Dephosphorylation of the Core Septin, AspB, in a Protein Phosphatase 2A-Dependent Manner Impacts Its Localization and Function in the Fungal Pathogen Aspergillus fumigatus.
Dephosphorylation of the Core Septin, AspB, in a Protein Phosphatase 2A-Dependent Manner Impacts Its Localization and Function in the Fungal Pathogen Aspergillus fumigatus.
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DOI：
10.3389/fmicb.2016.00997
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发表时间：
2016
影响因子：
5.2
通讯作者：
Steinbach WJ
中科院分区：
文献类型：
--
作者：
Vargas-Muñiz JM;Renshaw H;Richards AD;Waitt G;Soderblom EJ;Moseley MA;Asfaw Y;Juvvadi PR;Steinbach WJ
Septins是一个保守的GTP酶家族，在高等真核生物（植物除外）中形成异源寡聚复合物并执行多种功能。我们以前在人类真菌病原体烟曲霉的研究表明，核心隔蛋白，AspB，CDC 3的直系同源物，是所需的分隔，分生孢子，和分生孢子细胞壁组织。虽然AspB对这些细胞功能很重要，但对激酶或磷酸酶在A.烟熏。在这项研究中，我们评估了功能的Gin 4和Cla 4激酶和PP 2A调节亚基帕拉，在AspB的调节使用遗传和磷酸化蛋白质组学的方法。基因缺失分析表明，Cla 4和帕拉是菌丝延伸所必需的，而Gin 4、Cla 4和帕拉是分生孢子形成和正常分隔所必需的。虽然gin 4的缺失导致更大的间隔间距和高毒力，但模拟aspB缺失的表型、cla 4和帕拉的缺失导致高分隔而不影响毒力，表明在调节分隔中的不同作用。磷酸化蛋白质组学分析显示，AspB在野生型、Δ gin 4和Δ cla 4菌株中在GTdR结构域的5个残基（S134、S137、S247、T297和T301）和其C末端的2个残基（S416和S461）处被磷酸化。然而，伴随着AspB的差异定位模式和Δ帕拉菌株中的过度分隔，AspB在另外两个残基处保持磷酸化，N-末端多碱区的T68和卷曲螺旋结构域的S447。产生nonphosphorylatable和磷酸模拟菌株周围的每个差异磷酸化的残基显示，只有AspBmt-T68 E显示增加的间隔距离，这表明T68的去磷酸化是重要的适当septation。这项研究强调了septin磷酸化/去磷酸化在A.烟曲霉菌丝分隔。
Septins are a conserved family of GTPases that form hetero–oligomeric complexes and perform diverse functions in higher eukaryotes, excluding plants. Our previous studies in the human fungal pathogen Aspergillus fumigatus revealed that the core septin, AspB, a CDC3 ortholog, is required for septation, conidiation, and conidial cell wall organization. Although AspB is important for these cellular functions, nothing is known about the role of kinases or phosphatases in the posttranslational regulation and localization of septins in A. fumigatus. In this study, we assessed the function of the Gin4 and Cla4 kinases and the PP2A regulatory subunit ParA, in the regulation of AspB using genetic and phosphoproteomic approaches. Gene deletion analyses revealed that Cla4 and ParA are indispensable for hyphal extension, and Gin4, Cla4, and ParA are each required for conidiation and normal septation. While deletion of gin4 resulted in larger interseptal distances and hypervirulence, a phenotype mimicking aspB deletion, deletion of cla4 and parA caused hyperseptation without impacting virulence, indicating divergent roles in regulating septation. Phosphoproteomic analyses revealed that AspB is phosphorylated at five residues in the GTPase domain (S134, S137, S247, T297, and T301) and two residues at its C-terminus (S416 and S461) in the wild-type, Δgin4 and Δcla4 strains. However, concomitant with the differential localization pattern of AspB and hyperseptation in the ΔparA strain, AspB remained phosphorylated at two additional residues, T68 in the N-terminal polybasic region and S447 in the coiled-coil domain. Generation of nonphosphorylatable and phosphomimetic strains surrounding each differentially phosphorylated residue revealed that only AspBmt-T68E showed increased interseptal distances, suggesting that dephosphorylation of T68 is important for proper septation. This study highlights the importance of septin phosphorylation/dephosphorylation in the regulation of A. fumigatus hyphal septation.