Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) Isoform are Regulated through Binding to the Phosphatase Calcineurin.

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.