IL-10 signaling via IL-10E1 is dependent on tyrosine phosphorylation in the IL-10R alpha chain in human primary prostate cancer cell lines.
Interleukin 10 (IL-10) stimulates rapid nuclear translocation and binding of a 22 kDa protein, termed interleukin 10 enhancer 1 (IL-10E1), to a novel enhancer element (i.e. HTE-1) of the tissue inhibitor of metalloproteinase-1 (TIMP-1) gene to upregulate TIMP-1 expression. IL-10E1 signaling involves tyrosine phosphorylation of the IL-10R JAK1 (Janus kinase) and TYK2 (tyrosine kinase) receptor kinases and tyrosine phosphorylation of two tyrosine moieties (Y57 and Y62) of a LIM domain of the IL-10E1 protein. In this paper, the studies showed that two tyrosine residues (Tyr(446) and Tyr(496)) located in the cytoplasmic domain of the IL-10R alpha chain were required for receptor function, and for phosphorylation and activation of IL-10E1. Immunoprecipitation studies revealed that 12 amino-acid peptides encompassing either of these two tyrosine residues in phosphorylated form coprecipitated IL-10E1 and blocked ligand-dependent IL-10E1 phosphorylation in a cell-free system. In contrast, peptides containing serine substitutions for Tyr(446) and Tyr(496), and tyrosine-phosphorylated peptides containing Tyr(230) or Tyr(252/259) did not prevent IL-10E1 activation or signaling. To confirm these observations in vivo, fusion protein constructs were made between a modified form of green fluorescent protein or GFP and the intact IL-10E1 protein (IL-10E1-MmGFP) and n-terminal peptides of the IL-10E1 protein (i.e. nt-nls-MmGFP and mutant sequences identified as nt-nls mC61-MmGFP and nt-nls mY57/mY62-MmGFP peptides). Confocal microscopy revealed that IL-10 triggered transport to the nucleus of IL-10E1-MmGFP, nt-nls-MmGFP, and nt-nls mC61-MmGFP by 10-30 min in HPCA-10a (human prostrate cancer cells; derived from Gleason sum 10 tumor tissue) cells. IL-10 failed to induce nuclear translocation of the mY57/mY62-MmGFP peptides with point mutations of the two tyrosine groups. Coinjection of nt-nls-MmGFP with the IL-10R Tyr(446) and Tyr(496) amino-acid residues completely blocked ligand signaling. Coinjection of peptides containing either serine substitutions for Tyr(446) and Tyr(496) or Tyr(230) and Tyr(252/259) failed to block nt-nls-MmGFP signaling. The data demonstrate that IL-10E1 is directly recruited to the ligand-activated IL-10R by binding to specific phosphotyrosine groups which control tyrosine phosphorylation of the LIM domain of the IL-10E1 protein (i.e. Y57/Y62 groups) and IL-10E1 activation.
Stearns
Mark E
ME
Department of Pathology and Laboratory, Drexel University College of Medicine, 15th and Vine Sts, Philadelphia, PA 19102-1192, USA. Stearnsm1@aol.com
Hu
Youji
Y
Wang
Min
M
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D006801:Humans; D016753:Interleukin-10 / Q000494:pharmacology*; D015847:Interleukin-4 / Q000494:pharmacology; D008297:Male; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D018123:Receptors, Interleukin / Q000378:metabolism*; D053701:Receptors, Interleukin-10; D050800:STAT6 Transcription Factor; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D018123:Receptors, Interleukin; D053701:Receptors, Interleukin-10; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D016753:Interleukin-10; D015847:Interleukin-4; D014443:Tyrosine