Halfter Ursula M, Derbyshire Zachary E, Vaillancourt Richard R
Department of Pharmacology and Toxicology, University of Arizona, 1703 E. Mabel Street, Tucson, AZ 85721, USA.
Biochem J. 2005 May 15;388(Pt 1):17-28. doi: 10.1042/BJ20041236.
IFNgamma (interferon-gamma) binding to its cognate receptor results, through JAK (Janus kinase), in direct activation of receptor-bound STAT1 (signal transducer and activator of transcription 1), although there is evidence for additional activation of a MAPK (mitogen-activated protein kinase) pathway. In the present paper, we report IFNgamma-dependent activation of the MEKK4 (MAPK/extracellular-signal-regulated kinase kinase kinase 4) pathway in HaCaT human keratinocytes. MEKK4 is tyrosine-phosphorylated and the IFNgamma-dependent phosphorylation requires intracellular calcium. Calcium-dependent phosphorylation of MEKK4 is mediated by Pyk2. Moreover, MEKK4 and Pyk2 co-localize in an IFNgamma-dependent manner in the perinuclear region. Furthermore, the calcium-binding protein, annexin II, and the calcium-regulated kinase, Pyk2, co-immunoprecipitate with MEKK4 after treatment with IFNgamma. Immunofluorescence imaging of HaCaT cells shows an IFNgamma-dependent co-localization of annexin II with Pyk2 in the perinuclear region, suggesting that annexin II mediates the calcium-dependent regulation of Pyk2. Tyrosine phosphorylation of MEKK4 correlates with its activity to phosphorylate MKK6 (MAPK kinase 6) in vitro and subsequent p38 MAPK activation in an IFNgamma-dependent manner. Additional studies demonstrate that the SH2 (Src homology 2)-domain-containing tyrosine phosphatase SHP2 co-immunoprecipitates with MEKK4 in an IFNgamma-dependent manner and co-localizes with MEKK4 after IFNgamma stimulation in the perinuclear region in HaCaT cells. Furthermore, we provide evidence that SHP2 dephosphorylates MEKK4 and Pyk2, terminating the MEKK4-dependent branch of the IFNgamma signalling pathway.
γ干扰素（IFNγ）与其同源受体结合后，通过JAK（Janus激酶）直接激活受体结合的STAT1（信号转导和转录激活因子1），不过有证据表明丝裂原活化蛋白激酶（MAPK）途径也有额外激活。在本文中，我们报告了在HaCaT人角质形成细胞中IFNγ依赖性激活MEKK4（MAPK/细胞外信号调节激酶激酶激酶4）途径。MEKK4发生酪氨酸磷酸化，且IFNγ依赖性磷酸化需要细胞内钙。MEKK4的钙依赖性磷酸化由Pyk2介导。此外，MEKK4和Pyk2在核周区域以IFNγ依赖性方式共定位。此外，在用IFNγ处理后，钙结合蛋白膜联蛋白II和钙调节激酶Pyk2与MEKK4共同免疫沉淀。HaCaT细胞的免疫荧光成像显示，膜联蛋白II与Pyk2在核周区域以IFNγ依赖性方式共定位，表明膜联蛋白II介导Pyk2的钙依赖性调节。MEKK4的酪氨酸磷酸化与其在体外磷酸化MKK6（MAPK激酶6）以及随后以IFNγ依赖性方式激活p38 MAPK的活性相关。进一步的研究表明，含Src同源2（SH2）结构域的酪氨酸磷酸酶SHP2以IFNγ依赖性方式与MEKK4共同免疫沉淀，并在IFNγ刺激后在HaCaT细胞核周区域与MEKK4共定位。此外，我们提供证据表明SHP2使MEKK4和Pyk2去磷酸化，从而终止IFNγ信号通路中MEKK4依赖性分支。
