. Articles by Rebay, I.

CRM1-mediated nuclear export and regulated activity of the Receptor Tyrosine Kinase antagonist YAN require specific interactions with MAE

¹ Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
² Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA

View larger version (64K): [in a new window]   Fig. 1. Nuclear export of YAN is mediated by CRM1 and blocked by insertion of a NLS into YAN. (A-C,E-L) S2 cultured cells transfected with various YAN constructs and stained with anti-YAN. (A'-C',E'-L') DAPI staining of the same cells. (D) Schematic of YAN showing predicted domains and sites of SV40 Large T-antigen NLS insertions. For each experiment (A-C,E-L), the percentage of transfected cells exhibiting nuclear localization (A,C,E-K) or exclusively cytoplasmic localization (B,L) is indicated. n, number of cells scored in each experiment. (A-C') YANWT; (E-F') YANInt NLS; (G-H') YAN2x NLS; (I-J') YANN' NLS; (K-L') YANMut NLS. (A,E,G,I,K) YAN localization in the absence of RASV12 (B,F,H,J,L) YAN localization in the presence of RASV12. (C) YAN localization in the presence of RASV12 and RNAi of crm1. (C) YAN localization is restricted to the nucleus in the presence of RASV12 and RNAi of crm1. (F,H) Internal NLS insertions completely inhibit nuclear export of YAN in the presence of RASV12, while the N-terminal insertion only partially prevents export (J). (L) Insertion of a nonfunctional NLS into YAN has no effect on export.

View larger version (137K): [in a new window]   Fig. 2. NLS insertions restrict YAN to the nucleus in vivo. (A-E'') Confocal images of germband extended Drosophila embryos double labeled with anti-YAN (A-E,A'-E') and anti-ELAV (A''-E''). (A'-E') Higher magnification views of regions boxed in A-E with normal or failed YAN downregulation highlighted by bracket. ELAV GAL4 was used to drive expression of (A,A',A'') UAS YANACT; (B,B',B'') UAS YANWT; (C,C',C'') UAS YANInt NLS; (D,D',D'') UAS YAN2x Int NLS; (E,E',E'') UAS YANMut NLS. YANWT is downregulated normally in the ventral nerve cord (B,B'), allowing neuronal differentiation to proceed (B''). As with YANACT (A,A',A''), insertion of an NLS restricts YAN to the nucleus (C,C',D,D'), thereby blocking CNS development (C'',D'').

View larger version (58K): [in a new window]   Fig. 3. CRM1-mediated nuclear export of YAN requires both the NESs and the PD. (A-M) S2 cultured cells transfected with various YAN deletion constructs and stained with anti-YAN. (A'-M') DAPI staining of the same cells. For each experiment (A-M), the percentage of transfected cells exhibiting nuclear localization (A,C,D,F,G-I,L,M), both nuclear and cytoplasmic localization (J,K), or exclusively cytoplasmic localization (B,E) is indicated. n, number of cells scored in each experiment. (A-B,D-E) Deletion of the first or first and second NES has no effect on export. (G-H,J-K) Deletion of the third NES and majority of the PD results in inappropriate export in the absence of signaling, and impairs export in the presence of RASV12. (L-M) Deletion of the whole N terminus completely inhibits export. (C,F,I) RNAi-mediated knockdown of crm1 restricts YAN to the nucleus in the presence of RASV12. (O) Transcription assays with YANN' and YANN3+PD show that both deletions repress transcription and are responsive to RASV12.

View larger version (171K): [in a new window]   Fig. 4. MAE acts as a positive component of the RTK pathway and loss of mae function inhibits the downregulation of YAN. (A-D) Scanning electron micrographs of adult Drosophila eyes showing that loss of mae dominantly suppresses the rough eye phenotype of Sev-RASV12. (E-G) Tangential sections of adult Drosophila eyes. The average number of R7 photoreceptors per ommatidium is indicated below, with n referring to the total number of ommatidia scored. (A,E) Wild-type; (B,F) Sev-RASV12/+; (C,G) Sev-RASV12/l(2)k06602; (D,H) Sev-RASV12/Df(2R)PC4. Confocal images of germband extended embryos double labeled with anti-YAN (I,J, with high magnification of boxed region shown in I',J') and anti-ELAV (I'',J''). (I,I',I'') show that in mae mutants, YAN fails to be downregulated in the CNS (I', bracketed region) and ELAV expression is inhibited (I''). (I,I',I'') l(2)k06602/l(2)k06602; (J,J',J'') wild type.

