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Outstanding Early-Career Investigator Award Finalists 1 The MEKK 3 / MEK 5 / BMK 1 Signaling Cascade Regulates Calcineurin Activity by Phosphorylation of MCIP 1 in Cardiac Hypertrophy

Ca -dependent and Ca -independent signaling pathways are associated with cardiac hypertrophy. How these two pathways crosstalk with each other resulting in cardiac hypertrophy is poorly understood. To address this issue we designed a transfection bioassay to monitor NFAT activation in cultured rat neonatal cardiomyocytes. We determined that active MEKK3 was capable of stimulating calcineurin(Cn)/NFAT signaling in cardiac myocytes. Overexpression of active MEKK3 in cardiac myocytes and mouse hearts resulted in Cn/NFAT activation and reprogramming cardiac gene expression. In contrast, small interference RNA directed against MEKK3 and a dominant negative form of MEKK3 caused the loss of NFAT activation in response to angiotensin II (Ang II) in cardiac myocytes. Likewise, MEKK3-deficient mouse embryo fibroblasts failed to activate Cn/NFAT in response to Ang II, a potent NFAT activator. Conversely, restoring MEKK3 to the MEKK3-deficient cells restored Ang II-mediated Cn/NFAT activation. Thus, MEKK3 is sufficient and essential for Cn activation. Next we determined that MEK5 and BMK1 function downstream of MEKK3 to induce NFAT activation. Physical interaction assays showed that activated MEKK3/MEK5/BMK1 formed complex with MCIP1, a Cn interacting protein, resulting in its phosphorylation. In vivo labeling experiments and immunoprecipitation pulldown assays revealed that phospho-MCIP1 dissociated from Cn catalytic subunit and interacted with an unknown 31 KD protein. Sequence analysis of MCIP1 revealed a serine-proline (SP) repeat domain with the potential to bind with a 31 kDa protein termed 14 –3-3, a multifunctional phosphopeptide binding protein. Subsequent coimmunoprecipitation experiments demonstrated that phosphorylation of MCIP1 resulted in reduced affinity for Cn and increased affinity for 14–3-3, thereby displacing phospho-NFAT from it docking site on 14–3-3. The displaced phospho-NFAT interacted with Cn, where it was dephosphorylated allowing entry in the nucleus to contribute to the hypertrophic transcriptional response. Thus, our findings reveal a previously unrecognized novel essential regulatory role of MAP kinase signaling in Cn activation.