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First published online 12 February 2003
doi: 10.1242/jcs.00302

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Junctional protein MAGI-3 interacts with receptor tyrosine phosphatase ß (RPTPß) and tyrosine-phosphorylated proteins

Konstantin Adamsky^*, Katya Arnold^*, Helena Sabanay and Elior Peles

Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel

View larger version (69K): [in a new window]   Fig. 1. Amino-acid sequences and domain organization MAGI-3. (A) Amino-acid sequence of rat MAGI-3. (B) Schematic organization. MAGI-3 contains six PDZ domains, as well as two WW domains and a guanylate kinase domain (GUK), which are located between the first and second PDZ domains. This organization is similar for all members of the MAGI family and is distinct from the original MAGUK proteins that contain an SH3 domain instead of the WW domains and in which the GUK domain is located at the C-terminal end of the protein.

View larger version (39K): [in a new window]   Fig. 2. Expression of MAGI-3 mRNA and protein. (A) A northern blot containing mRNA from various rat tissues hybridized with a DNA fragment containing the third and fourth PDZ domains of MAGI-3 as a probe. The autoradiogram is shown along with the location of molecular weight markers in kb. A single 6.4 kb transcript was detected in the indicated tissues. (B) RT-PCR analyses peformed with MAGI-3-specific primers on the indicated rat tissues, GH3 neuroendocrine cell line, neuronal stem cells (NSC) and primary astrocytes. MAGI-3 cDNA (MAGI-3) and a reaction containing no DNA (—DNA) were used as positive and negative controls, respectively. (C) Expression of MAGI-3 protein in various cell lines. Cell lysates made from the indicated cell lines or primary astrocytes were subjected to immunoprecipitation and western blot analysis using an antibody to MAGI-3. Molecular weight markers in kDa are shown on the right. Note that the lower two bands shown in C6 are degradation products of MAGI-3, which were not detected in other experiments. (D) Rat brain membrane lysates were immunoprecipitated with specific antibodies to MAGI-3 or with the preimmune serum (PI) as indicated, followed by immunoblotting with anti-MAGI-3 antibody.

View larger version (27K): [in a new window]   Fig. 3. Association of MAGI-3 with RPTPß. (A) HEK-293T cells were transfected with MAGI-3, HA-tagged RPTPß or with both MAGI-3 and RPTPß as indicated (-/+). Cell lysates were subjected to immunoprecipitation with an antibody against the HA-tag that recognizes the extracellular domain of RPTPß (IP:HA) or an antibody to MAGI-3 (IP:MAGI-3) as indicated on the right. Washed immunocomplexes were separated on an SDS gel and immunoblotted with an antibody to HA-tag (left panels) or to MAGI-3 (right panels). (B) Pulldown of MAGI-3 by the cytoplasmic domain of RPTPß. Lysates of HEK-293T cells expressing MAGI-3 were mixed with agarose-bound GST or GST-fusion proteins containing the entire cytoplasmic domain of RPTPß (ßD12), the first phosphatase domain (ßD1), the second phosphatase domain (ßD2) or a mutant form lacking the C-terminus (ßD12dCT) as indicated. Bound proteins were immunoblotted with an antibody to MAGI-3. Immunoprecipitation with an antibody to MAGI-3 (MAGI-3) was used as a control. (C) Interaction of the C-terminal tail of RPTPß with MAGI-3. Rat brain membrane lysate was mixed with immobilized peptides corresponding to the C-terminal tails of RPTPß (RPTPßCT), a peptide containing a scrambled sequence (ßCTS), the C-terminal peptide of Caspr2 (CSP2CT) or a similar peptide lacking the last amino acid (CSP2dCT). Bound proteins were separated on SDS gels and blotted with an antibody to MAGI-3. As a positive control, a lysate sample used in the pulldown experiment was immunoprecipitated with an antibody to MAGI-3 (MAGI-3). Molecular weight markers are shown in kDa on the right. (D) Two-hybrid analysis. The ability of the different domains of RPTPß to interact with a MAGI-3 construct, containing its third and a fourth PDZ domains, was examined using the two-hybrid method (-/+). All RPTPß constructs containing the C-terminal tail interact with MAGI-3, unlike those that lacked this region.

View larger version (119K): [in a new window]   Fig. 4. Subcellular distribution of MAGI-3 in epithelial cells. Caco2 colon carcinoma cells were double labeled using a polyclonal antibody to MAGI-3 (A,D,G,J) and monoclonal antibodies to ß-catenin (B), desmoplakin (E), cingulin (H) or ZO-1 (K) as indicated. MAGI-3 is shown in red, all other proteins in green, and DAPI-labeled nuclei are blue. Merge images of each set are shown on the right. Higher magnification of the region labeled with an asterisk from the merge image is shown in the inset of each panel. Note that MAGI-3 colocalized with cingulin and ZO-1 but not with ß-catenin or desmoplakin. Similar cellular distribution of MAGI-3 in Caco2 cells was also detected using a distinct monoclonal antibody (M). The localization of MAGI-3 in transfected MDCK cells, which do not express endogenous MAGI-3, is presented in panels N, or as a double staining with Claudin-1 (O; green). Bars, A-L, 15 µm; M, 20 µm; N-O, 40 µm.

