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

First published online 6 February 2003
doi: 10.1242/jcs.00275

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 Hatzfeld, M. Articles by Sauter, H. Search for Related Content PubMed PubMed Citation Articles by Hatzfeld, M. Articles by Sauter, H. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Genetics Home Reference Social Bookmarking                         What's this?

Targeting of p0071 to desmosomes and adherens junctions is mediated by different protein domains

¹ Institute of Physiological Chemistry, Medical Faculty of the University of Halle, 06097 Halle/Saale, Germany
² Departments of Pathology and Dermatology and the Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, IL 60611, USA

View larger version (32K): [in a new window]   Fig. 1. (A) Western blot analysis with recombinant protein fragments expressed in E. coli BL21DE3 cells. BL21DE3 cells not expressing exogenous protein (BL21) or expressing the p0071 tail (p0071), the NPRAP/-catenin tail (NPRAP), the p120ctn tail (p120ctn) or the plakophilin 1 repeat + tail domains (PKP1) were lysed in SDS sample buffer, separated on 15% gels and transferred to membranes. Membranes were probed with 6D1-10-, p120ctn- and His-tag monoclonal antibodies as indicated. 6D1-10 antibody recognized NPRAP and p0071, but not p120ctn or plakophilin 1, the p120ctn antibody was specific for the p120ctn tail and the His-tag antibody detected all recombinant proteins. (B) Total cell extracts from kidney were prepared in SDS sample buffer, separated on 7% gels and transferred to membranes. Individual lanes were probed with antibodies to p0071 (6D1-10), p120ctn and plakophilin 1, 2 and 3, as indicated. 6D1-10 detected a single band of 135 kDa that differs from protein bands detected by p120ctn- and plakophilin antibodies. (C) Intracellular localization of endogenous p0071 in MCF-7 cells as detected by antibody 6D1-10. MCF-7 cells were extracted with Triton-X 100 prior to fixation and double labeled with the 6D1-10 monoclonal antibody (p0071) and rabbit anti-desmoplakin (serum 616) and rabbit polyclonal anti-Pan-cadherin (cadherin). Plasma membrane regions denoted by arrows are shown in detail at higher magnification. Bars, 3 µm.

View larger version (51K): [in a new window]   Fig. 2. Overexpression of p0071 wt in MCF-7 cells. P0071 wt with a C-terminal DsRed tag or an N-terminal EGFP tag was expressed in MCF-7 cells. Cells were fixed in methanol and labeled with desmoplakin (serum 616) or E-cadherin antibodies. Plasma membrane regions denoted by arrows are shown in detail at higher magnification. Note that desmoplakin staining disappears from the membrane regions enriched in p0071, whereas E-cadherin staining is increased. Bars, 3 µm.

View larger version (27K): [in a new window]   Fig. 3. (A) p0071 constructs used in this study. All constructs contain the short splice variant of the tail domain. (B) Plakoglobin (pg) constructs used in this study. Localization of tag sequences is indicated on top. Boxes represent the armadillo repeat units. For details see Materials and Methods.

View larger version (64K): [in a new window]   Fig. 4A. Intracellular localization of the p0071 domains in MCF-7 cells. (A,B) Expression of the p0071 head domain (A) and a N-terminally deleted fragment (head 149-509, B) in the pDsRed vector. Cells were Triton-extracted, PFA-fixed and stained with monoclonal desmoplakin antibody (A) or methanol-fixed and labeled with polyclonal desmoplakin antibody (B, serum 616). Plasma membrane regions denoted by arrows are shown in detail at higher magnification. (C,D) Intracellular targeting of the p0071 tail domain. Cells transfected with the p0071 tail pDsRed were fixed in methanol and stained with anti-E-cadherin (C) or desmoplakin antibodies (D). Membrane regions denoted by arrows are shown at higher magnification. (E,F,G) Intracellular localization of p0071 headless pDsRed. Cells were fixed in methanol and labeled with E-cadherin (E,F) or desmoplakin (G, serum 616) antibodies. Enlargement of membrane regions denoted by arrows shows colocalization with E-cadherin (E,F), whereas desmoplakin staining is distinct from p0071 headless localization (G). Note that desmoplakin staining is sparse along the membrane enriched in p0071 headless (G). (H,I,J) Localization of the p0071 repeats pDsRed. Cells were fixed in methanol and labeled with E-cadherin (H,I) or desmoplakin (J, serum 616) antibodies. Enlargement of membrane regions denoted by arrows shows colocalization with E-cadherin (H,I), whereas desmoplakin staining is distinct from p0071 repeat domain localization (J). Bars, 3 µm.

