Zhang M Y, Barber D L, Alessi D R, Bell L L, Stine C, Nguyen M H, Beattie B K, Cheung J Y, Miller B A
Department of Pediatrics, The Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, PA, USA
Exp Hematol. 2001 Apr;29(4):432-40. doi: 10.1016/s0301-472x(00)00681-0.
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 required for cell cycle progression. Here, the minimal cytoplasmic domains of the EPO receptor (EPO-R) required for p70 S6K activation were determined.Ba/F3 cells were stably transfected with wild-type (WT) EPO-R or EPO-R carboxyl-terminal deletion mutants, designated by the number of amino acids deleted from the cytoplasmic tail (-99, -131, -221). Transfected cells were growth factor deprived and then stimulated with EPO. p70 S6K, JAK2, IRS-2, and ERK1/2 phosphorylation/activation were examined. The ability of transfected 3-phosphoinositide-dependent protein kinase 1 (PDK1) to reconstitute p70 S6K phosphorylation in EPO-R mutants also was determined. Phosphorylation and activation of p70 S6K, JAK2, IRS-2, and ERK1/2 in Ba/F3 cells transfected with EPO-R-99 or EPO-R-99Y343F were similar to WT 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 transfected with EPO-R-131 or EPO-R-221. JAK2 phosphorylation was reduced significantly in cells transfected with EPO-R-131 and abolished with EPO-R-221. 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 constitutively active PDK1 restored p70 S6K phosphorylation in Ba/F3 EPO-R-131 cells but not in Ba/F3 EPO-R-221 cells. The results demonstrate that a minimal cytoplasmic subdomain of the EPO-R extending between -99 and -131 is required for p70 S6K phosphorylation and activation. The results also demonstrate that PDK1 is a critical component in this signaling pathway, which requires the presence of domains between -131 and -221 for its activation of p70 S6K.
促红细胞生成素（EPO）是一种谱系限制性生长因子，是红细胞增殖和分化所必需的。EPO刺激p70 S6激酶（p70 S6K）的磷酸化和激活，而p70 S6K是细胞周期进展所必需的。在此，确定了激活p70 S6K所需的促红细胞生成素受体（EPO-R）的最小胞质结构域。用野生型（WT）EPO-R或EPO-R羧基末端缺失突变体（根据从胞质尾端缺失的氨基酸数量命名为-99、-131、-221）稳定转染Ba/F3细胞。将转染后的细胞去除生长因子，然后用EPO刺激。检测p70 S6K、JAK2、IRS-2和ERK1/2的磷酸化/激活情况。还确定了转染的3-磷酸肌醇依赖性蛋白激酶1（PDK1）在EPO-R突变体中重建p70 S6K磷酸化的能力。用EPO-R-99或EPO-R-99Y343F转染的Ba/F3细胞中，p70 S6K、JAK2、IRS-2和ERK1/2的磷酸化和激活情况与WT EPO-R相似。相比之下，用EPO-R-131或EPO-R-221转染的细胞中，EPO依赖性p70 S6K磷酸化/激活以及IRS-2和ERK1/2磷酸化极少或不存在。用EPO-R-131转染的细胞中JAK2磷酸化显著降低，而用EPO-R-221转染的细胞中JAK2磷酸化则完全消失。为了研究PDK1（一种已知可磷酸化p70 S6K的激酶）的作用，将PDK临时转染到Ba/F3 EPO-R-131细胞中。野生型组成型活性PDK1可恢复Ba/F3 EPO-R-131细胞中的p70 S6K磷酸化，但不能恢复Ba/F3 EPO-R-221细胞中的p70 S6K磷酸化。结果表明，EPO-R在-99至-131之间延伸的最小胞质亚结构域是p70 S6K磷酸化和激活所必需的。结果还表明，PDK是该信号通路中的关键成分，其激活p70 S6K需要存在-131至-221之间的结构域。
