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.
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.
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DOI：
10.3389/fmicb.2015.00175
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发表时间：
2015
影响因子：
5.2
通讯作者：
Steinbach WJ
中科院分区：
文献类型：
--
作者：
Juvvadi PR;Ma Y;Richards AD;Soderblom EJ;Moseley MA;Lamoth F;Steinbach WJ
钙调磷酸酶是烟曲霉菌丝生长和毒力所需的关键蛋白磷酸酶，是一个有吸引力的抗真菌靶点。然而，目前可用的钙调磷酸酶抑制剂FK506和环孢素A具有免疫抑制作用，限制了在侵袭性曲霉病患者治疗中的使用。因此，鉴定内源性钙调磷酸酶抑制剂属于钙加压素家族是一个重要的平行策略。我们之前确定了cbpA基因是烟曲霉钙加压素的成员，并表明其参与菌丝生长和钙稳态。然而，其通过磷酸化及其与钙调磷酸酶相互作用的激活/抑制机制尚不清楚。在这里，我们发现a . fumigatus CbpA在三个不同的结构域被磷酸化，包括保守的SP重复结构域（磷酸化结构域- 1；PD-I），丝状真菌特异性结构域（PD-II）和c端CIC结构域（Calcineurin的Calcipressin Inhibitor； PD-III）。虽然PD-II中三个磷酸化残基（Ser208, Ser217, Ser223）的突变在体内不影响CbpA功能，但SP重复基序中两个磷酸化丝氨酸（Ser156, Ser160）的突变导致菌丝生长减少和对氧化应激的敏感性降低。calcineurin A （CnaA）关键结构域的突变分析和蛋白质组学相互作用研究证实，CbpA与CnaA结合需要PxIxIT基序结合残基（352-NIR-354）和calcineurin B （CnaB）结合螺旋残基（V371）。此外，虽然钙调素结合残基（442-RVF-444）不影响CbpA与CnaA的结合，但在CnaA催化和cnab结合螺旋之间聚集的三个突变（T359P， H361L和L365S）也是CbpA结合所必需的。本研究首次分析了丝状真菌中钙加压素的磷酸化状态，并确定了与钙调磷酸酶结合所需的结构域。
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.