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Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling.

Holmes DA, Suto E, Lee WP, Ou Q, Gong Q, Smith HR, Caplazi P, Chan AC - J. Exp. Med. (2015)

Bottom Line: Pep(-/-) hematopoietic progenitors demonstrate increased IFNAR signaling, increased IFN-inducible gene expression, and enhanced proliferation and activation compared to Pep(+/+) progenitors in response to IFN-α.In addition, Pep(-/-) mice treated with IFN-α display a profound defect in hematopoiesis, resulting in anemia, thrombocytopenia, and neutropenia when compared to IFN-α-treated Pep(+/+) mice.As SLE patients carrying the PTPN22(C1858T) risk variant have higher serum IFN-α activity, these data provide a molecular basis for how type I IFNs and PTPN22 may cooperate to contribute to lupus-associated cytopenias.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Department of Translational Immunology, and Department of Pathology, Genentech, Inc., South San Francisco, CA 94080.

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PEP interacts with the IFNAR signaling complex to negatively regulate IFNAR signaling. (A) 3T3 cells were transfected with vector or Pep cDNAs. 24 h after transfection, cells were stimulated with rIFN-α4 (2,000 U/ml) for indicated times and whole-cell extracts were analyzed by immunoblotting for phosphorylated STAT1 and total STAT1 (top), phosphorylated STAT2 and total STAT2 (middle), PEP, and ACTIN (bottom). (B) Relative band intensity by densitometry of pSTAT1 (α-isoform) and pSTAT2 normalized to relative band intensity of total STAT1 (α-isoform) and total STAT2, respectively, from A are shown. (C) 3T3 cells were transfected with vector, Pep-C227S, or Pep cDNAs. 24 h after transfection, cells were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times and whole-cell extracts analyzed by immunoblotting for phosphorylated STAT1 and total STAT1 (top) and ACTIN (bottom) are shown. (D) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from C are shown. (E) 3T3 cells transfected with Pep or vector cDNAs were immunoprecipitated with anti-JAK1, -TYK2, -SOCS1, -IFNAR, -STAT1, or IgG control antibodies. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting for PEP (P1 antibody). 1% cell lysates are shown for comparison. Data shown A, C, E, and F are representative of three independent experiments.
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fig6: PEP interacts with the IFNAR signaling complex to negatively regulate IFNAR signaling. (A) 3T3 cells were transfected with vector or Pep cDNAs. 24 h after transfection, cells were stimulated with rIFN-α4 (2,000 U/ml) for indicated times and whole-cell extracts were analyzed by immunoblotting for phosphorylated STAT1 and total STAT1 (top), phosphorylated STAT2 and total STAT2 (middle), PEP, and ACTIN (bottom). (B) Relative band intensity by densitometry of pSTAT1 (α-isoform) and pSTAT2 normalized to relative band intensity of total STAT1 (α-isoform) and total STAT2, respectively, from A are shown. (C) 3T3 cells were transfected with vector, Pep-C227S, or Pep cDNAs. 24 h after transfection, cells were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times and whole-cell extracts analyzed by immunoblotting for phosphorylated STAT1 and total STAT1 (top) and ACTIN (bottom) are shown. (D) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from C are shown. (E) 3T3 cells transfected with Pep or vector cDNAs were immunoprecipitated with anti-JAK1, -TYK2, -SOCS1, -IFNAR, -STAT1, or IgG control antibodies. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting for PEP (P1 antibody). 1% cell lysates are shown for comparison. Data shown A, C, E, and F are representative of three independent experiments.

Mentions: To further explore the biochemical nature by which PEP regulates IFNAR signaling, we expressed PEP in 3T3 cells that are IFN-α responsive. 3T3 cells expressing transfected PEP demonstrated reduced STAT1 and STAT2 phosphorylation after rIFN-α4 stimulation compared to vector control cDNA-transfected cells (Fig. 6, A and B). The phosphatase activity of PEP was required for maximal reduction of STAT1 phosphorylation because expression of a catalytically inactive PEP(C227S) did not reduce STAT1 phosphorylation to the same extent as wild-type PEP (Fig. 6, C and D). Finally, we investigated whether PEP could co-localize with the IFNAR signaling complex. In 3T3 cells transfected with a Pep cDNA, PEP co-immunoprecipitated with multiple components of the IFNAR signaling complex, including JAK1, TYK2, SOCS1, IFNAR, and STAT1 (Fig. 6 E). Hence, PEP is physically positioned to regulate IFNAR signaling.


Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling.

