Association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with an essential erythropoietin receptor subdomain.
Using an active, HAI epitope-tagged form of the murine erythropoietin (EPO) receptor and via direct coimmunoprecipitation, the p85 regulatory subunit of phosphatidyl inositol-3 kinase (p85/PI3-K) is shown to associate with the EPO receptor in transfected FDC-P1 cell lines. Coimmunoprecipitation of p85 with epitope-tagged EPO receptors was observed initially in FDC-HER cells labeled metabolically with [32P]orthophosphate, and association of these factors was confirmed by Western analyses of receptor immunoprecipitates using p85 antiserum. Interestingly, this association occurred in the absence of ligand, and exposure of FDC-HER cells to EPO did not detectably affect levels of receptor-associated p85 or overall levels of p85 phosphorylation. However, EPO was observed to stimulated the rapid formation of phosphatidylinositol 32P-phosphate in FDC-HER and FDC-ER cells. Through baculovirus-mediated expression of epitope-tagged EPO receptor forms in SF9 cells, domains for p85 association were mapped. Analyses of receptor forms with cytosolic truncations and deletions delineated a candidate subdomain for p85 binding to an essential extended box-2 region (P329-E374; including a putative motif for SH2 binding, Y343LVL). These findings extend a mechanistic alignment between the EPO receptor and protein tyrosine kinase-encoding receptors that likewise activate PI3-K, and expand the importance of further defining pathways to PI3-K activation.
He
T C
TC
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Zhuang
H
H
Jiang
N
N
Waterfield
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MD
Wojchowski
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D000939:Epitopes / Q000032:analysis; D004921:Erythropoietin / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D016296:Mutagenesis; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017467:Receptors, Erythropoietin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D014162:Transfection
D017931:DNA Primers; D000939:Epitopes; D046911:Macromolecular Substances; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)