Rosorius O, Mieskes G, Issinger O G, Körner C, Schmidt B, von Figura K, Braulke T
Institute of Biochemistry II, University of Göttingen, Germany.
Biochem J. 1993 Jun 15;292 ( Pt 3)(Pt 3):833-8. doi: 10.1042/bj2920833.
The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR 300 domain at Ser20 and at a non-identified site, neither of which are phosphorylated in vivo, and that (ii) the two sites phosphorylated by CK II in vivo and in vitro are Ser82 and Ser157. The results indicate that the human MPR 300 is a physiological substrate of either CK II or a related kinase which may play a role in the transport function of MPR 300 and/or interaction with other proteins.
人300kDa甘露糖-6-磷酸受体（MPR 300）在其胞质结构域的丝氨酸残基上发生体内磷酸化。二维分离可将胰蛋白酶磷酸肽解析为四个主要种类。为了鉴定参与MPR 300磷酸化的激酶以及磷酸化位点，在大肠杆菌中表达了胞质尾的整个编码序列。分离的胞质结构域用作四种纯化的丝氨酸/苏氨酸激酶[酪蛋白激酶II（CK II）、蛋白激酶A（PKA）、蛋白激酶C和Ca2+/钙调蛋白激酶]的底物。所有激酶仅在丝氨酸残基上磷酸化胞质尾。使用合成肽的抑制研究、分离的胰蛋白酶磷酸肽的部分测序以及与体内标记的MPR 300的胰蛋白酶磷酸肽的共迁移表明：（i）PKA在Ser20和一个未鉴定的位点磷酸化胞质MPR 300结构域，这两个位点在体内均未被磷酸化；（ii）CK II在体内和体外磷酸化的两个位点是Ser82和Ser157。结果表明，人MPR 300是CK II或相关激酶的生理底物，其可能在MPR 300的转运功能和/或与其他蛋白质的相互作用中发挥作用。
