The Cell Surface Receptor Tartan Is a Potential In Vivo Substrate for the Receptor Tyrosine Phosphatase Ptp52F

Tartan is tyrosine phosphorylated in v-Src-transfected S2 cells. (A) Anti-GFP immunoblot of lysates (L) and anti-GFP IPs (I) from cells transfected with the Trn cyto-GFP plasmid. As indicated by “+” and “−” signs above the blot, cells were left untreated (lanes 1 and 2), treated with pervanadate (lanes 3 and 4), or cotransfected with D-Abl (lanes 5 and 6) or D-Src64B plasmids (lanes 7 and 8). The immunoglobulin G heavy chain (IgG HC), unphosphorylated Trn-cyto-GFP, and tyrosine-phosphorylated Trn-cyto-GFP bands (P-Trn) are labeled. (B) Anti-PY immunoblot of the same samples. The IgG HC and P-Trn bands are labeled. (C) Anti-PY immunoblot of lysates and anti-GFP IPs from cells transfected with the Trn cyto-GFP plasmid. Cells were left untreated (lane 1), cotransfected with the v-Src plasmid (lanes 2 and 3), or treated with pervanadate (lane 4). The IgG HC and P-Trn bands are labeled. Note that the P-Trn band in lane 3 migrates more rapidly than that in lane 4, suggesting that Trn is less heavily tyrosine phosphorylated in v-Src-expressing cells than in pervanadate-treated cells.

Phosphorylated Trn binds to the Ptp52F substrate-trapping mutant and is dephosphorylated by wild-type Ptp52F in S2 cells. (A) An anti-Myc immunoblot of lysates (L), anti-Myc IPs (I), and post-IP supernatants (P) from cells that were cotransfected with the Trn-FL and v-Src plasmids. As indicated by “+” and “−” signs above the blot, these cells were also transfected with the Ptp52F-trap-Myc plasmid (lanes 1 to 3) or the Ptp52F-wild-type-Myc plasmid (lanes 4 to 6). The region of the blot containing the ∼65-kDa Ptp52F-Myc band is shown. (B) Anti-Trn immunoblot of the same samples. Note that a strong Trn band was present in anti-Myc IPs of cells cotransfected with Ptp52F-trap-Myc (lane 2), but only a very faint signal was seen when Ptp52F-wild type-Myc was cotransfected (lane 5). This shows that the substrate-trapping mutant bound selectively to Trn-FL. (C) Anti-GFP immunoblot of lysate (L), an anti-GFP IP, and an anti-Trn IP from cells transfected with the Trn-FL-GFP plasmid. An ∼100-kDa band was present (labeled as Trn FL-GFP), as expected. An ∼60-kDa band that is likely to represent a cleavage product was also present (labeled as cleaved Trn-GFP). The cleavage site must be in the XC domain, close to the membrane, based on the size of this product. The IgG heavy chain (IgG HC) band is also labeled. (D) Anti-PY blot of anti-GFP IPs from S2 cells cotransfected with the Trn FL-GFP and v-Src plasmids, together with either the Ptp52F-trap-Myc (lane 1) or Ptp52F-wild type-Myc (lane 2) plasmids. Note that both the Trn-FL-GFP and cleaved Trn-GFP bands were tyrosine phosphorylated in lane 1; similar levels of phosphorylation were observed when the trap was not cotransfected. When Ptp52F-wild type-Myc was cotransfected, the PY signal was barely detectable for Trn-FL-GFP and undetectable for the cleaved product. This shows that Ptp52F caused dephosphorylation of v-Src-phosphorylated Trn-FL-GFP and the cleavage product. (E) Anti-Myc immunoblot of lysates (L), anti-GFP IPs (GI), and anti-Myc IPs (MI) from cells transfected with Trn-FL-GFP. Cells were also transfected with Ptp52F-trap-Myc (lanes 1 to 6) or Ptp52F-wild type-Myc (lanes 7 to 12) plasmids, with (lanes 4 to 6 and lanes 10 to 12) or without (lanes 1 to 3 and lanes 7 to 9) the v-Src plasmid. The region of the blot containing the ∼65-kDa Ptp52F-Myc band is shown. Note that this band was detected in anti-GFP IPs from cells cotransfected with Ptp52F-trap-Myc and v-Src (lane 5) but was not detectable in anti-GFP IPs from trap-transfected cells lacking the kinase (lane 2). A very faint signal was observed in anti-GFP IPs from cells transfected with Ptp52F-wild type-Myc and the kinase (lane 11). This shows that the substrate-trapping mutant bound to Trn-FL-GFP in a phosphorylation-dependent manner. (F) Anti-PY immunoblot of the same anti-GFP (GI) or anti-Myc (MI) IPs. Note that, as in panel D, the phosphorylated Trn-FL-GFP and cleavage product bands were present when Ptp52F-trap-Myc and v-Src were both transfected (lane 3) but were absent when Ptp52F-wild type-Myc and v-Src were transfected (lane 7). Faint signals were present for both the Trn-FL-GFP and cleavage product bands in lane 4, which is an anti-Myc IP from cells transfected with Ptp52F-trap-Myc and v-Src; this confirms that phosphorylated Trn-FL-GFP associated with the trap. The IgG HC band is also labeled.

Phosphorylated Trn binds selectively to the Ptp52F substrate-trapping mutant in vitro. (A) Anti-PY immunoblot of a pervanadate-treated S2 cell lysate incubated with the indicated amounts (in μg) of Ptp52F-wild type-GST. (B) Anti-PY immunoblot of an anti-GFP IP from a lysate of pervanadate-treated S2 cells transfected with the Trn-cyto-GFP plasmid. The bead-bound protein was incubated with the indicated amounts (in μg) of Ptp52F-wild type-GST (100-μl reaction volume, 1 h, 20°C). The Trn-cyto-GFP band is indicated; other bands on the gel are presumably background. Note that the PY signal for Trn-cyto-GFP was eliminated by 1 μg of Ptp52F-wild type-GST (lane 2), showing that it dephosphorylated Trn, but many bands in Fig. 4A (lane 2) were also diminished in intensity by this same amount of fusion protein. (C) Anti-PY immunoblot of a GST “pulldown” experiment in which lysate from pervanadate-treated S2 cells transfected with the Trn-cyto-GFP plasmid was incubated with the indicated amounts of Ptp52F-trap-GST (in μg; 100-μl volume), followed by precipitation of the trap-bound proteins with glutathione-agarose beads. Only a single prominent band, of ∼85 kDa, was observed. The Trn-cyto-GFP band (∼60 kDa) was not detected. (D) Anti-PY immunoblot of a GST pulldown experiment in which lysate from pervanadate-treated untransfected or Trn-FL-transfected S2 cells was incubated with 10 μg of Ptp52F-trap-GST or Ptp52F-wild type-GST, followed by precipitation of the bound proteins as in panel C. Note that lysate from these cells produces a continuous smear but that only a single band is pulled down by Ptp52F-trap-GST, and the intensity of this band is increased when Trn-FL is overexpressed. (E) An anti-Trn immunoblot of transfected cell pulldowns from the same experiment. The Trn band is observed when either Ptp52F-trap-GST or Ptp52F-wild type-GST is used for pulldown, showing that wild-type Ptp52F dephosphorylates Trn but can then remain bound to it.