Role of methionine adenosyltransferase α2 and β phosphorylation and stabilization in human hepatic stellate cell trans-differentiation.
Role of methionine adenosyltransferase α2 and β phosphorylation and stabilization in human hepatic stellate cell trans-differentiation.
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DOI：
10.1002/jcp.24839
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发表时间：
2015-05
影响因子：
5.6
通讯作者：
Park S
中科院分区：
文献类型：
--
作者：
Ramani K;Donoyan S;Tomasi ML;Park S
肝星状细胞（HSC）向肌纤维母细胞转分化是肝纤维化发生发展的重要环节。这些变化涉及基因组和蛋白质组的关键调节因子的调节。甲硫氨酸腺苷转移酶（MAT）催化甲硫氨酸合成甲基供体S-腺苷甲硫氨酸（SAMe）。我们以前已经证明，两个MAT基因，MAT 2A和MAT 2B（分别编码MATα2和MATβ蛋白），是HSC激活所必需的，MAT 2A转录控制的丧失有利于其在转分化过程中的上调。因此，MAT基因在激活过程中与HSC机制内在相关。在目前的研究中，我们首次鉴定了MATα2和MATβ蛋白的翻译后修饰，这些修饰使它们稳定并有利于人HSC转分化。人HSC的培养活化诱导MATα2和MATβ蛋白。使用质谱法，我们鉴定了MATα2和MATβ中的磷酸化位点，这些位点预计被丝裂原活化蛋白激酶（MAPK）家族成员[ERK 1/2，V-Raf鼠肉瘤病毒癌基因同源物B1（B-Raf），MEK]磷酸化。这两种蛋白质的磷酸化在HSC活化过程中增强。阻断MEK活化降低了MAT蛋白的磷酸化和稳定性，而不影响其mRNA水平。沉默ERK 1/2或B-Raf降低了MATβ的磷酸化和稳定性，但不影响MATα2。激活的人HSC细胞系LX 2静止的抑制降低了磷酸化并使MAT蛋白不稳定。MATα2和MATβ磷酸化位点的突变使其不稳定并阻止HSC转分化。数据显示，在HSC活化期间MAT蛋白的磷酸化使其稳定，从而正向调节转分化。
Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members [ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (B-Raf), MEK]. Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation.