Detection of Phospho-Sites Generated by Protein Kinase CK2 in CFTR: Mechanistic Aspects of Thr1471 Phosphorylation

By mass spectrometry analysis of mouse Cystic Fibrosis Transmembrane-conductance Regulator (mCFTR) expressed in yeast we have detected 21 phosphopeptides accounting for 22 potential phospho-residues, 12 of which could be unambiguously assigned. Most are conserved in human CFTR (hCFTR) and the majority cluster in the Regulatory Domain, lying within consensus sequences for PKA, as identified in previous mammalian studies. This validates our yeast expression model. A number of phospho-residues were novel and human conserved, notably mouse Ser670, Ser723, Ser737, and Thr1467, that all lie in acidic sequences, compatible with their phosphorylation by protein kinase CK2. Thr1467 is localized in the C-terminal tail, embedded in a functionally important and very acidic sequence (EETEEE) which displays an optimal consensus for protein kinase CK2. Herein, we show that Thr1467, homologous to human Thr1471 is readily phosphorylated by CK2. Indeed a 42 amino acid peptide encompassing the C-terminal segment of human CFTR is readily phosphorylated at Thr1471 with favorable kinetics (Km 1.7 µM) by CK2 holoenzyme, but neither by its isolated catalytic subunit nor by other acidophilic Ser/Thr kinases (CK1, PLK2/3, GCK/FAM20C). Our finding that by treating CFTR expressing BHK cells with the very specific CK2 inhibitor CX4945, newly synthesized wild type CFTR (and even more its Phe508del mutant) accumulates more abundantly than in the absence of CK2 inhibitor, supports the conclusion that phosphorylation of CFTR by CK2 correlates with decreased stability of the protein.本研究通过对酵母中表达的小鼠囊性纤维化跨膜电导调节因子（Cystic Fibrosis Transmembrane-conductance Regulator, mCFTR）进行质谱分析，共检测到21种磷酸肽，对应22个潜在磷酸化残基，其中12个残基可被明确鉴定。其中多数磷酸化位点在人CFTR（human CFTR, hCFTR）中保守，且大部分聚集于调节结构域（Regulatory Domain）内，位于蛋白激酶A（Protein Kinase A, PKA）的共识磷酸化序列中，这与此前哺乳动物研究的结果一致，验证了本研究采用的酵母表达模型。本研究还发现了多个全新且在人源CFTR中保守的磷酸化残基，包括小鼠Ser670、Ser723、Ser737及Thr1467，这些位点均处于酸性序列中，符合蛋白激酶CK2（Protein Kinase CK2, CK2）的磷酸化底物特征。其中Thr1467定位于C末端尾区，嵌入一段功能重要且高度酸性的序列（EETEEE）中，该序列具备蛋白激酶CK2的最优共识磷酸化位点特征。本研究证实，与人Thr1471同源的小鼠Thr1467可被CK2高效磷酸化。具体而言，一段包含人CFTR C末端片段的42个氨基酸肽段，可被CK2全酶（CK2 holoenzyme）以良好的动力学特性（米氏常数Km=1.7 μM）在Thr1471位点发生磷酸化，但无法被其单独的催化亚基或其他嗜酸性丝氨酸/苏氨酸激酶（CK1、PLK2/3、GCK/FAM20C）磷酸化。本研究还发现，用高特异性CK2抑制剂CX4945处理表达CFTR的幼仓鼠肾细胞（Baby Hamster Kidney, BHK）后，新合成的野生型CFTR（甚至包括其Phe508del突变体）的积累量较未添加CK2抑制剂时显著升高，这一结果支持"CK2对CFTR的磷酸化与蛋白质稳定性降低相关"的结论。