Understanding signaling through the 'striatin interacting phosphatase and kinase' (STRIPAK) complex in eukaryotic development

• Applicants: Professor Dr. Ulrich Kück; Professor Dr. Albert Sickmann
• Subject Area: Plant Biochemistry and Biophysics ; Plant Cell and Developmental Biology
• Term: from 2016 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 321675624

Striatin-interacting phosphatase and kinase (STRIPAK) complexes are supramolecular structures that are evolutionary conserved from yeast to man. Molecular genetic analysis has shown that diverse processes are STRIPAK complex dependent, such as mammalian cell proliferation, differentiation, and embryonic development, autophagosome transport in Drosophila neurons, viability of Caenorhabditis elegans embryos, and cellular fusion and organ formation in microbial systems. Further, medical studies revealed that dysregulation of the STRIPAK complex is correlated with diverse human diseases, including cancer.Recently, several reports have provided evidence that STRIPAK complexes are regulators or mediators of vital cell signaling pathways including the Hippo signaling pathway in Drosophila, the septation initiation network (SIN) in fungal systems or mitogen-activated kinases (MAPK) pathways in various eukayotes. However, the precise targets of phosphorylation and dephosphorylation by STRIPAK associated kinases and phosphatases have not been identified. Previously, we have established the filamentous fungus Sordaria macrospora as a model system for eukaryotic multicellular development. Studying mutant strains that show defects in hyphal fusion and fruiting body development, we discovered subunits of a fungal STRIPAK complex. To get a detailed insight into the regulatory mechanisms of the STRIPAK complex, we will identify specific phosphorylation sites of target proteins, which are differentially phosphorylated or dephosphorylated in developmental mutants. Included in our study are strains that lack one or two subunits of the STRIPAK complex. We have established a multi-level protocol to isolate protein extract in a reproducible manner, in particular we tested different media and growth conditions to get optimal protein yields. Further, we have tested different protease inhibitors to prevent semi-tryptic cleavage of proteins. Finally, we reduced the rate of semi-tryptic peptides by about 46%. Using a modified protocol for iTRAQ-labelling of peptides combined with a 2D LC-MS/MS in 24 experiments, we identified 228 differentially phosphorylated proteins. These will be the subjects for further functional analysis in S. macrospora. The identification of targets of the STRIPAK complex will contribute to our understanding of STRIPAK complex functions and mechanisms in the context of cell signaling.

• DFG Programme: Research Grants