IL-10 signaling via IL-10E1 is dependent on tyrosine phosphorylation in the IL-10R α 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 (Tyr446 and Tyr496) located in the cytoplasmic domain of the IL-10R α 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 Tyr446 and Tyr496, and tyrosine-phosphorylated peptides containing Tyr230 or Tyr252/259 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 Tyr446 and Tyr496 amino-acid residues completely blocked ligand signaling. Coinjection of peptides containing either serine substitutions for Tyr446 and Tyr496 or Tyr230 and Tyr252/259 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.