Avalos B R, Parker J M, Ware D A, Hunter M G, Sibert K A, Druker B J
Division of Bone Marrow Transplantation, The Ohio State University, Columbus 43210, USA.
Exp Hematol. 1997 Feb;25(2):160-8.
Activation of the granulocyte colony-stimulating factor receptor (G-CSFR) induces rapid tyrosine phosphorylation of multiple intracellular substrates in proliferating cells and nonproliferating, terminally differentiated neutrophils. The kinases that couple ligand binding to tyrosine phosphorylation of cellular substrates by the G-CSFR with activation of specific functional programs are largely unknown. In this study, we examined early signaling events in proliferating and terminally differentiated cells following G-CSF stimulation to determine whether identical signaling cascades are activated. In murine Ba/F3 cells transfected with the human G-CSFR and NFS-60 cells constitutively expressing the murine G-CSFR, G-CSF induced tyrosine phosphorylation and activation of Jak1, Jak2, and Tyk2. Tyrosine phosphorylation of Stat3 and, to a lesser extent, Stat1 was also detected following G-CSF stimulation. Using a mitogenically incompetent human G-CSFR mutant in which Pro639 and Pro641 were substituted by alanine, the box 1 PDP motif was found to be required for activation of Jak kinases, tyrosine phosphorylation of the G-CSFR, and recruitment of Stat proteins. Notably, no activation of Jak1, Jak2, Tyk2, Stat1, or Stat3 was observed in neutrophils following G-CSF stimulation. In addition, there was no detectable activation in neutrophils of the recently cloned Jak3 kinase, which has been reported to be expressed at high levels as myeloid cells undergo terminal neutrophilic maturation. These results indicate a lack of involvement of Jak kinases in signaling by the G-CSFR in neutrophils, and suggest utilization of alternative signal transduction pathways distinct from those in proliferating cells. Activation of the Jak-Stat pathway correlates with proliferative signaling by the G-CSFR and requires the membrane-proximal box 1 PXP motif, which is conserved in members of the cytokine receptor superfamily.
粒细胞集落刺激因子受体（G-CSFR）的激活可诱导增殖细胞和非增殖性终末分化中性粒细胞中多种细胞内底物的快速酪氨酸磷酸化。将配体结合与G-CSFR介导的细胞底物酪氨酸磷酸化以及特定功能程序激活相偶联的激酶在很大程度上尚不清楚。在本研究中，我们检测了G-CSF刺激后增殖细胞和终末分化细胞中的早期信号事件，以确定是否激活了相同的信号级联反应。在转染了人G-CSFR的小鼠Ba/F3细胞和组成性表达小鼠G-CSFR的NFS-60细胞中，G-CSF诱导Jak1、Jak2和Tyk2的酪氨酸磷酸化和激活。G-CSF刺激后还检测到Stat3的酪氨酸磷酸化，Stat1的酪氨酸磷酸化程度较低。使用将Pro639和Pro641替换为丙氨酸的无促有丝分裂活性的人G-CSFR突变体，发现框1 PDP基序是激活Jak激酶、G-CSFR的酪氨酸磷酸化以及Stat蛋白募集所必需的。值得注意的是，G-CSF刺激后在中性粒细胞中未观察到Jak1、Jak2、Tyk2、Stat1或Stat3的激活。此外，在中性粒细胞中未检测到最近克隆的Jak3激酶的激活，据报道，随着髓样细胞经历终末嗜中性粒细胞成熟，Jak3激酶会高水平表达。这些结果表明Jak激酶不参与G-CSFR在中性粒细胞中的信号传导，并提示利用了与增殖细胞中不同的替代信号转导途径。Jak-Stat途径的激活与G-CSFR的增殖信号传导相关，并且需要膜近端框1 PXP基序，该基序在细胞因子受体超家族成员中保守。
