I. JAK kinases與IL-3受體α和βc次單位結合之探討 II. p38訊息傳遞於細胞分化與生長抑制之角色探討

I. Determinants for the interactions between the cytoplasmic regions of the Interleukin-3 receptor subunits α and βc and JAK kinases and JAK kinases II.Roles of the p38 pathway in cell differentiation and growth inhibition

李雅莉(Ya-Li Lee)

指導教授 ： 黃惠美 博士

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2006年)

摘要

中文摘要 IL-3為細胞激素家族的一員，調控著細胞的生長、增生、分化與凋亡。IL-3受體上沒有酪胺酸激?，必須藉由JAK蛋白激?傳遞訊息。我們實驗室過去以免疫沉澱法證實JAK1會與IL-3受體β次單位結合，而JAK2則與IL-3受體α次單位結合。而JAK1和JAK2與IL-3受體的結合是不受細胞激素刺激影響的。JAK蛋白上有七個同源的區域，分別命名JH1至JH7。目前已知JH1具有酵素活性，而JH2調控JAK的活性，而JH3至JH7片段則與受體的結合有關。但JAK1和JAK2究竟以哪個區域與IL-3受體結合？我利用實驗室先前建構好的GST-IL-3受體融合質體及一系列的JAK1和JAK2缺失質體，以GST-pull down方法分析。實驗結果顯示：JAK1的JH3至JH7片段與IL-3受體β次單位結合，與α次單位有微弱的結合。JAK2的JH7或JH6至JH7區域缺失的情況下，皆不影響與IL-3受體β次單位結合的能力，與α次單位有微弱的結合。 中文摘要 p38 pathway對於調控細胞增生、分化及細胞凋亡扮演重要的角色。本實驗室過去研究發現，Activin A誘導globin gene promoter活性及抑制細胞生長的作用會被p38抑制劑SB203580所抑制。SB203580是專一性抑制p38α和p38β。為了想更進一步了解p38α或p38β參與細胞激素調控細胞增生與分化的情形，首先我利用RT-PCR分析在K562及Jurkat細胞內四個p38 isoforms表現的情形。在K562及Jurkat細胞中p38α,p38β,p38γ和p38δ皆有表現。我們的實驗結果發現Activin A所誘導的? globin和? globin gene promoters活性會被p38 dominant negative mutants p38αAF和p38βAF所抑制，顯示Activin A誘導??globin和??globin gene promoters活性的作用是透過p38α和p38β。另外，c-Jun，紅血球分化的抑制者，其蛋白表現量於K562細胞中會受SB203580正向調控。最後，我利用Interferon alpha（IFN-α）刺激Jurkat細胞，發現IFN-α會透過p38 pathway抑制Jurkat細胞生長。綜合這些結果，於K562和Jurkat細胞中p38扮演調控細胞生長和分化的重要角色。

關鍵字

受體結合 ； 細胞生長 ； 分化

並列摘要

Abstract IL-3, a member of cytokine family, is involved in the cell growth, proliferation, differentiation and apoptosis. IL-3 stimulates its biological effects by binding to a heterodimeric receptor composed of α and β subunits. JAK1 and JAK2 are rapidly activated after binding of IL-3 to their receptors, and are essential for the initiation of intracellular signaling. Previously, we reported that JAK1 and JAK2 were associated with the IL-3 receptor β and α subunits, respectively. In addition to the JH1 domain（kinase domain）, the JAK family contains the JH2-JH7 domains. JH2 has been shown to have a negative regulatory effect on JAK2 kinase activity. JH3-JH7 domains have been implicated in receptor association. However, which regions of JAKs interact with IL-3 receptors is now unclear. In this study, I demonstrated that the JH3-JH7 domains of JAK1 interacted with βc subunit, but had weak interaction with IL-3 receptor α subunit by GST-pull down assay. The deletion of JH7 domain or JH6-JH7 domains of JAK2 did not affect the interaction with IL-3 receptors. Abstract p38 play a central role in cellular responses such as cell proliferation, differentiation and apoptosis. We have previously reported that Activin A-induced globin promoters and cell growth inhibition were inhibited by p38 inhibitor SB203580. The p38 inhibitor SB203580 targets both p38α and p38β. To further investigate that p38α, p38β or both kinases are involved in cytokine-mediated cell proliferation and differentiation, we performed the RT-PCR to analyze the expression of four p38 isoforms on K562 and Jurkat cells. We showed that p38α, p38β, p38γ and p38δ transcripts are expressed in K562 and Jurkat cells. Expression of the exogenous p38 dominant negative mutants, p38αAF and p38βAF, reduced Activin A-induced α- and ζ-globin promoters activity, indicating that Activin A activated α- and ζ-globin gene promoters through p38α and p38β. In addition, the expression of c-Jun, a blocker of erythroid differentiation, was up-regulated by SB203580 in K562 cells. At last IFN-α inhibited Jurkat cell growth through p38 pathway. Together, these results suggest that p38 plays an important role in regulating cell proliferation and differentiation.

並列關鍵字

JAK ； GST-pull down ； p38 isoforms

參考文獻

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