View larger version (83K): [in a new window]   Fig. 4E. Intracellular localization of the p0071 domains in MCF-7 cells. (A,B) Expression of the p0071 head domain (A) and a N-terminally deleted fragment (head 149-509, B) in the pDsRed vector. Cells were Triton-extracted, PFA-fixed and stained with monoclonal desmoplakin antibody (A) or methanol-fixed and labeled with polyclonal desmoplakin antibody (B, serum 616). Plasma membrane regions denoted by arrows are shown in detail at higher magnification. (C,D) Intracellular targeting of the p0071 tail domain. Cells transfected with the p0071 tail pDsRed were fixed in methanol and stained with anti-E-cadherin (C) or desmoplakin antibodies (D). Membrane regions denoted by arrows are shown at higher magnification. (E,F,G) Intracellular localization of p0071 headless pDsRed. Cells were fixed in methanol and labeled with E-cadherin (E,F) or desmoplakin (G, serum 616) antibodies. Enlargement of membrane regions denoted by arrows shows colocalization with E-cadherin (E,F), whereas desmoplakin staining is distinct from p0071 headless localization (G). Note that desmoplakin staining is sparse along the membrane enriched in p0071 headless (G). (H,I,J) Localization of the p0071 repeats pDsRed. Cells were fixed in methanol and labeled with E-cadherin (H,I) or desmoplakin (J, serum 616) antibodies. Enlargement of membrane regions denoted by arrows shows colocalization with E-cadherin (H,I), whereas desmoplakin staining is distinct from p0071 repeat domain localization (J). Bars, 3 µm.

View larger version (50K): [in a new window]   Fig. 5. Yeast two-hybrid interaction analysis using p0071 head 1-198, p0071 head 209-509 and p0071 repeats as baits. (A) Cotransformations of p0071 constructs in pGBKT7 with non-desmosomal cadherin-, dsg and dsc cytoplasmic domains, pg domains, PKP2 and the desmoplakin N-terminus (DP-NTP) were streaked in parallel on plates lacking tryptophane and leucine (-TL) and tryptophane, leucine and histidine (-TLH). Colonies from -TLH plates were blotted on filters and analyzed for lacZ reporter gene activation (lac-Z). The p0071 head 1-198 interacted with dsc3a and pg-3-13+C, but not pg-4-9 or pg-N+1-6. Cotransformants expressing DP-NTP activate the his reporter gene, but not the lac-Z reporter gene. The p0071 head 209-509 interacted with pg-3-13+C and DP-NTP although lac-Z reporter gene activation was weak for the latter interaction. p0071 arm repeats interacted with E-, N-, OB-cadherin/cadherin 11, pg-3-13+C, pg-N+1-6 and PKP2. Again, cotransformants with p0071 repeats and DP-NTP activated the his reporter gene but not the lac-Z reporter gene. E-cadherin interacted with p0071 rep1-5, but not p0071 rep2-10 or p0071 rep4-10, indicating that repeat1 is important for the interaction. (B) Quantitation of lac-Z reporter gene activation by p0071 arm-repeat—cadherin interactions and p0071-head (aa 1-198)—cadherin interactions using ONPG. Lac-Z activity was measured for >5 independent transformants. Lac-Z activation of the N-cadherin-p0071 cotransformants is 3.5-fold higher compared with that of E- and OB-cadherin-p0071 cotransformants.

View larger version (75K): [in a new window]   Fig. 6. Mom-targeting assay. Dscs 1a, 2a and 3a in the mom flag vector were contransfected with the p0071 head domain in PtK2 cells and visualized by anti-flag antibody. Dsc3a and p0071 head colocalize on mitochondria, whereas dsc1a and dsc2a localization is distinct from p0071 head distribution. E-cadherin in the mom-vector with 4A6 tag was cotransfected with the p0071 head, repeat and tail domains. Whereas the p0071 repeat domain was recruited to E-cadherin-coated mitochondria, head and tail domains of p0071 were not recruited. Bars, 3 µm.

View larger version (94K): [in a new window]   Fig. 7. Cotransfection studies of p0071 domains and plakoglobin domains. Cells were fixed in paraformaldehyde and labeled with the myc antibody recognizing plakoglobin constructs and desmoplakin antibody (serum 616) to visualize desmosomes. P0071wt DsRed and plakoglobin-myc colocalize along the plasma membrane. This localization partially overlaps with desmoplakin staining as shown in detail at higher magnification. p0071 headless DsRed colocalizes with pg-CN along the membrane, whereas the same plakoglobin construct localizes preferentially in the cytoplasm when cotransfected with the p0071 head domain. In contrast, cotransfection of p0071 headless and pg-Sac shows membrane association of p0071 headless as noted before and cytoplasmic and nuclear localization of pg-Sac. Double transfection of pg-Sac with the p0071 head domain showes only partial recruitment of plakoglobin end domains to the membrane. Bars, 3 µm.

View larger version (44K): [in a new window]   Fig. 8. (A) Binding sites of cell contact proteins in the p0071 molecule. (B,C) Model depicting p0071 interactions in desmosomes (B) and adherens junctions (C).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter