PHOSPHOPROTEOME ANALYSIS OF SELF-FUSION REGULATION IN NEUROSPORA CRASSA

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell fusion is an essential process in eukaryotes. It occurs during fertilization and the biogenesis of tissues including muscle, bone and placenta. Defects in fusion also have consequences for liver detoxification and cancer metastasis. In fungi, fusion between mating cells has been extensively studied, while self-fusion between genetically identical cells remains largely uncharacterized. The filamentous fungus Neurospora crassa is a model organism for molecular biology, and is now used to study self-fusion. In N. crassa, self-fusion occurs between genetically identical cells that are in the same developmental state, the mechanisms of which are unknown in any system. Recent data has shown that N. crassa cells alternate between two different physiological states in order to facilitate communication. The conserved MAP kinase MAK2 is essential for self-fusion in N. crassa and has a direct role in chemotropic attraction between fusing cells. In addition to cell fusion, mak-2 mutants show growth defects, highlighting the importance of this pathway in filamentous fungi. Furthermore, orthologs of MAK2 are important regulators of plant and animal pathogenesis caused by a variety of ascomycete species. The molecular details of self-fusion are still unknown in N. crassa, including the identity of the chemoattractant molecule and any targets of the kinase pathway. We constructed allele MAK2Q100G, whose kinase activity can be specifically inhibited through the addition of the ATP analog 1NM-PP1. Using this allele, we showed that MAK2 kinase activity is required for MAK2 complex dynamics in the partner cell, and also for the dynamic activity of SO, a protein of unknown molecular function. The proposed collaboration aims to identify targets of MAK2, which will not only reveal proteins involved in self-fusion, but also some required for vegetative growth. Homolog comparisons will reveal targets of the orthologous kinases in related pathogenic species. Specific aims include: (1) Comparative phosphoproteome analysis of kinase active/inactive MAK2Q100G cells. (2) Identify specific phosphorylation motifs on MAK2 targets. (3) Biological characterization of the roles of identified MAK2 targets. This collaboration has the potential to significantly increase the knowledge of self-fusion mechanisms in filamentous fungi, and can be easily expanded to studies of fusion in other multicellular eukaryotes, potentially including mammals. The data obtained from this study will also have other implications, since MAK2 homologs are essential for the invasion of several pathogens.