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

Praveen R Juvvadi,M Arthur Moseley,William J Steinbach,Amber D Richards,Frédéric Lamoth,Erik J Soderblom,Yan Ma

doi:10.3389/fmicb.2015.00175

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.

Highlights

Calcineurin is an important protein phosphatase essential for hyphal growth, development, and virulence in Aspergillus fumigatus, making it an attractive antifungal target (Steinbach et al, 2007)
We previously demonstrated the importance of the RCAN1 ortholog, CbpA, for hyphal growth and calcium homeostasis in A. fumigatus (Pinchai et al, 2009)
In order to validate this finding and more clearly verify the phosphorylation status of A. fumigatus CbpA in vivo, a strain expressing the cbpA–egfp fusion construct under the control of its native promoter was generated in the akuBKU80 strain

Summary

Introduction

Calcineurin ( known as protein phosphatase 2B) is an important protein phosphatase essential for hyphal growth, development, and virulence in Aspergillus fumigatus, making it an attractive antifungal target (Steinbach et al, 2007). While the phosphorylation of the FxISPPxSPP motif in the PD-I (Ser156, Ser160; Supplementary Figure S2) is well-known from mammalian and the yeast RCANs (Vega et al, 2002; Hilioti et al, 2004; Abbasi et al, 2006), the identification of three residues phosphorylated in PD-II (Ser208, Ser217, Ser223) seems to be specific to A. fumigatus as this region was not conserved in the C. neoformans Cbp1 or in the Human MCIP1 (Figure 1).

Results

Conclusion