View larger version (180K): [in a new window]   Fig. 5. Ultrastructural localization of MAGI-3. Caco-2 cells were labeled with an antibody to MAGI-3 and a colloidal gold-conjugated secondary goat anti-rabbit antibody. (A) An immunoelectron micrograph showing labeling of the gold particles at tight junctional complex region (red arrows), nucleus (n; red arrowheads) and microvilli (mv: red asterisks) are presented. (B,C) Higher magnification images, showing the localization of MAGI-3 at tight junctions. Bar, A, 200 nm; B,C, 35 nm.

View larger version (143K): [in a new window]   Fig. 6. Immunolocalization of MAGI-3 in primary cultured astrocytes. Primary rat astrocytes (A-F) or older cultures (G-L) were double labeled with antibodies to MAGI-3 (red) and ß-catenin (green), phosphotyrosine (PY, green), vinculin (green) or Alexa-488 phalloidin to stain actin filaments as indicated in each panel. Merged images for each corresponding set of staining are shown in the right panels. Colocalization of MAGI-3 and tyrosine phosphorylated proteins at cell-cell contact sites are labeled with an arrow. Arrowheads in D-I mark focal adhesion sites, which contain tyrosine-phosphorylated proteins but not MAGI-3. Localization of MAGI-3 at the end of actin filaments is shown at higher magnification in the inset of the last panel.

View larger version (30K): [in a new window]   Fig. 7. Association of MAGI-3 with tyrosine-phosphorylated proteins. (A) SF763T cells treated with (+) or without (-) vanadate were immunoprecipitated using antibodies to MAGI-3 (MAGI-3 or M23), preimmune serum (PI) or anti-phosphotyrosine (PY). The immunocomplexes were separated on SDS gels and immunoblotted with anti-MAGI-3 (upper panel) or anti-phosphotyrosine (lower panel) antibodies, as indicated. The immunocomplexes from one set of samples were treated with alkaline phosphatase (MAGI-3 + AP) before immunoblotting. MAGI-3 was neither detected by anti-phosphotyrosine after immunoprecipitation with an anti-MAGI-3 antibody nor by anti-MAGI-3 immunoblotting after immunoprecipitation with anti-phosphotyrosine antibodies. The location of molecular weight markers is shown in kDa. (B) MAGI-3 is associated with different tyrosine-phosphorylated proteins in several cell lines. MAGI-3 was immunoprecipitated from untreated (-) or vanadate-treated (+) SF763T and C6 glioblastoma cells or Caco2 colon carcinoma cells, followed by immunoblotting with anti-phosphotyrosine (lower panel) or anti-MAGI-3 (upper panel) antibodies.

View larger version (30K): [in a new window]   Fig. 8. Dephosphorylation of p130 by RPTPß. (A) MAGI-3 immunocomplexes from vanadate-treated SF763T cells were incubated with GST or GST-fusion proteins containing the cytoplasmic domain of RPTPß (ßD12), a catalytic inactive mutant (ßD12DA), the first phosphatase domain (ßD1) or a mutant form lacking the C-terminus (ßD12dCT) as indicated. The reaction was terminated by the addition of SDS gel loading buffer, and the samples were immunoblotted with anti-phosphotyrosine (upper panel) or anti-MAGI-3 (lower panel) antibodies. Treatment of the immunocomplexes with alkaline phosphatase (AP) was used as a positive control. (B) Coommassie blue staining of the RPTPß constructs used in panel A. (C) Phosphatase activity. The indicated RPTPß GST-fusion proteins were incubated with p-nitrophenilphosphate as described in experimental procedures, and the absorbance of the reaction product was measured at 450 nm.

View larger version (10K): [in a new window]   Fig. 9. Schematic model of RPTPß/Slipr complex. The C-terminal tail of RPTPß binds to the third PDZ domain of Slipr/MAGI-3. This interaction brings the phosphatase into proximity with its tyrosine phosphorylated p130 substrate, resulting in dephosphorylation of p130. Since the molecular identity of p130 is not known, it was arbitrarily drawn bound to one PDZ and one WW domain. It is likely that the multi-domain organization of MAGI-3 allows it to bind simultaneously to other proteins in addition to RPTPß and p130, generating a signaling complex at the plasma membrane.