Erythropoietin-dependent association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated erythropoietin receptor.
Erythropoietin (Epo) regulates the proliferation and differentiation of erythroid precursors. The Epo receptor (EpoR) belongs to the cytokine receptor family and lacks a tyrosine kinase domain. However, Epo induces tyrosine phosphorylation of cellular substrates including the EpoR. To explore the functional significance of receptor tyrosine phosphorylation, we examined the possible interaction of the receptor with the 85-kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3-kinase. After Epo stimulation, p85 was found to associate with the tyrosine-phosphorylated 72-kDa form of EpoR as well as a 92-kDa phosphotyrosyl protein, and PI 3-kinase activity was detectable in anti-EpoR immunoprecipitates. Anti-EpoR blotting of anti-p85 immunoprecipitates revealed that p85 binds specifically to the 72-kDa form of the EpoR and not to unphosphorylated 66- and 64-kDa forms. Association of p85 with the EpoR was Epo dose- and time-dependent and correlated with tyrosine phosphorylation of the receptor. Consistent with a role for tyrosine phosphorylation of the EpoR, PI 3-kinase did not associate with a mitogenically active receptor mutant that lacked tyrosine-phosphorylation sites in the carboxyl-terminal region. A recombinant fusion protein containing the carboxyl-terminal SH-2 domain of p85 was shown to bind to tyrosine-phosphorylated EpoR in vitro. Taken together, these results indicate that, following Epo stimulation, the EpoR recruits PI 3-kinase to the cell membrane by binding between the carboxyl-terminal SH-2 domain of p85 and the tyrosine-phosphorylated carboxyl-terminal region of the receptor. The association with PI 3-kinase is, however, not required for the growth signal transduction from the EpoR.
Miura
O
O
First Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
Nakamura
N
N
Ihle
J N
JN
Aoki
N
N
D001665:Binding Sites; D002460:Cell Line; D004921:Erythropoietin / Q000494:pharmacology*; D007377:Interleukin-3 / Q000494:pharmacology; D007700:Kinetics; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000187:drug effects* / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*
D007377:Interleukin-3; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)