Immunoreceptor tyrosine-based inhibitory motif of the IL-4 receptor associates with SH2-containing phosphatases and regulates IL-4-induced proliferation.
Immunoreceptor tyrosine-based inhibitory motifs (ITIM) have been implicated in the negative modulation of immunoreceptor signaling pathways. The IL-4R alpha-chain (IL-4Ralpha) contains a putative ITIM in the carboxyl terminal. To determine the role of ITIM in the IL-4 signaling pathway, we ablated the ITIM of IL-4Ralpha by deletion and site-directed mutagenesis and stably expressed the wild-type (WT) and mutant hIL-4Ralpha in 32D/insulin receptor substrate-2 (IRS-2) cells. Strikingly, 32D/IRS-2 cells expressing mutant human (h)IL-4Ralpha were hyperproliferative in response to IL-4 compared with cells expressing WT hIL-4Ralpha. Enhanced tyrosine phosphorylation of Stat6, but not IRS-2, induced by hIL-4 was observed in cells expressing mutant Y713F. Using peptides corresponding to the ITIM of hIL-4Ralpha, we demonstrate that tyrosine-phosphorylated peptides, but not their nonphosphorylated counterparts, coprecipitate SH2-containing tyrosine phosphatase-1, SH2-containing tyrosine phosphatase-2, and SH2-containing inositol 5'-phosphatase. The in vivo association of SH2-containing inositol 5'-phosphatase with IL-4Ralpha was verified by coimmunoprecipitation with anti-IL-4Ralpha Abs. These results demonstrate a functional role for ITIM in the regulation of IL-4-induced proliferation.
Kashiwada
M
M
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Giallourakis
C C
CC
Pan
P Y
PY
Rothman
P B
PB
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D002460:Cell Line; D003593:Cytoplasm / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D015847:Interleukin-4 / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D053613:Janus Kinase 1; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010750:Phosphoproteins / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D019948:Receptors, Interleukin-4 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D050800:STAT6 Transcription Factor; D017384:Sequence Deletion; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D015534:Trans-Activators / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains / Q000276:immunology*
C526297:IRS2 protein, human; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D011971:Receptors, Immunologic; D019948:Receptors, Interleukin-4; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015534:Trans-Activators; D015847:Interleukin-4; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C507904:JAK1 protein, human; D053613:Janus Kinase 1; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D010744:Phosphoric Monoester Hydrolases; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases