title: Identification and mutational analyses of phosphorylation sites of the calcineurin-binding protein CbpA and the identification of domains required for calcineurin binding in Aspergillus fumigatus.
abstract: Calcineurin is a key protein phosphatase required for hyphal growth and virulence in Aspergillus fumigatus, making it an attractive antifungal target. However, currently available calcineurin inhibitors, FK506 and cyclosporine A, are immunosuppressive, limiting usage in the treatment of patients with invasive aspergillosis. Therefore, the identification of endogenous inhibitors of calcineurin belonging to the calcipressin family is an important parallel strategy. We previously identified the gene cbpA as the A. fumigatus calcipressin member and showed its involvement in hyphal growth and calcium homeostasis. However, the mechanism of its activation/inhibition through phosphorylation and its interaction with calcineurin remains unknown. Here we show that A. fumigatus CbpA is phosphorylated at three distinct domains, including the conserved SP repeat motif (phosphorylated domain-I; PD-I), a filamentous fungal-specific domain (PD-II), and the C-terminal CIC motif (Calcipressin Inhibitor of Calcineurin; PD-III). While mutation of three phosphorylated residues (Ser208, Ser217, Ser223) in the PD-II did not affect CbpA function in vivo, mutation of the two phosphorylated serines (Ser156, Ser160) in the SP repeat motif caused reduced hyphal growth and sensitivity to oxidative stress. Mutational analysis in the key domains in calcineurin A (CnaA) and proteomic interaction studies confirmed the requirement of PxIxIT motif-binding residues (352-NIR-354) and the calcineurin B (CnaB)-binding helix residue (V371) for the binding of CbpA to CnaA. Additionally, while the calmodulin-binding residues (442-RVF-444) did not affect CbpA binding to CnaA, three mutations (T359P, H361L, and L365S) clustered between the CnaA catalytic and the CnaB-binding helix were also required for CbpA binding. This is the first study to analyze the phosphorylation status of calcipressin in filamentous fungi and identify the domains required for binding to calcineurin.
authors.affiliation: Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center Durham, NC, USA.
authors.forename: Praveen R
authors.initials: PR
authors.lastname: Juvvadi
authors.affiliation: Department of Dermatology and Venereology, The Second Hospital of Shanxi Medical University Taiyuan, Shanxi, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Department of Dermatology and Venereology, The Second Hospital of Shanxi Medical University Taiyuan, Shanxi, China.
authors.forename: Amber D
authors.initials: AD
authors.lastname: Richards
authors.affiliation: Department of Dermatology and Venereology, The Second Hospital of Shanxi Medical University Taiyuan, Shanxi, China.
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Soderblom
authors.affiliation: Duke Proteomics and Metabolomics Core Facility, Center for Genomic and Computational Biology, Duke University Durham, NC, USA.
authors.forename: M Arthur
authors.initials: MA
authors.lastname: Moseley
authors.affiliation: Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center Durham, NC, USA ; Infectious Diseases Service, Department of Medicine, Lausanne University Hospital Lausanne, Switzerland ; Institute of Microbiology, Lausanne University Hospital Lausanne, Switzerland.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Lamoth
authors.affiliation: Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center Durham, NC, USA ; Department of Molecular Genetics and Microbiology, Duke University Medical Center Durham, NC, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Steinbach
keywords: Aspergillus fumigatus; calcineurin; calcineurin-binding protein (CbpA); mutation; phosphorylation
references.citation: Eukaryot Cell. 2009 Apr;8(4):511-9
references.pmid: 19252123
references.citation: Hum Mol Genet. 2000 Jul 1;9(11):1681-90
references.pmid: 10861295
references.citation: Biochem J. 2003 Sep 1;374(Pt 2):567-75
references.pmid: 12809556
references.citation: Mol Cell Biol. 2009 May;29(10 ):2777-93
references.pmid: 19273587
references.citation: Clin Immunol Immunopathol. 1996 Sep;80(3 Pt 2):S40-5
references.pmid: 8811062
references.citation: J Biol Chem. 1998 Oct 16;273(42):27412-9
references.pmid: 9765270
references.citation: Eukaryot Cell. 2006 Jul;5(7):1091-103
references.pmid: 16835453
references.citation: Proc Natl Acad Sci U S A. 2007 Oct 30;104(44):17418-23
references.pmid: 17954914
references.citation: Infect Immun. 2001 Oct;69(10):6411-8
references.pmid: 11553585
references.citation: J Biol Chem. 2002 Aug 16;277(33):30401-7
references.pmid: 12063245
references.citation: Genes Dev. 2004 Jan 1;18(1):35-47
references.pmid: 14701880
references.citation: Biochim Biophys Acta. 2013 Oct;1833(10):2311-21
references.pmid: 23732701
references.citation: PLoS Pathog. 2013;9(8):e1003564
references.pmid: 23990785
references.citation: Proc Natl Acad Sci U S A. 2006 May 9;103(19):7327-32
references.pmid: 16648267
references.citation: Eukaryot Cell. 2005 Sep;4(9):1526-38
references.pmid: 16151246
references.citation: J Biol Chem. 2009 Apr 3;284(14 ):9394-401
references.pmid: 19189965
references.citation: J Biol Chem. 2006 Mar 24;281(12 ):7717-26
references.pmid: 16415348
references.citation: Cell Signal. 2006 Sep;18(9):1430-8
references.pmid: 16406492
references.citation: Biochim Biophys Acta. 2007 Mar;1773(3):330-41
references.pmid: 17270291
references.citation: Trends Cardiovasc Med. 2003 Jan;13(1):15-21
references.pmid: 12554096
references.citation: Science. 1995 Jan 6;267(5194):108-11
references.pmid: 7528941
references.citation: Nat Rev Microbiol. 2007 Jun;5(6):418-30
references.pmid: 17505522
references.citation: J Cell Sci. 2011 Jan 1;124(Pt 1):82-90
references.pmid: 21172821
references.citation: Genes Dev. 2000 Jul 1;14(13):1595-604
references.pmid: 10887154
references.citation: Proc Natl Acad Sci U S A. 2009 Apr 14;106(15):6117-22
references.pmid: 19332797
references.citation: J Biol Chem. 2000 Mar 24;275(12):8719-25
references.pmid: 10722714
references.citation: Arch Biochem Biophys. 2000 Jul 15;379(2):221-8
references.pmid: 10898938
references.citation: J Biol Chem. 1998 Jul 17;273(29):18325-31
references.pmid: 9660798
references.citation: EMBO J. 2000 Jul 17;19(14):3618-29
references.pmid: 10899116