Identification of phosphorylated sites in the mouse glucocorticoid receptor.
Identification of phosphorylated sites in the mouse glucocorticoid receptor.
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DOI：
10.1016/s0021-9258(20)89482-4
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发表时间：
1991-04
期刊：
影响因子：
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通讯作者：
J. Bodwell;E. Ortí;J. Coull;D. Pappin;L. I. Smith;F. Swift
中科院分区：
文献类型：
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作者：
J. Bodwell;E. Ortí;J. Coull;D. Pappin;L. I. Smith;F. Swift
体内糖皮质激素受体在没有激素的情况下磷酸化，在糖皮质激素激动剂而不是拮抗剂存在时变得过度磷酸化(Ortí， E., Mendel, d.b., Smith, l.i., and Munck， A. (1989) J. Biol。化学，264,9728-9731)。作为阐明受体磷酸化功能意义的初步步骤，我们已经确定了小鼠受体上的七个磷酸化位点。从糖皮质激素处理的小鼠胸腺瘤细胞（WEHI-7）和稳定转染的表达大量小鼠受体的中国仓鼠卵巢细胞（WCL2）中纯化了32p标记受体的胰蛋白酶肽。这两种细胞类型的受体的磷酸肽图几乎无法区分。固相测序显示122、150、212、220、234和315丝氨酸和159苏氨酸被磷酸化。丝氨酸122、150、212、220和234及其周围的序列在大鼠和人类受体的同源区域是保守的，但苏氨酸159和丝氨酸315在人类受体中没有同源物。这7个磷酸化位点位于受体的氨基末端区域。除丝氨酸315外，其余均位于人类和/或大鼠受体中鉴定的转激活域内。丝氨酸212、220和234位于高酸性区域，在小鼠受体中，这是完全转录起始活性和减少非特异性DNA结合所必需的。丝氨酸212、220、234和苏氨酸159是脯氨酸定向激酶和/或p34cdc2激酶的一致序列。丝氨酸122在酪蛋白激酶II的一致序列中，而丝氨酸150和315似乎不在任何已知的激酶一致序列中。许多这些位点的位置表明磷酸化在反活化中的作用。
Glucocorticoid receptors in vivo are phosphorylated in the absence of hormone and become hyperphosphorylated in the presence of glucocorticoid agonist but not antagonists (Ortí, E., Mendel, D.B., Smith, L.I., and Munck, A. (1989) J. Biol. Chem. 264, 9728-9731). As a preliminary step to elucidating the functional significance of receptor phosphorylation, we have identified seven phosphorylated sites on the mouse receptor. Tryptic phosphopeptides from 32P-labeled receptors were purified from glucocorticoid-treated mouse thymoma cells (WEHI-7) and from stably transfected Chinese hamster ovary cells (WCL2) that express large numbers of mouse receptors. Phosphopeptide maps of receptors from these two cell types were almost indistinguishable. Solid phase sequencing revealed phosphorylation at serines 122, 150, 212, 220, 234, and 315 and threonine 159. Serines 122, 150, 212, 220, and 234 and the sequences surrounding them are conserved in the homologous regions of the rat and human receptors, but threonine 159 and serine 315 have no homologues in the human receptor. The seven phosphorylated sites are in the amino-terminal domain of the receptor. All but serine 315 are within transactivation domains identified in the human and/or rat receptors. Serines 212, 220, and 234 are in a highly acidic region that in the mouse receptor is necessary for full transcription initiation activity and reduces nonspecific DNA binding. Serines 212, 220, and 234 and threonine 159 are in consensus sequences for proline-directed kinase and/or p34cdc2 kinase. Serine 122 is in a consensus sequence for casein kinase II whereas serines 150 and 315 do not appear to be in any known kinase consensus sequence. The location of many of these sites suggests a role of phosphorylation in transactivation.