A minimal cytoplasmic subdomain of the erythropoietin receptor mediates p70 S6 kinase phosphorylation

Objective. Erythropoietin (EPO) is a lineage-restricted growth factor that is required for erythroid proliferation and differentiation. EPO stimulates the phosphorylation and activation of p70 S6 kinase (p70 S6K), which is re quired for cell cycle progression. Here, the minimal cytoplasmic domains of the EPO receptor (EPO-R) required for p70 S6K activation were determined. Materials and Methods, Ba/F3 cells were stably transfected with wild-type ( WT) EPO-R or EPO-R carboxyl-terminal deletion mutants, designated by the nu mber of amino acids deleted from the cytoplasmic tail (-99, -131, -221). Tr ansfected cells were growth factor deprived and then stimulated with EPO. p 70 SGE, JAK2 IRS-2, and ERK1/2 phosphorylation/activation were examined. Th e ability of transfected 3-phosphoinositide-dependent protein kinase I (PDK 1) to reconstitute p70 S6K phosphorylation in EPO-R mutants also was determ ined. Results. Phosphorylation and activation of p70 S6K, JAK2, IRS-5 and ERK1/2 in Ba/F3 cells transfected with EPO-R-99 or EPO-R-99Y343F were similar to N T EPO-R. In contrast, EPO-dependent p70 S6K phosphorylation/activation, as well as IRS-2 and ERK1/2 phosphorylation, were minimal or absent in cells t ransfected with EPO-R-131 or EPO-R-221. JAK2 phosphorylation was reduced si gnificantly in cells transfected with EPO-R-131 and abolished with EPO-R-22 1. To examine the role of PDK1, a kinase known to phosphorylate p70 S6K, Ba /F3 EPO-R-131 cells were transiently transfected with PDK1. WT constitutive ly active PDK1 restored p70 S6K phosphorylation in Ba/F3 EPO-R-131 cells bu t not in Ba/F3 EPO-R-221 cells. Conclusions. The results demonstrate that a minimal cytoplasmic subdomain o f tile EPO-R extending between -99 and -131 is required for p70 S6K phospho rylation and activation, The results also demonstrate that PDK1 is a critic al component in this signaling pathway, which requires the presence of doma ins between -131 and -221 far its activation of p70 S6K. (C) 2001 Internati onal Society for Experimental Hematology. published by Elsevier Science Inc .