THE 50 KDA PROTEIN SUBUNIT OF ASSEMBLY POLYPEPTIDE (AP) AP-2 ADAPTER FROM CLATHRIN-COATED VESICLES IS PHOSPHORYLATED ON THREONINE-156 BY AP-1 AND A SOLUBLE AP50 KINASE WHICH COPURIFIES WITH THE ASSEMBLY POLYPEPTIDES
THE 50 KDA PROTEIN SUBUNIT OF ASSEMBLY POLYPEPTIDE (AP) AP-2 ADAPTER FROM CLATHRIN-COATED VESICLES IS PHOSPHORYLATED ON THREONINE-156 BY AP-1 AND A SOLUBLE AP50 KINASE WHICH COPURIFIES WITH THE ASSEMBLY POLYPEPTIDES
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DOI：
10.1042/bj2960409
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发表时间：
1993-12-01
影响因子：
4.1
通讯作者：
THURIEAU, C
中科院分区：
文献类型：
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作者：
PAULOIN, A;THURIEAU, C
AP50 是来自牛脑包被囊泡的组装多肽 (AP) 亚类 AP-2 的一个亚基。它可以通过与 AP 相关的 AP50 激酶活性在体内和体外在苏氨酸残基上磷酸化。我们对 AP50 磷酸化位点周围的氨基酸序列进行了分析。 AP50 体外磷酸化后进行胰蛋白酶裂解，经过 Mono-Q 离子交换 f.p.l.c. 仅分离出一种放射性肽。和反相 hplc.该肽的氨基酸序列：Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gln-Ile-Gly-Trp-Arg162，显示两个苏氨酸残基。对自动 Edman 降解产物的产率和放射性分析表明，只有 Thr-156 被磷酸化，反映了 AP50 中存在单个磷酸化位点。 AP 在相同的苏氨酰残基上磷酸化相应的合成肽。我们证明 AP50 是一种不能自磷酸化的磷酸化底物。参与 AP50 磷酸化的酶被证明与 AP-1 以及与 AP 共纯化的可溶性蛋白质复合物相关，但通过羟基磷灰石柱排阻色谱法从后者中分离出来。根据凝胶过滤数据，该 AP50 激酶活性对应于 280 kDa 的蛋白质复合物。
AP50 is a subunit of the assembly polypeptide (AP) subclass AP-2 from bovine brain coated vesicles. It can be phosphorylated in vivo and in vitro on a threonine residue by means of the AP50 kinase activity associated with AP. We have undertaken an analysis of the amino acid sequence around the AP50 phosphorylation site. After phosphorylation in vitro of AP50 followed by tryptic cleavage, only one radioactive peptide was isolated following Mono-Q ion-exchange f.p.l.c. and reverse-phase h.p.l.c. The amino acid sequence of this peptide: Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gln-Ile-Gly-Trp-Arg162, displayed two threonine residues. Analysis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflecting the presence of a single phosphorylation site in AP50. AP phosphorylated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to autophosphorylate. The enzyme involved in the AP50 phosphorylation was shown to be associated with AP-1 and with a soluble protein complex co-purified with APs but resolved from the latter by hydroxyapatite-column exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gelfiltration data.