PHOSPHORYLATION BY PROTEIN-KINASE-C AND CYCLIC-AMP-DEPENDENT PROTEIN-KINASE OF SYNTHETIC PEPTIDES DERIVED FROM THE LINKER REGION OF HUMAN P-GLYCOPROTEIN
PHOSPHORYLATION BY PROTEIN-KINASE-C AND CYCLIC-AMP-DEPENDENT PROTEIN-KINASE OF SYNTHETIC PEPTIDES DERIVED FROM THE LINKER REGION OF HUMAN P-GLYCOPROTEIN
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DOI：
10.1042/bj2990309
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发表时间：
1994-04-01
影响因子：
4.1
通讯作者：
KUO, JF
中科院分区：
文献类型：
--
作者：
CHAMBERS, TC;POHL, J;KUO, JF
用合成的多肽底物PG-2鉴定了被蛋白激酶C(PKC)磷酸化的人P-糖蛋白连接子区域的特定位点，该底物对应于该区域的656-689残基。由于PG-2有几个由环状AMP依赖的蛋白激酶(PKA)识别的序列，PG-2也被测试为PKA的底物。PG-2被纯化的PKC以钙/磷脂依赖的方式磷酸化，其K-m为1.3微米，最大化学计量比为2.9+/-0.1摩尔磷酸盐/摩尔多肽。PE磷酸化PG-2胰酶片段的序列分析C鉴定Ser-661、Ser-667和Ser-671为三个磷酸化位点。PG-2还被纯化的PKA以环状AMP依赖的方式磷酸化，其K-m为21微米，最大化学计量比为2.6+/-0.2摩尔磷酸盐/摩尔多肽。Ser-667、Ser-671和Ser-683被PKA磷酸化。利用PG-2的截短肽证实Ser-661是PKC特异的，Ser-683是PKA特异的。进一步的研究表明，PG-2是多药耐药的人KB细胞膜上存在的P-糖蛋白激酶的竞争性底物。膜蛋白激酶主要在Ser-661、Ser-667和Ser-671上磷酸化PG-2。这些结果表明，在不同的第二信使系统的刺激下，人的P-糖蛋白可以被至少两种蛋白激酶磷酸化，这两种第二信使系统对分子连接区多个位点的磷酸化表现出重叠和独特的特异性。
Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a K-m of 1.3 mu M, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PE;C identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation. PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a K-m of 21 mu M, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2, were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells. The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule.