Watanabe S, Itoh T, Arai K
Department of Molecular and Developmental Biology, University of Tokyo, Japan.
J Allergy Clin Immunol. 1996 Dec;98(6 Pt 2):S183-91. doi: 10.1016/s0091-6749(96)70065-9.
The receptors for human interleukin-3 (IL-3) and human granulocyte-macrophage colony-stimulating factor (GM-CSF), hIL-3R, hGM-CSFR, respectively, consists of two subunits, alpha and beta, both of which are members of the cytokine receptor superfamily. Phosphorylation of tyrosine residues in the hGMR beta subunit and several cellular proteins is observed after hGM-CSF stimulation. We analyzed the role of tyrosine residues in the hGMR beta subunit and the nature of tyrosine kinase, JAK2, in hGMR signal transduction using several hGMR beta subunit mutants. In addition to the box1 region, a membrane distal region (a.a. 544-589) of the hGMR beta was required for c-fos activation. Only one tyrosine residue (Tyr577) existed within the region 544 to 589, and substitution of Tyr577 to phenylalanine in GMR beta 589 resulted in loss of c-fos activation. In contrast, the same substitution in a wild type receptor did not affect GM-CSF induced activities such as c-fos messenger RNA (mRNA) induction and proliferation, but the substitution abolished Shc phosphorylation. These results suggest that the activation of Shc is not essential for c-fos activation and several tyrosine residues cooperate for c-fos activation. It is well documented that IL-3 or GM-CSF activate JAK2 in BA/F3 cells. The role of JAK2 in IL-3/GM-CSF functions, however, is largely unknown. We examined the role of JAK2 in GM-CSF induced signaling pathways. Dominant negative JAK2 (delta JAK2) lacking the C-terminus kinase domain suppressed IL-3/GM-CSF induced c-fos activation and c-myc activation and proliferation, suggesting that JAK2 was involved in both signaling pathways. Protein tyrosine phosphatase SHP-2 (also called PTP 1D) and Shc were phosphorylated by IL-3/GM-CSF in BA/F3 cells; however, these phosphorylation events were inhibited by the expression of delta JAK2. Taken together, these results indicate the JAK2 is a primary kinase regulating all the known activities of GM-CSF. JAK2 mediates GM-CSF induced c-fos activation through receptor phosphorylation and Shc/PTP 1D activation.
人白细胞介素-3（IL-3）和人粒细胞-巨噬细胞集落刺激因子（GM-CSF）的受体，分别为hIL-3R和hGM-CSFR，由α和β两个亚基组成，二者均为细胞因子受体超家族成员。hGM-CSF刺激后可观察到hGMRβ亚基及几种细胞蛋白中酪氨酸残基的磷酸化。我们使用几种hGMRβ亚基突变体分析了hGMRβ亚基中酪氨酸残基的作用以及酪氨酸激酶JAK2在hGMR信号转导中的性质。除box1区域外，hGMRβ的膜远端区域（氨基酸544 - 589）对于c-fos激活是必需的。在544至589区域内仅存在一个酪氨酸残基（Tyr577），将GMRβ589中的Tyr577替换为苯丙氨酸会导致c-fos激活丧失。相反，野生型受体中的相同替换并不影响GM-CSF诱导的活性，如c-fos信使核糖核酸（mRNA）诱导和增殖，但该替换消除了Shc磷酸化。这些结果表明，Shc的激活对于c-fos激活并非必需，并且几个酪氨酸残基协同作用以实现c-fos激活。有充分文献记载，IL-3或GM-CSF可在BA/F3细胞中激活JAK2。然而，JAK2在IL-3/GM-CSF功能中的作用在很大程度上尚不清楚。我们研究了JAK2在GM-CSF诱导的信号通路中的作用。缺乏C末端激酶结构域的显性负性JAK2（δJAK2）抑制了IL-3/GM-CSF诱导的c-fos激活、c-myc激活和增殖，表明JAK2参与了这两条信号通路。蛋白酪氨酸磷酸酶SHP-2（也称为PTP 1D）和Shc在BA/F3细胞中被IL-3/GM-CSF磷酸化；然而，这些磷酸化事件被δJAK2的表达所抑制。综上所述，这些结果表明JAK2是调节GM-CSF所有已知活性的主要激酶。JAK2通过受体磷酸化以及Shc/PTP 1D激活介导GM-CSF诱导的c-fos激活。
