Jayant Kumar Ojha, Nikita (2016-05). Characterizing the Role of a Phosphorelay in the Circadian Regulation of the OS Mapk Pathway in Neurospora crassa. Master's Thesis.
Circadian regulation of Mitogen Activated Protein Kinase (MAPK) pathways provides a priming mechanism to allow organisms to anticipate daily environmental stresses such as heat, light, desiccation and osmotic stress. In Neurospora crassa, the OS MAPK pathway, which is homologous to the p38 MAPK pathway in mammals and the Hog pathway in yeast, is important for adaptation to osmotic stress. The OS pathway consists of two modules: a sensory phosphorelay and a downstream MAPK module. Previous work in our lab demonstrated that under constant environmental conditions, the circadian clock controls daily activation of the MAPK OS-2, through direct transcriptional regulation of the MAPKKK, OS-4. Although this signaling is necessary for rhythmic activation of OS-2, it is not known if it is sufficient for robustness in the amplitude of the rhythms. An upstream phosphorelay, consisting of a sensor histidine kinase (OS-1), a phosphotransferase (HPT-1) and a response regulator (RRG-1), is required for acute stress signaling to the MAPK cascade to activate OS-2. We discovered that the levels of RRG-1 are clock-controlled, suggesting that the phosphorelay functions to regulate rhythms in activation of OS MAPK cascade. To test this idea, I generated constructs with mutations in the phosphorylation site of RRG-1 that will abolish rhythmic activation of the phosphorelay components. These strains will be used to determine if loss of temporal activation of the phosphorelay alters rhythmic accumulation of active OS-2. In addition, transcriptional reporter fusion construct was generated to test if rrg-1 transcription is under the control of the circadian clock. To detect phosphorylation of RRG-1, phos-tag coupled with SDS-PAGE gels was used. This procedure will be utilized in future studies to test if phosphorylation of RRG-1 is rhythmic. Further, two upstream histidine kinases (NCU07221 and NCU00939) were validated as direct targets of the white collar complex (WCC). Tagged versions of these proteins were generated, which will be used in future studies to determine if these histidine kinases accumulate with a circadian rhythm to further understand their role in the clock regulation of the OS MAPK phosphorelay.