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Competitive inhibition of calcineurin phosphatase activity by its autoinhibitory domain.

ABSTRACT: Calcineurin (protein phosphatase 2B), a calmodulin- and calcium-dependent serine/threonine phosphatase, appears to be regulated by a C-terminal autoinhibitory domain. A 25 amino acid peptide derived from this domain inhibits calcineurin phosphatase activity in vitro. Here we show that a 97 amino acid fragment of the calcineurin A alpha C-terminus is approx. 8-fold more potent than the shorter peptide in calcineurin inhibition experiments. Mutation of an evolutionarily conserved Asp to Asn, previously shown to disrupt calcium-dependent signalling and calcineurin regulation in T-lymphocytes, greatly reduced inhibition by the autoinhibitory domain in vitro. Kinetic analysis of wild-type and mutated autoinhibitory domains show that both are competitive inhibitors of calcineurin phosphatase activity with Ki values of 5.0 +/- 0.2 microM and 36.0 +/- 3.7 microM respectively. This suggests intrasteric regulation of calcineurin, with the autoinhibitory domains interacting at the active site of the enzyme. The competitive behaviour of the autoinhibitory domains contrasts with the mechanism of calcineurin inhibition by immunosuppressant-immunophilin complexes, which have been shown to bind to calcineurin at a region removed from the active site.

SUBMITTER: Sagoo JK

PROVIDER: S-EPMC1218010 | biostudies-other | 1996 Dec

REPOSITORIES: biostudies-other

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Project description:Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin pathogenesis based on studies with gB and gH hyperfusogenic VZV mutants. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Two structurally related non-antibiotic macrolides, tacrolimus and pimecrolimus, both significantly increased VZV gB/gH-gL mediated cell fusion. These compounds form a drug-protein complex with FKBP1A, which binds to calcineurin and specifically inhibits calcineurin phosphatase activity. Inhibition of calcineurin phosphatase activity also enhanced both herpes simplex virus-1 fusion complex and syncytin-1 mediated cell fusion, indicating a broad role of calcineurin in modulating this process. To characterize the role of calcineurin phosphatase activity in VZV gB/gH-gL mediated fusion, a series of biochemical, biological and infectivity assays was performed. Pimecrolimus-induced, enhanced cell fusion was significantly reduced by shRNA knockdown of FKBP1A, further supporting the role of calcineurin phosphatase activity in fusion regulation. Importantly, inhibition of calcineurin phosphatase activity during VZV infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with the previously reported phenotypes of gB and gH hyperfusogenic VZV mutants. Seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited were identified as potential downstream factors involved in fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis.

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Competitive inhibition of calcineurin phosphatase activity by its autoinhibitory domain.

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