Phosphoproteomic analysis of STRIPAK mutants identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important in fungal sexual development and polarized growth
Phosphoproteomic analysis of STRIPAK mutants identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important in fungal sexual development and polarized growth
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DOI：
10.1111/mmi.14475
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发表时间：
2020-02-16
影响因子：
3.6
通讯作者：
Kueck, Ulrich
中科院分区：
文献类型：
--
作者：
Maerker, Ramona;Blank-Landeshammer, Bernhard;Kueck, Ulrich
高度保守的条纹蛋白相互作用磷酸酶和蛋白激酶(STRIPAK)复合体调节真核微生物、动物和人类发育蛋白的磷酸化/去磷酸化。为了首先确定STRIPAK的潜在靶点，我们对丝状真菌Sordaria macpora进行了广泛的等压标记，进行了基于相对和绝对量化的蛋白质组和磷蛋白质组分析。我们总共鉴定了4,193个蛋白质和2,489个磷酸蛋白，它们由10,635个磷酸肽代表。通过比较野生型和突变体的磷酸化数据，我们确定了228个在三个STRIPAK突变体中都被调控的磷酸蛋白，从而代表了STRIPAK的潜在靶点。为了提供STRIPAK依赖的磷酸化蛋白的典型功能分析，我们选择了CLA4，它是保守的p21激活蛋白家族的成员。对增强型CLA4缺失菌株的功能鉴定表明，CLA4控制性发育和极化生长。为了确定CLA4磷酸化的功能相关性和特定磷酸化位点对发育的影响，我们接下来产生了CLA4的仿磷和缺陷型变体。这项分析发现，CLA4催化域中高度保守的丝氨酸(S685)残基的(去)磷酸化对真菌细胞的发育是重要的。总之，这些分析有助于理解STRIPAK作为磷酸酶和激酶信号复合体的机制功能。
The highly conserved striatin-interacting phosphatases and kinases (STRIPAK) complex regulates phosphorylation/dephosphorylation of developmental proteins in eukaryotic microorganisms, animals and humans. To first identify potential targets of STRIPAK, we performed extensive isobaric tags for relative and absolute quantification-based proteomic and phosphoproteomic analyses in the filamentous fungus Sordaria macrospora. In total, we identified 4,193 proteins and 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By comparing phosphorylation data from wild type and mutants, we identified 228 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing potential targets of STRIPAK. To provide an exemplarily functional analysis of a STRIPAK-dependent phosphorylated protein, we selected CLA4, a member of the conserved p21-activated kinase family. Functional characterization of the increment cla4 deletion strain showed that CLA4 controls sexual development and polarized growth. To determine the functional relevance of CLA4 phosphorylation and the impact of specific phosphorylation sites on development, we next generated phosphomimetic and -deficient variants of CLA4. This analysis identified (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic domain of CLA4 as being important for fungal cellular development. Collectively, these analyses significantly contribute to the understanding of the mechanistic function of STRIPAK as a phosphatase and kinase signaling complex.