Pleiotrophin signals increased tyrosine phosphorylation of beta beta-catenin through inactivation of the intrinsic catalytic activity of the receptor-type protein tyrosine phosphatase beta/zeta

Abstract

Pleiotrophin (PTN) is a platelet-derived growth factor-inducible, 18-kDa heparin-binding cytokine that signals diverse phenotypes in normal and deregulated cellular growth and differentiation. To seek the mechanisms of PTN signaling, we studied the interactions of PTN with the receptor protein tyrosine phosphatase (RPTP) beta/zeta in U373-MG cells. Our results suggest that PTN is a natural ligand for RPTP beta/zeta. PTN signals through "ligand-dependent receptor inactivation" of RPTP beta/zeta and disrupts its normal roles in the regulation of steady-state tyrosine phosphorylation of downstream signaling molecules. We have found that PTN binds to and functionally inactivates the catalytic activity of RPTP beta/zeta. We also have found that an active site-containing domain of RPTP beta/zeta both binds beta-catenin and functionally reduces its levels of tyrosine phosphorylation when added to lysates of pervanidate-treated cells. In contrast, an (inactivating) active-site mutant of RPTP beta/zeta also binds beta-catenin but fails to reduce tyrosine phosphorylation of beta-catenin. Finally, in parallel to its ability to inactivate endogenous RPTP beta/zeta, PTN sharply increases tyrosine phosphorylation of beta-catenin in PTN-treated cells. The results suggest that in unstimulated cells, RPTP beta/zeta is intrinsically active and functions as an important regulator in the reciprocal control of the steady-state tyrosine phosphorylation levels of beta-catenin by tyrosine kinases and phosphatases. The results also suggest that RPTP beta/zeta is a functional receptor for PTN; PTN signals through ligand-dependent receptor inactivation of RPTP beta/zeta to increase levels of tyrosine phosphorylation of beta-catenin to initiate downstream signaling. PTN is the first natural ligand identified for any of the RPTP family; its identification provides a unique tool to pursue the novel signaling pathway activated by PTN and the relationship of PTN signaling with other pathways regulating beta-catenin.

Figure 1

Association of RPTP β/ζ with PTN. (A) Lysates of U373-MG glioblastoma cells were immunoprecipitated with anti-RPTP β/ζ monoclonal antibodies. The immunoprecipitates were separated on 6% acrylamide gel, transferred to a poly(vinylidene difluoride) membrane, and probed with anti-RPTP β/ζ antibodies. The arrowheads indicate the RPTP β/ζ spliced products of ≈230, 130, and 85 kDa. (B) Western analysis of RPTP β/ζ captured by PTN-Fc. Lysates of U373-MG cells were incubated with PTN-Fc and proteins interactive with PTN-Fc (right lane) were captured with protein A Sepharose-4B beads for 2 h. The beads were washed in cold lysis buffer, boiled in SDS/PAGE sample buffer, and the eluted proteins were separated on an 8% acrylamide gel and analyzed by Western blots probed with anti-RPTP β/ζ monoclonal antibodies. As a control, PTN-Fc was replaced with an equal amount of human IgG (left lane). The arrowheads indicate the ≈130- and ≈85-kDa spliced products of RPTP β/ζ. (C) Western analysis of RPTP β/ζ captured by endogenous PTN. Lysates of U373-MG cells were incubated with anti-PTN monoclonal antibodies (right lane) and the complexes were captured with protein A Sepharose-4B beads for 2 h. The beads were washed in cold lysis buffer and boiled in SDS/PAGE sample buffer, and the eluted proteins were separated on an 8% acrylamide gel and analyzed by Western blots probed with anti-RPTP β/ζ monoclonal antibodies. As a control, mouse IgG replaced the anti-PTN antibody (left lane). The arrowheads indicate the ≈130- and ≈85-kDa spliced products of RPTP β/ζ.

Figure 2

PTN-dependent inhibition of the intrinsic tyrosine phosphatase activity of RPTP β/ζ. (A) Inhibition of the endogenous RPTP β/ζ tyrosine phosphatase activity in PTN-treated U373-MG cells. The left bar represents tyrosine phosphatase activity in immunoprecipitates from lysates of untreated cells with mouse IgG (control) to replace the anti-RPTP β/ζ antibodies. The center bar represents tyrosine phosphatase activity in immunoprecipitates with anti-RPTP β/ζ antibodies from lysates of untreated cells, and the right bar represents tyrosine phosphatase activity of immunoprecipitates with anti-RPTP β/ζ antibodies from lysates of cells treated with recombinant PTN (50 ng/ml.) (B) Inhibition of recombinant RPTP β/ζ phosphatase activity in Sf9 cell membranes. Membrane fractions of Sf9 cells that were infected by a baculovirus containing a cDNA-encoding RPTP β/ζ (right two bars), or uninfected (left two bars) that were untreated (− PTN) or treated (+ PTN) with 50 ng/ml PTN were assayed as described in Materials and Methods. (C). Time course of PTN-dependent inactivation of RPTP β/ζ in PTN-treated (50 ng/ml) Sf9 cell membranes expressing RPTP β/ζ (solid bars) and Sf9 cell membranes without RPTP β/ζ (open bar, t = 0 only).

Figure 3

Physical and functional association of β-catenin with PTN/RPTP β/ζ. (A). PTN-Fc is in complex with RPTP β/ζ and β-catenin. PTN-Fc-treated confluent U373-MG cells from 60-mm dish were chemically cross-linked with 3,3′-dithiobis(sulfosuccinimidyl propionate). Lysates from PTN-Fc-treated, chemically cross-linked cells (lanes 1) or Fc- (alone) treated (control) U373-MG cells (lanes 2) were incubated with protein A Sepharose, washed, eluted with SDS sample buffer with 5% 2-mercaptoethanol, and analyzed in 6% SDS gels and Western blots. Lysates from untreated U373-MG cells alone (lanes 3) were also analyzed as a control. Western blots were analyzed with anti-β-catenin (Right) or anti-RPTP β/ζ antibodies (Left). Arrowheads identify RPTP β/ζ spliced products of ≈250, 230, 130, and 85 kDa (Left) and β-catenin (94 kDa) (Right). (B) β-Catenin interacts with proximal (catalytic) domain of RPTP β/ζ. The GST-D1-RPTP β/ζ wild-type, GST-D1-Cys-1925 → Ser (inactivating) mutant fusion protein or GST alone were expressed and immobilized with glutathione-Sepharose-4B beads, incubated with U373-MG cell lysates, washed, and analyzed in Western analysis with the α-phosphotyrosine antibodies and visualized with the enhanced chemiluminescence ECL-PLUS system (Lower). The same blot was reprobed with α-β-catenin antibodies and detected as above (Upper).

Figure 4

Increased β-catenin tyrosine phosphorylation. (A) Time course of the tyrosine phosphorylation of β-catenin in response to PTN-Fc treatment. Cells were treated with 10 ng/ml PTN-Fc for the times indicated. Lysates were immunoprecipitated with α-β-catenin antibodies and analyzed in Western blots probed with α-phosphotyrosine antibodies (Upper) and the blots were reprobed with α-β-catenin antibodies (Lower). (B) U373-MG cells were treated with different doses of PTN-Fc for 20 min. Cells were grown to near confluence, and then were serum-starved for 48 h. PTN-Fc was added up to the indicated concentrations. The Fc fragment alone (20 ng/ml) was added as a control. Lysates were immunoprecipitated and analyzed in Western blots with antiphosphotyrosine antibodies as described above. Parallel immunoblots were probed for β-catenin.