Holmes DA, Suto E, Lee WP, Ou Q, Gong Q, Smith HR, Caplazi P, Chan AC - J. Exp. Med. (2015)

PEP interacts with the IFNAR signaling complex to negatively regulate IFNAR signaling. (A) 3T3 cells were transfected with vector or Pep cDNAs. 24 h after transfection, cells were stimulated with rIFN-α4 (2,000 U/ml) for indicated times and whole-cell extracts were analyzed by immunoblotting for phosphorylated STAT1 and total STAT1 (top), phosphorylated STAT2 and total STAT2 (middle), PEP, and ACTIN (bottom). (B) Relative band intensity by densitometry of pSTAT1 (α-isoform) and pSTAT2 normalized to relative band intensity of total STAT1 (α-isoform) and total STAT2, respectively, from A are shown. (C) 3T3 cells were transfected with vector, Pep-C227S, or Pep cDNAs. 24 h after transfection, cells were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times and whole-cell extracts analyzed by immunoblotting for phosphorylated STAT1 and total STAT1 (top) and ACTIN (bottom) are shown. (D) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from C are shown. (E) 3T3 cells transfected with Pep or vector cDNAs were immunoprecipitated with anti-JAK1, -TYK2, -SOCS1, -IFNAR, -STAT1, or IgG control antibodies. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting for PEP (P1 antibody). 1% cell lysates are shown for comparison. Data shown A, C, E, and F are representative of three independent experiments.
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fig6: PEP interacts with the IFNAR signaling complex to negatively regulate IFNAR signaling. (A) 3T3 cells were transfected with vector or Pep cDNAs. 24 h after transfection, cells were stimulated with rIFN-α4 (2,000 U/ml) for indicated times and whole-cell extracts were analyzed by immunoblotting for phosphorylated STAT1 and total STAT1 (top), phosphorylated STAT2 and total STAT2 (middle), PEP, and ACTIN (bottom). (B) Relative band intensity by densitometry of pSTAT1 (α-isoform) and pSTAT2 normalized to relative band intensity of total STAT1 (α-isoform) and total STAT2, respectively, from A are shown. (C) 3T3 cells were transfected with vector, Pep-C227S, or Pep cDNAs. 24 h after transfection, cells were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times and whole-cell extracts analyzed by immunoblotting for phosphorylated STAT1 and total STAT1 (top) and ACTIN (bottom) are shown. (D) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from C are shown. (E) 3T3 cells transfected with Pep or vector cDNAs were immunoprecipitated with anti-JAK1, -TYK2, -SOCS1, -IFNAR, -STAT1, or IgG control antibodies. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting for PEP (P1 antibody). 1% cell lysates are shown for comparison. Data shown A, C, E, and F are representative of three independent experiments.
Mentions: To further explore the biochemical nature by which PEP regulates IFNAR signaling, we expressed PEP in 3T3 cells that are IFN-α responsive. 3T3 cells expressing transfected PEP demonstrated reduced STAT1 and STAT2 phosphorylation after rIFN-α4 stimulation compared to vector control cDNA-transfected cells (Fig. 6, A and B). The phosphatase activity of PEP was required for maximal reduction of STAT1 phosphorylation because expression of a catalytically inactive PEP(C227S) did not reduce STAT1 phosphorylation to the same extent as wild-type PEP (Fig. 6, C and D). Finally, we investigated whether PEP could co-localize with the IFNAR signaling complex. In 3T3 cells transfected with a Pep cDNA, PEP co-immunoprecipitated with multiple components of the IFNAR signaling complex, including JAK1, TYK2, SOCS1, IFNAR, and STAT1 (Fig. 6 E). Hence, PEP is physically positioned to regulate IFNAR signaling.

Bottom Line: Pep(-/-) hematopoietic progenitors demonstrate increased IFNAR signaling, increased IFN-inducible gene expression, and enhanced proliferation and activation compared to Pep(+/+) progenitors in response to IFN-α.In addition, Pep(-/-) mice treated with IFN-α display a profound defect in hematopoiesis, resulting in anemia, thrombocytopenia, and neutropenia when compared to IFN-α-treated Pep(+/+) mice.As SLE patients carrying the PTPN22(C1858T) risk variant have higher serum IFN-α activity, these data provide a molecular basis for how type I IFNs and PTPN22 may cooperate to contribute to lupus-associated cytopenias.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Department of Translational Immunology, and Department of Pathology, Genentech, Inc., South San Francisco, CA 94080.

Show MeSH
Related in: MedlinePlus