POSTTRANSLATIONALLY PROCESSED STRUCTURE OF THE HUMAN PLATELET PROTEIN SMG P21B - EVIDENCE FOR GERANYLGERANYLATION AND CARBOXYL METHYLATION OF THE C-TERMINAL CYSTEINE
POSTTRANSLATIONALLY PROCESSED STRUCTURE OF THE HUMAN PLATELET PROTEIN SMG P21B - EVIDENCE FOR GERANYLGERANYLATION AND CARBOXYL METHYLATION OF THE C-TERMINAL CYSTEINE
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DOI：
10.1073/pnas.87.22.8960
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发表时间：
1990-11-01
影响因子：
11.1
通讯作者：
TAKAI, Y
中科院分区：
文献类型：
--
作者：
KAWATA, M;FARNSWORTH, CC;TAKAI, Y
smg p21 A和smg p21-B是小的GTP结合蛋白，其与rasp 21蛋白共享推定的效应子和共有的C末端序列。在本报告中，我们表明，人血小板smg p21 B成为标记时，完整的血小板与外源性[3 H]甲羟戊酸内酯孵育时，smg p21 B的纯化制剂与牛脑膜和S-腺苷-L-[甲基-3H]蛋氨酸孵育。此外，我们通过气相色谱/质谱证明，用雷尼镍处理smg p21 B释放了摩尔比约为1：1的香叶基香叶基部分。相比之下，用NH 2 OH或KOH处理smg p21 B没有得到棕榈酰硫酯存在的证据。用无色杆菌蛋白酶I广泛消化smg p21 B产生两个C-末端三肽，其含有摩尔比为2：1的丝氨酸和半胱氨酸。这两种肽都被硫醚连接的香叶基香叶基基团修饰。其中一个肽与甲基化的smg p21 B的3 H标记的蛋白水解产物在反相HPLC上共迁移，并且该肽在用KOH处理后与另一个肽的保留时间相同。由于smg p21 B的cDNA预测的C-末端序列在其C-末端结构域内含有独特的Ser-Ser-Cys肽，-Lys-Lys-Ser-Ser-Cys-Gln-Leu-Leu 184，这些结果表明，smg p21 B是通过Cys-181的香叶基香叶基化后修饰的，并提示进一步的修饰导致三个预测的C-末端氨基酸，接着是半胱氨酰羧基的部分甲基化。
smg p21A and -B are small GTP-binding proteins that share putative effector and consensus C-terminal sequences with ras p21 proteins. In the present report, we showed that human platelet smg p21B became labeled when intact platelets were incubated with exogenous [3H]mevalonolactone and when a purified preparation of smg p21B was incubated with bovine brain membranes and S-adenosyl-L-[methyl-3H]methionine. In addition, we demonstrated by gas chromatography/mass spectrometry that treatment of smg p21B with Raney nickel released a geranylgeranyl moiety in a molar ratio of about 1:1. In contrast, treatment of smg p21B with NH2OH or KOH yielded no evidence for the presence of a palmitoyl thioester. Extensive digestion of smg p21B with Achromobacter protease I yielded two C-terminal tripeptides that contained serine and cysteine in a molar ratio of 2:1. Both peptides were modified by a thioether-linked geranylgeranyl group. One of the peptides comigrated with a 3H-labeled proteolytic product of methylated smg p21B on reverse-phase HPLC and this peptide appeared at the same retention time as that of the other peptide after being treated with KOH. Since the cDNA-predicted C-terminal sequence of smg p21B contains a unique Ser-Ser-Cys peptide within its C-terminal domain, -Lys-Lys-Ser-Ser-Cys-Gln-Leu-Leu184, these results indicate that smg p21B is posttranslationally modified by geranylgeranylation of Cys-181 and suggest that further modifications cause proteolytic removal of the three predicted C-terminal amino acids followed by partial methylation of the cysteinyl carboxyl group.