Schematic representation of the protein segments used in the two-hybrid interaction trap. Shown are the regions of PTP-BL (A) used as baits and the RIL segments [hRIL(164–330) and hRIL(188–330)] (B) as they were retrieved from the G0-fibroblast cDNA library and the RIL-PDZ domain used as bait to assay for intramolecular interactions. In parentheses, the numbers corresponding to the first and last aa positions in the PTP-BL and RIL segments are shown. Band 4.1-like domain, hatched box; PTPase, open box; PDZ motifs, black ovals; LIM domain, stippled box.

Sequence alignment of RIL protein sequences from mouse, rat, and human. Protein sequences of mouse (accession number Y08361), rat (Kiess et al., 1995; accession number X76454), and hRIL (Schaefer, unpublished data; accession number X93510) were aligned using MULTALIGN. aas identical to those of the mouse sequence are indicated with a dash. The PDZ motif is double underlined and the LIM domain is indicated by a single underline. Vertical arrows indicate the start positions of clones hRIL(164–330) and hRIL(188–330), which were originally identified in the two-hybrid interaction screening with the BL-PDZ-(I–V) bait. A potential tyrosine phosphorylation site is shaded.

Multiple alignment of PDZ motifs. The PDZ motif of mouse RIL is aligned with the PDZ motifs of Drosophila DlgA (DLG1, DLG2, and DLG3; accession number M73529), mouse PTP-BL (BL1 through BL5; accession number Z32740), rat CLP36 (accession number U23769), human enigma (PIR:A55050), mouse nNOS (accession number D14552), and mouse LIMK1 (accession number X86569). The position number of the initial and last aa of the protein segments that were used are shown on either side of the sequence. Sequences presented below RIL are ordered by descending homology. Aas similar (score >0.7 in Dayhoff table; Schwartz and Dayhoff, 1979) in 9 of 13 sequences are shown on a gray background, and the most frequent aa at these positions is indicated in the consensus line. Aas identical in more than half of the sequences are indicated in white on a black background. Dashes represent sequence gaps.

Determination of the relative strength of two-hybrid interactions. β-Galactosidase activities (arbitrary units) were measured in total lysates of yeast containing an LacZ-reporter plasmid. The yeast cells contained plasmids encoding baits as indicated below the bars, and the empty prey vector pJG4–5 (A, gray bars), hRIL(164–330 (B, filled bars), or mRIL-FL (C, open bars). Error bars indicate SD (n = 5).

Coprecipitation of RIL with PDZ motifs. VSV epitope-tagged PDZ motifs of PTP-BL and RIL were transiently coexpressed in COS-1 cells with the C-terminal part of RIL, hRIL(164–330). α-VSV mAbs were used to immunoprecipitate the various PDZ proteins. Coprecipitated RIL was detected by Western blotting using a pAb (α-RIL) directed against the C-terminal part of hRIL. The labels on top refer to the protein segments outlined in Figure 1 and the dash indicates mock-transfected cells.

Colocalization of PTP-BL and RIL in mouse tissues. Visualization of the presence of PTP-BL and RIL proteins in mouse lung, stomach, and skin was done using immunofluorescence labeling with affinity-purified polyclonal antiserum directed against PTP-BL (α-BL-PDZ-I and α-BL-PTP) and RIL (α-RIL). Corresponding hematoxylin/eosin-stained sections (HE) are shown for comparison. Bars, 25 μm.

PDZ-mediated interactions with truncated RIL peptides. Schematic representation of the regions of RIL used as prey in the two-hybrid interaction trap are shown on the left. Interaction results using these constructs combined with BL-PDZ-II, BL-PDZ-IV, and mRIL-PDZ as baits are represented by + and −. +++, very strong interaction (blue colonies within 2 d on X-gal assay plates); ++, interaction (blue colonies after 3 d); +, weak but detectable interaction (blue colonies after 4–6 d); −, no interaction detectable (no blue colonies after 7 d).

RIL is phosphorylated on tyrosines in vitro and in vivo and is an in vitro substrate for PTP-BL. (A) GST-fusion proteins were purified from bacterial TKX cells expressing an inducible tyrosine kinase. Purified protein was detected by Western blotting using an anti-GST antibody (top panel) and analyzed for tyrosine phosphorylation using the phosphotyrosine specific antibody PY20 (bottom panel). Only GST-RIL-LIM (aas 209–330) is phosphorylated on tyrosines and mutation of tyrosine 294 to phenylalanine (GST-RIL-LIM-Y-F) clearly diminishes the phosphorylation level. (B) Purified phosphorylated GST-RIL-LIM was incubated for 15 min with purified GST or GST-BL-PTP (aas 2101–2460) and analyzed for its phosphotyrosine content. GST-BL-PTP can dephosphorylate the RIL-LIM domain, whereas GST alone does not affect the tyrosine phosphorylation. Furthermore, the specific tyrosine phosphatase inhibitor vanadate can completely inhibit the dephosphorylation by GST-BL-PTP. (C) COS-1 cells were transfected with an expression construct encoding the C-terminal half of RIL [hRIL(164–330)] or mRIL-FL. The presence of transiently expressed RIL in total cell lysates and anti-RIL immunoprecipitates was detected by Western blotting using an anti-RIL antibody. (D) The tyrosine phosphorylation content of the same samples as depicted in C was determined by Western blotting using the phosphotyrosine-specific antibody PY20. Both the immunoprecipitated hRIL(164–330) and the mRIL-FL are found to be phosphorylated on tyrosine residues.