Interaction between TAK1–TAB1–TAB2 and RCAN1–calcineurin defines a signalling nodal control point

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Abstract
RCAN1, the regulator of calcineurin phosphatase, interacts with TAK1 kinase binding protein 2 (TAB2). This leads to formation of the signalling complex TAK1-TAB1-TAB2, which phosphorylates RCAN1 and converts it to an activator of the calcineurin-NFAT pathway. Activated calcineurin then switches the signal off by dephopshorylating TAK1 and TAB1. The calcium-activated protein phosphatase calcineurin is controlled by regulator of calcineurin (RCAN) in organisms ranging from yeast to mammals. Here we performed a yeast two-hybrid screen with RCAN1 as bait, identifying TAK1 binding protein 2 (TAB2) as an interacting partner. TAB2 interacted directly with RCAN1 in vitro and in vivo, recruiting TAK1, TAB1 and calcineurin, forming a macromolecular signalling complex. Overexpression of TAK1 and TAB1, or active TAK1ΔN, promoted direct phosphorylation of RCAN1 in vitro and in vivo. TAK1 phosphorylated RCAN1 at Ser 94 and Ser 136, converting RCAN1 from an inhibitor to a facilitator of calcineurin–NFAT signalling, and enhancing NFATc1 nuclear translocation, NFAT transcriptional activation and the hypertrophic growth of cultured cardiomyocytes. The TAK1–TAB1–TAB2 and the calcineurin–NFAT signalling modules did not interact in Rcan1/2- or Tab2-deficient mouse embryonic fibroblast (MEF) cultures. Calcineurin activation also dephosphorylated and inhibited TAK1 and TAB1, an effect that was absent in Rcan1/2 deficient MEFs. Functionally, TAK1 was indispensable for the cardiomyocyte growth response induced by pro-hypertrophic stimuli through calcineurin. These results describe a signalling relationship between two central regulatory pathways in which TAK1–TAB1–TAB2 selectively induces calcineurin–NFAT signalling through direct phosphorylation of RCAN1, while calcineurin activation diminishes TAK1 signalling by dephosphorylation of TAK1 and TAB1.