Subcellular Localization and Activity of the Mitogen-activated Protein Kinase Kinase 7 (MKK7) γ 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 docking of CaN to the γ isoform of MKK7, a component of the c-Jun N-terminal kinase (JNK) pathway. Due to alternative splicing of a single exon within the N-terminal domain, MKK7γ encodes a unique PxIxIT motif (PIIVIT) that is not present in MKK7 α or β. We found that MKK7γ 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 the nucleus of living cells. In contrast, MKK7α and β exhibited no direct binding or FRET with CaN and were localized more in the nucleus than the cytoplasm. Furthermore, inhibition of CaN phosphatase activity increased the basal phosphorylation of MKK7γ but not β. Deletion of the MKK7γ PIIVIT motif eliminated FRET with CaN and promoted MKK7γ redistribution to the nucleus; however, inhibition of CaN activity did not alter MKK7γ localization, indicating that MKK7γ cytoplasmic retention by CaN is phosphatase activity-independent. Finally, inhibition of CaN phosphatase activity in vascular smooth muscle cells, which express MKK7γ mRNA, enhances JNK activation. Overall, we conclude that the MKK7γ-specific PxIxIT motif promotes high-affinity CaN binding that could promote novel crosstalk between CaN and JNK signaling by limiting MKK7γ phosphorylation and restricting its localization to the cytoplasm.