Chakraborty A, Saha D, Bose A, Chatterjee M, Gupta N K
Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
Biochemistry. 1994 May 31;33(21):6700-6. doi: 10.1021/bi00187a041.
An eIF-2 associated 67-kDa protein (p67) protects eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation and promotes protein synthesis in the presence of active eIF-2 kinase(s). p67 is a glycoprotein and contains multiple O-linked GlcNAc moieties. We have now studied the roles of hemin, p67, and the glycosyl residues on p67 in the regulation of eIF-2 alpha-subunit phosphorylation in reticulocyte lysates. The results are as follows: (i) Both hemin and p67 inhibited HRI (heme-regulated protein synthesis inhibitor) and dsI (double-stranded RNA activated protein synthesis inhibitor) catalyzed phosphorylation of eIF-2 alpha-subunit in vitro. However, only hemin, and not p67, inhibited casein kinase catalyzed phosphorylation of eIF-2 beta-subunit. (ii) Only p67, and not hemin, inhibited eIF-2 alpha-subunit phosphorylation by eIF-2 kinase(s) in reticulocyte lysate. Significant eIF-2 alpha-subunit phosphorylation was observed even in the presence of hemin when p67 in the reticulocyte lysate was removed by treatment with p67 antibodies. (iii) Reticulocyte lysate contains a p67-deglycosylase in latent form, and hemin prevents activation of this deglycosylase. In the absence of hemin, this p67-deglycosylase is activated. Once activated in the absence of hemin, the activated deglycosylase deglycosylates p67, even in the presence of hemin. This inactivates p67 and allows eIF-2 kinase to phosphorylate eIF-2 alpha-subunit and inhibit protein synthesis. Protein synthesis in reticulocyte lysate is thus regulated by two novel cascades of covalent modifications: protein deglycosylation leading to protein phosphorylation.
一种与真核起始因子2（eIF-2）相关的67 kDa蛋白（p67）可保护eIF-2α亚基免受eIF-2激酶催化的磷酸化作用，并在有活性eIF-2激酶存在的情况下促进蛋白质合成。p67是一种糖蛋白，含有多个O-连接的N-乙酰葡糖胺部分。我们现在研究了血红素、p67以及p67上的糖基残基在网织红细胞裂解物中eIF-2α亚基磷酸化调节中的作用。结果如下：（i）血红素和p67均在体外抑制血红素调节蛋白合成抑制剂（HRI）和双链RNA激活蛋白合成抑制剂（dsI）催化的eIF-2α亚基磷酸化。然而，只有血红素而非p67抑制酪蛋白激酶催化的eIF-2β亚基磷酸化。（ii）在网织红细胞裂解物中，只有p67而非血红素抑制eIF-2激酶催化的eIF-2α亚基磷酸化。当用p67抗体处理去除网织红细胞裂解物中的p67时，即使存在血红素也观察到显著的eIF-2α亚基磷酸化。（iii）网织红细胞裂解物含有一种潜伏形式的p67去糖基化酶，血红素可阻止该去糖基化酶的激活。在没有血红素的情况下，这种p67去糖基化酶被激活。一旦在没有血红素的情况下被激活，即使存在血红素，激活的去糖基化酶也会使p67去糖基化。这使p67失活，并使eIF-2激酶磷酸化eIF-2α亚基并抑制蛋白质合成。因此，网织红细胞裂解物中的蛋白质合成受两种新的共价修饰级联调节：蛋白质去糖基化导致蛋白质磷酸化。
