RESUMO
The protein kinase MpkA plays a prominent role in the cell wall integrity signaling (CWIS) pathway, acting as the terminal MAPK activating expression of genes which encode cell wall biosynthetic enzymes and other repair functions. Numerous studies focus on MpkA function during cell wall perturbation. Here, we focus on the role MpkA plays outside of cell wall stress, during steady state growth. In an effort to seek other, as yet unknown, connections to this pathway, an mpkA deletion mutant (ΔmpkA) was subjected to phosphoproteomic and transcriptomic analysis. When compared to the control (isogenic parent of ΔmpkA), there is strong evidence suggesting MpkA is involved with maintaining cell wall strength, branching regulation, and the iron starvation pathway, among others. Particle-size analysis during shake flask growth revealed ΔmpkA mycelia were about 4 times smaller than the control strain and more than 90 cell wall related genes show significantly altered expression levels. The deletion mutant had a significantly higher branching rate than the control and phosphoproteomic results show putative branching-regulation proteins, such as CotA, LagA, and Cdc24, have a significantly different level of phosphorylation. When grown in iron limited conditions, ΔmpkA had no difference in growth rate or production of siderophores, whereas the control strain showed decreased growth rate and increased siderophore production. Transcriptomic data revealed over 25 iron related genes with altered transcript levels. Results suggest MpkA is involved with regulation of broad cellular functions in the absence of stress.
Assuntos
Aspergillus nidulans/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Fosfoproteínas/genética , Transcriptoma/genética , Aspergillus nidulans/enzimologia , Aspergillus nidulans/crescimento & desenvolvimento , Proteínas de Ciclo Celular/genética , Parede Celular/genética , Parede Celular/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Ferro/metabolismo , Deleção de Sequência/genética , Transdução de Sinais/genéticaRESUMO
Protein phosphorylation is a major means of regulation for cellular processes, and is important in cell signaling, growth, and cell proliferation. To study phosphorylated proteins, high throughput phosphoproteomic technologies, such as reverse phase protein array, phospho-specific flow cytometry, and mass spectrometry (MS) based technologies, have been developed. Among them, mass spectrometry has become the primary tool employed for the identification of phosphoproteins and phosphosites in fungi, leading to an improved understanding of a number of signaling pathways. Using mass spectrometry techniques, researchers have discovered new kinase substrates, established connections between kinases and fungal pathogenicity, and studied the evolutionary lineage of kinases between different fungal species. Further, many specific phosphorylation sites recognized by individual kinases have been described. In this review, we will focus on recent discoveries made in yeast and filamentous fungi using phosphoproteomic analysis.