View larger version (104K): [in a new window]   Fig. 5. MAE localization in S2 cells depends on the distribution of its binding partners. (A,B) Immunoblots of MYC-IPs visualized with anti-MYC (MAE), anti-YAN and anti-FLAG (PNT-P2). MAE complexes with YAN in the absence of RAS/MAPK signaling (A) and with PNT in the both the absence and presence of signaling (B). Lanes are from the same gel and immunoblot, but have been rearranged. Lanes 1, 3, 5 are non-IPed lysates; lanes 2, 4, 6 are the corresponding IPs. Specificity of the anti-MYC IP is demonstrated in lane 6 of A and B, where in the absence of MAE, YAN or PNT-P2 are not precipitated. (C-O) Anti-MYC staining of S2 cells transfected with MYC-mae. (C'-O') DAPI staining of the same cells. (C,E,G,I,K,M) Absence of RASV12. (D,F,H,J,L,N,O) Presence of RASV12. For each experiment (C-O), the percentage of transfected cells exhibiting nuclear localization (G,K-O), or both nuclear and cytoplasmic localization (C-F,H-J) is indicated. n, number of cells scored in each experiment. (C,D) MAE; (E,F) MAE+RNAi crm1; (G,H) MAE+YAN; (I,J) MAE+YANN'; (K,L) MAE+YANACT; (M,N) MAE+PNT-P2; (O) MAE+YAN+RNAi crm1. MAE is ubiquitously expressed in S2 cells (C,D), except when YAN or PNT-P2 is co-transfected. When MAE is co-transfected with wild-type YAN, MAE is nuclear in the absence of signaling (G) and becomes nuclear and cytoplasmic in the presence of RASV12 (H). CRM1 does not mediate the export of MAE (E,F). However, when YAN is co-transfected with MAE, and CRM1-mediated export is inhibited by RNAi, MAE remains nuclear (O). MAE interacts with YAN via the PD, as MAE is ubiquitously expressed when YANN' is co-expressed (I,J). Co-transfection of MAE with YANACT restricts MAE to the nucleus in the absence and presence of RASV12 (K,L). Similarly, PNT-P2 restricts MAE to the nucleus (M,N).

View larger version (20K): [in a new window]   Fig. 6. MAE inhibits the ability of both YAN and PNT-P2 to regulate transcription. (A) Transcriptional repression assays with YAN. (B) Transcriptional activation assays with PNT-P2. Overexpression of MAE inhibits YAN-mediated transcriptional repression (A) and PNT-P2-mediated activation (B).

View larger version (58K): [in a new window]   Fig. 7. (A-C) The model for the downregulation of YAN. (D,E) The model for the activation and subsequent downregulation of PNT-P2. (A) In the absence of signaling, YAN localizes to DNA, repressing transcription. (B) Upon RTK signaling, phosphorylated MAPK enters the nucleus, interacts with YAN-MAE complex and phosphorylates YAN. YAN is removed from the DNA, although the exact timing of this event is not yet clear. (C) The YAN-MAE complex then interacts with CRM1, causing release of MAE and CRM1 mediated export of YAN through the nuclear pore. (D) In the absence of signaling, PNT-P2 can bind to MAE and is prevented from activating transcription, either as a consequence of its interaction with MAE or because it is out competed by YAN, or both. (E) Upon RTK activation, phosphorylated MAPK enters the nucleus and phosphorylates PNT-P2. This allows PNT-P2 to bind DNA and activate transcription of the target genes now freed from YAN repression. (F) To prevent runaway signaling, a negative feedback loop may occur in which MAE binds to PNT-P2 and inhibits transcriptional activation. This could occur by MAE binding causing PNT-P2 to no longer bind DNA (1), or by MAE binding resulting in dephosphorylation of PNT-P2 (2), resulting in inhibition of transcriptional activation.