Erythropoietin induces p21ras activation and p120GAP tyrosine phosphorylation in human erythroleukemia cells.
Erythropoietin is the major regulator of the proliferation and differentiation of erythroid precursors, but little is known about its molecular mechanism of action. Using a human erythroleukemic cell line (HEL), we investigated whether p21ras is involved in erythropoietin signal transduction. We found that stimulation of HEL cells with erythropoietin induces a 5-fold increase in the amount of GTP bound to the endogenous p21ras. This effect is dose-dependent and occurs very rapidly. We also observed that erythropoietin causes tyrosine phosphorylation of several proteins in a time-dependent manner that correlates with the p21ras activation. Moreover, inhibition of tyrosine kinases by genistein totally prevents the erythropoietin-induced accumulation of a p21ras.GTP complex. By using an antiserum against the GTPase-activating protein, we found that p120GAP is rapidly phosphorylated in tyrosine in response to erythropoietin. Furthermore, the ability of a lysate from erythropoietin-stimulated HEL cells to induce in vitro hydrolysis of GTP bound to p21ras was strongly reduced. These results demonstrate that activation of p21ras is an early event in the erythropoietin signal transduction pathway, and they suggest that accumulation of the p21ras.GTP complex may be triggered by inhibition of GTPase-activating protein activity.
Torti
M
M
Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Marti
K B
KB
Altschuler
D
D
Yamamoto
K
K
Lapetina
E G
EG
D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000494:pharmacology*; D020690:GTPase-Activating Proteins; D019833:Genistein; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007529:Isoflavones / Q000494:pharmacology; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism*; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D007529:Isoflavones; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D004921:Erythropoietin; D014443:Tyrosine; D006160:Guanosine Triphosphate; D019833:Genistein; D015689:Oncogene Protein p21(ras)