Department of Pharmacology (E.S.G., K.M.W., M.L.D.) and Department of Medicine, Division of Renal Diseases and Hypertension (R.A.N.), University of Colorado School of Medicine, Aurora, Colorado; Department of Craniofacial Biology, University of Colorado School of Dental Medicine, Aurora, Colorado (L.E.H.); Immune Disease Institute, Harvard Medical School, Boston, Massachusetts (H.L.); and La Jolla Institute for Allergy and Immunology, La Jolla, California (P.G.H.).
Department of Pharmacology (E.S.G., K.M.W., M.L.D.) and Department of Medicine, Division of Renal Diseases and Hypertension (R.A.N.), University of Colorado School of Medicine, Aurora, Colorado; Department of Craniofacial Biology, University of Colorado School of Dental Medicine, Aurora, Colorado (L.E.H.); Immune Disease Institute, Harvard Medical School, Boston, Massachusetts (H.L.); and La Jolla Institute for Allergy and Immunology, La Jolla, California (P.G.H.)
Mol Pharmacol. 2019 Jan;95(1):20-32. doi: 10.1124/mol.118.113159. Epub 2018 Nov 7.
Calcineurin (CaN) phosphatase signaling is regulated by targeting CaN to substrates, inhibitors, and scaffold proteins containing docking motifs with the consensus sequence of PxIxIT. Here, we identify the docking of CaN to the isoform of MKK7, a component of the c-Jun N-terminal kinase (JNK) pathway. Because of alternative splicing of a single exon within the N-terminal domain, MKK7 encodes a unique PxIxIT motif (PIIVIT) that is not present in MKK7 or We found that MKK7 bound directly to CaN through this PIIVIT motif in vitro, immunoprecipitated with CaN from cell extracts, and exhibited fluorescence resonance energy transfer (FRET) with CaN in the cytoplasm but not in the nucleus of living cells. In contrast, MKK7 and exhibited no direct binding or FRET with CaN and were localized more in the nucleus than the cytoplasm. Furthermore, the inhibition of CaN phosphatase activity increased the basal phosphorylation of MKK7 but not MKK7 Deletion of the MKK7 PIIVIT motif eliminated FRET with CaN and promoted MKK7 redistribution to the nucleus; however, the inhibition of CaN activity did not alter MKK7 localization, indicating that MKK7 cytoplasmic retention by CaN is phosphatase activity independent. Finally, the inhibition of CaN phosphatase activity in vascular smooth muscle cells, which express MKK7 mRNA, enhances JNK activation. Overall, we conclude that the MKK7-specific PxIxIT motif promotes high-affinity CaN binding that could promote novel cross talk between CaN and JNK signaling by limiting MKK7 phosphorylation and restricting its localization to the cytoplasm.
钙调神经磷酸酶（CaN）磷酸酶信号转导受靶向 CaN 到底物、抑制剂和支架蛋白的调节，这些蛋白含有与 PxIxIT 共识序列的衔接基序。在这里，我们确定 CaN 与 JNK 途径的组成部分 MKK7 的 同工型的对接。由于 N 端结构域内单个外显子的选择性剪接，MKK7 编码一个独特的 PxIxIT 基序（PIIVIT），该基序不存在于 MKK7 或 中。我们发现 MKK7 可通过该 PIIVIT 基序在体外直接与 CaN 结合，从细胞提取物中用 CaN 免疫沉淀，并在活细胞的细胞质中而不是核中表现出荧光共振能量转移（FRET）。相比之下，MKK7 和 与 CaN 没有直接结合或 FRET，并且更多地定位于核内而不是细胞质。此外，CaN 磷酸酶活性的抑制增加了 MKK7 的基础磷酸化，但不增加 MKK7 的基础磷酸化。MKK7 的 PIIVIT 基序缺失消除了与 CaN 的 FRET，并促进了 MKK7 向核内的重新分布；然而，CaN 活性的抑制并没有改变 MKK7 的定位，表明 CaN 通过 CaN 独立于磷酸酶活性将 MKK7 保留在细胞质中。最后，在表达 MKK7 mRNA 的血管平滑肌细胞中抑制 CaN 磷酸酶活性增强了 JNK 的激活。总体而言，我们得出结论，MKK7 特异性 PxIxIT 基序促进了高亲和力的 CaN 结合，通过限制 MKK7 磷酸化和限制其定位到细胞质，可以促进 CaN 和 JNK 信号之间的新的串扰。
