Abstract
RCAN1, also known as DSCR1, is an endogenous regulator of calcineurin, a serine/threonine protein phosphatase that plays a critical role in many physiological processes. In this report, we demonstrate that p38α MAP kinase can phosphorylate RCAN1 at multiple sites in vitro and show that phospho-RCAN1 is a good protein substrate for calcineurin. In addition, we found that unphosphorylated RCAN1 noncompetitively inhibits calcineurin protein phosphatase activity and that the phosphorylation of RCAN1 by p38α MAP kinase decreases the binding affinity of RCAN1 for calcineurin. These findings reveal the molecular mechanism by which p38α MAP kinase regulates the function of RCAN1/calcineurin through phosphorylation.
Highlights
Calcineurin (CN) is a Ca2+/calmodulin-dependent serine/ threonine protein phosphatase with roles in myriad physiological functions [1 3], including T cell activation [4], apoptosis [5], skeletal myocyte differentiation [6], neuronal synaptic plasticity [7], memory formation [8], and cardiac health [9]
We found that unphosphorylated RCAN1 noncompetitively inhibits calcineurin protein phosphatase activity and that the phosphorylation of RCAN1 by p38 MAP kinase decreases the binding affinity of RCAN1 for calcineurin. These findings reveal the molecular mechanism by which p38 MAP kinase regulates the function of RCAN1/calcineurin through phosphorylation
We further show that the phosphorylation of RCAN1 by p38 MAP kinase decreases its binding affinity for calcineurin, suggesting that phosphorylation may relieve the inhibition of calcineurin signaling by RCAN1
Summary
Calcineurin (CN) is a Ca2+/calmodulin-dependent serine/ threonine protein phosphatase with roles in myriad physiological functions [1 3], including T cell activation [4], apoptosis [5], skeletal myocyte differentiation [6], neuronal synaptic plasticity [7], memory formation [8], and cardiac health [9]. The most thoroughly characterized calcineurin substrates are the nuclear factor of activated T cells (NFAT) family transcription factors. The PxIxIT motif is proposed to be the main calcineurin binding site, and it is present in many other calcineurin substrates and regulatory proteins. Modulatory calcineurin interacting proteins ( called RCAN proteins) are unique among these proteins in terms of their expression pattern, and they function in a negative feedback loop to regulate calcineurin activity. The RCAN proteins comprise a family of endogenous calcineurin regulators that are conserved from yeast to humans and are essential for normal calcineurin signaling. We demonstrate for the first time that p38 MAP kinase directly phosphorylates RCAN1 at five sites (S93, S108, S112, T124 and T153) in vitro. We conducted kinetic studies on the phospho-RCAN1 protein and demonstrated that it is a good substrate for calcineurin. We further show that the phosphorylation of RCAN1 by p38 MAP kinase decreases its binding affinity for calcineurin, suggesting that phosphorylation may relieve the inhibition of calcineurin signaling by RCAN1
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