Direct interaction of STAT4 with the IL-12 receptor.
Signal transduction by interleukin-12 (IL-12) requires phosphorylation and activation of STAT4. Direct interaction of the SH2 domain of STAT4 with a phosphotyrosine residue in the IL-12 receptor has been proposed to be required for the subsequent STAT4 phosphorylation. The IL-12 receptor beta2 subunit contains three tyrosine residues in its cytoplasmic domain. To test the hypothesis that one of these tyrosines is involved in binding STAT4, phosphopeptides were synthesized according to the amino acid sequences surrounding each of these tyrosine residues. Only the phosphopeptide containing pTyr800 strongly bound to STAT4 in a cell-free binding assay. When this phosphopeptide was introduced into TALL-104 cells, it blocked IL-12-induced STAT4 phosphorylation by competing with the IL-12 receptor for binding to STAT4. A series of alanine replacements was performed in this phosphopeptide to elucidate which amino acids surrounding the pTyr800 residue are critical for STAT4 binding. To summarize, the site on the IL-12 receptor which binds STAT4 can be described as -T-X-X-G-pY(800)-L-, where the core G-pY(800)-L motif is critical for the binding; the threonine at the pY-4 position has only a minor contribution and X represents amino acids not critical for the binding. These results demonstrate that only a small region of the IL-12 receptor is critically involved in binding STAT4 and suggest the feasibility that small molecule inhibitors could be identified which interfere with IL-12 signal transduction for treatment of autoimmune diseases.
Pharmaceutical Products Division, Pharmaceutical Discovery, Abbott Park, Illinois, 60064, USA. betty.yao@abbott.com
B B
BB
Yao
P
P
Niu
C S
CS
Surowy
C R
CR
Faltynek
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D018123:Receptors, Interleukin / Q000737:chemistry / Q000378:metabolism*; D053707:Receptors, Interleukin-12; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D050797:STAT4 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry; D018909:src Homology Domains
D004268:DNA-Binding Proteins; C508439:IL12RB2 protein, human; D010748:Phosphopeptides; D018123:Receptors, Interleukin; D053707:Receptors, Interleukin-12; D011994:Recombinant Proteins; D050797:STAT4 Transcription Factor; C494090:STAT4 protein, human; D015534:Trans-Activators; D014443:Tyrosine