Studies for the phosphorylation and signaling pathway of a transcription factor induced by neuronal defferentiation

神经元分化诱导的转录因子磷酸化及信号通路研究

• 批准号: 08680852
• 负责人: HIRATA Yoko
• 金额: $ 0.77万
• 依托单位: The Institute of Physical and Chemical Research (RIKEN)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680852/
• 关键词: Calpain; Limited proteolysis; NGFI-B(Nur77); Nurr1; Protein Kinase Cbeta; Rat brain; NGFI-B kinase; Protein kinase C beta

The NGFI-B, also called nur77, is an immediate-early gene that encodes a member of the steroid-thyroid hormone receptor superfamily, a class of ligand-dependent transcriptional regulators, although a ligand for the NGFI-B protein has not been identified. It is rapidly synthesized and phosphorylated in PC12 cells in response to nerve growth factor (NGF). We have demonstrated previously that in vitro phosphorylation of NGFI-B at Ser35O reduces it's ability to bind to the NBRE.NGFI-B kinase I has been identified as the kinase that phosphorylates NGFI-B at Ser35O following NGF treatment of PC12 cells. In this study, we found that NGFI-B kinase I activity appears in rat brain during embryogenesis and that the major postnatal form (approximately 50 kDa) is different from the embryonic type (90 kDa). We purified NGFI-B kinase (p50 NGFI-B kinase) from rat brain (8-10 weeks, 60 g). NH_2-terminal sequencing of the enzyme revealed identity to amino acids 312 to 324 of protein kinase C (PKC) beta. Furthermore, anti-PKC beta antibodies raised against COOH-terminal peptides of the two PKC beta isoforms recognized p50 NGFI-B kinase, suggesting that the adult form of NGFI-B kinase is a COOH-terminal fragment of PKC beta, a constitutive active form due to a lack of the regulatory domain. Finally, enzymes immunoprecipitated from rat brain by anti-PKC beta I and II antibodies phosphorylate NGFI-B at Ser35O in a Ca^<2+> and phospholipid-independent manner. A significant role for this enzyme fragment was originally suggested by the report that Ca^<2+>- dependent neutral proteases I and II (calpain I and II) cleave PKC beta to produce a catalytically active fragment that is free of the regulatory domain. These results indicate that multiple protein kinases could be involved in the post-translational modification of NGFI-B and that limited proteolysis of PKC beta may be important in the regulation of NGFI-B activity after the birth.

NGFI-B，也被称为Nur77，是一个即刻早期基因，编码类固醇-甲状腺激素受体超家族的成员，这是一类配体依赖的转录调节因子，尽管NGFI-B蛋白的配体尚未确定。在神经生长因子(NGF)的作用下，它在PC12细胞中迅速合成和磷酸化。我们以前已经证明，在体外，Ser35O处的NGFI-B的磷酸化降低了它与NBRE的结合能力。NGFI-B激酶I已被确定为在NGF处理PC12细胞后，在Ser35O处磷酸化NGFI-B的激酶。在本研究中，我们发现NGFI-B激酶I活性在胚胎发育过程中出现在大鼠脑中，并且主要的出生后形式(约50 kDa)与胚胎型(90 KDa)不同。我们从大鼠(8-10周，60g)的脑组织中提纯了NGFI-B激酶(P50)。氨基末端测序表明该酶与蛋白激酶C(PKC)β的312~324位氨基酸具有同源性。此外，针对两种PKCβ亚型的COOH末端多肽产生的抗PKCβ抗体识别p50 NGFI-B激酶，表明成体形式的NGFI-B激酶是PKCβ的COOH末端片段，由于缺乏调节结构域，PKCβ是一种结构性活性形式。最后，由抗PKCβI和II抗体从大鼠脑中免疫沉淀的酶以钙和磷脂不依赖的方式磷酸化Ser35O处的NGFI-B。这个酶片段的一个重要作用最初是由报告提出的，即依赖于钙的中性蛋白水解酶I和II(calain I和II)裂解PKCβ，产生一个没有调节域的催化活性片段。这些结果表明，多种蛋白激酶可能参与了NGFI-B的翻译后修饰，PKCβ的有限蛋白分解可能在出生后对NGFI-B活性的调节中起重要作用。