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

NGFI-B，也称为nur 77，是一种立即早期基因，编码类固醇-甲状腺激素受体超家族的成员，这是一类配体依赖性转录调节因子，尽管NGFI-B蛋白的配体尚未被鉴定。它在PC 12细胞中响应神经生长因子（NGF）而快速合成和磷酸化。我们以前已经证明，在体外磷酸化NGFI-B的Ser 35 O降低其与NBRE结合的能力。NGFI-B激酶I已被鉴定为在NGF处理的PC 12细胞后磷酸化NGFI-B的Ser 35 O的激酶。在这项研究中，我们发现，NGFI-B激酶I活性出现在大鼠大脑胚胎发育过程中，主要的产后形式（约50 kDa）是不同的胚胎型（90 kDa）。我们从大鼠脑（8-10周，60 g）中纯化NGFI-B激酶（p50 NGFI-B激酶）。NH_2末端序列分析表明，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PKC β同种型的COOH末端肽产生的抗PKC β抗体识别p50 NGFI-B激酶，表明NGFI-B激酶的成人形式是PKC β的COOH末端片段，由于缺乏调节结构域，是组成性活性形式。最后，用抗PKC β I和II抗体免疫沉淀的酶以Ca^2+和磷脂非依赖的方式磷酸化NGFI-B的Ser 35 O。该酶片段的一个重要作用最初是由Ca^2+依赖性中性蛋白酶I和II（钙蛋白酶I和II）切割PKC β产生无调节结构域的催化活性片段的报道提出的。这些结果表明，多种蛋白激酶可能参与NGFI-B的翻译后修饰，并且PKC β的有限蛋白水解可能在出生后NGFI-B活性的调节中是重要的。

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.