Protein Tyrosine Phosphorylation
Figure 11A is representative of three different experiments showing that the EPO-R of Jar trophoblast-derived choriocarcinoma cells undergoes phosphorylation on tyrosine residues in response to treatment with exogenous rhuEPO for 2 and 5 min (40 U/ml). After immunoprecipi-tation of cell lysates with monoclonal anti-EPO-R antibody clone mh2er 7.9.2, and probing of the immunoblot with anti-phosphotyrosine antibody, a prominent band of > 80 kDa was observed. Low amounts of basal tyrosine phosphorylation of the receptor were also detectable in cells prior to EPO treatment. Analysis of tyrosine phosphorylation of the EPO-R in immunopurified third-trimester placental cells was also performed (Fig. 11B).
As with Jar cells, purified trophoblast cells express a phosphoprotein that can be immunoprecipitated with an anti-EPO-R antibody, and the degree of intensity is increased with exogenous EPO treatment. In this representative blot of three experiments, an intense signal was observed in the freshly isolated trophoblast cells (time zero); however, in subsequent experiments, a band of lesser intensity was detected. In addition, a higher molecular weight band was observed, but this band was also apparent when IgG1K was substituted for the 7.9.2 monoclonal antibody in the immunopre-cipitation procedure (data not shown). Finally, immunopur-ification of the EPO-R was performed with either the 7.9.2 or the 16.5.1 anti-EPO-R antibody, which recognize distinct epitopes, and a phosphotyrosine protein was found in Jar cells treated with exogenous EPO (Fig. 11C).
FIG. 11. A) EPO-induced tyrosine phosphorylation of the EPO-R in tro-phoblast-derived choriocarcinoma Jar cells. The EPO-R was immunopre-cipitated with anti-EPO-R antibody (mh2er 7.9.2) from Jar cell lysates at various times after treatment with recombinant human EPO (40 U/ml) and separated on 5-20% gradient SDS-PAGE; then immunoblots were probed with anti-phosphotyrosine antibody (PY20). See Materials and Methods for further details. Nonstimulated Jar cells after an overnight preincubation in serum-free conditions were considered to be Time 0 (Lane 1). Lanes 2 and 3 are Jar cells after 2 min without (lane 2) or with EPO treatment (lane 3), and after 5 min without (lane 4) and with EPO treatment (lane 5). Lane 6 represents Jar cells at Time 0 that were immunoprecipitated with a mouse IgG1 к isotypic control. B) EPO-induced tyrosine phosphorylation of the EPO-R in freshly isolated, immunopurified trophoblast cells from the term placenta. EPO-R was immunoprecipitated with anti-EPO-R antibody (7.9.2) from cell lysates, and immunoblots were probed with an anti-phosphotyrosine antibody (PY99). Lanes 1-6 are as follows: negative control immunoprecipitation with mouse IgG1 к, untreated trophoblast cells at time zero, untreated trophoblast cells after 5-min incubation at 37°C, trophoblast cells incubated for 5 min with rhEPO (40 U/ ml), Jar cells incubated 5 min with no treatment, Jar cells incubated 5 min with rhEPO (40 U/ml). C) EPO-induced tyrosine phosphorylation of the EPO-R in trophoblast-derived choriocarcinoma Jar cells. Cultured Jar cells were treated for 5 min with rhEPO (40 U/ml), and the EPO-R was immunoprecipitated from lysates with either monoclonal antibody mh2er 7.9.2 (lane 1) or 16.5.1 (lane 2). Immunoblots were probed with anti-phosphotyrosine antibody, PY99. Molecular weight standards X 10~3 are indicated on the left.