Oscillatory Phosphorylation of Yeast Fus3 MAP Kinase Controls Periodic Gene Expression and Morphogenesis

Abstract

SUMMARY Signal transduction networks can display complex dynamic behavior such as oscillations in the activity of key components [1-6], but it is often unclear if such dynamic complexity is actually important for the network's regulatory functions [7, 8]. Here we found that the mitogen-activated protein kinase (MAPK) Fus3, a key regulator of the yeast mating pheromone response undergoes sustained oscillations in its phosphorylation/activation state during continuous pheromone exposure. These MAPK activity oscillations led to corresponding oscillations in mating gene expression. Oscillations in MAPK activity and gene expression required the negative regulator of G-protein signaling Sst2, and partially required the MAPK phosphatase Msg5. Peaks in Fus3 activation correlated with periodic rounds of cell morphogenesis, with each peak preceding the formation of an additional mating projection. Preventing projection formation did not eliminate MAPK oscillation, but preventing MAPK oscillation blocked the formation of additional projections. A mathematical model was developed that reproduced several features of the observed oscillatory dynamics. These observations demonstrate a role for MAPK activity oscillation in driving a periodic downstream response, and explain how the pheromone signaling pathway, previously thought to desensitize after 1-3 hours, controls morphology changes that continue for a much longer time.

Oscillatory signaling processes: the how, the why and the where

“…Whether an artifactual augmentation of the oscillation propensity indeed takes place should be determined by independent control experiments not involving genetic or other perturbations of the cell. For instance, if the oscillatory responses are synchronized across the cell population, biochemical methods can and have been used to detect them, e.g., common Western blots [10–13]. If, on the other hand, the oscillations are asynchronous, and thus would be masked in the average cell population response, one can resort to the analysis, such as flow cytometry or immunocytochemistry, that would allow taking snapshots of responses of single cells within a population at different times [14].…”

“…When unable to sense the gradient of the pheromone secreted by the cells of the opposite mating type, but sensing the presence of the pheromone in the immediate environment, yeast cells attempt to find the mating partner by trying to grow several projections sequentially in random directions. This periodic morphogenesis processes, that may ultimately leave a cell resembling a starfish, is controlled by a periodic activation and expression of a MAPK molecule regulating a multitude of the requisite genes [10]. The oscillation thus can control the duration of sequential phases of cell growth, with the MAPK activity localized to each new projection in preparation for a potential mating event.…”

Band-pass processing in a GPCR signaling pathway selects for NFAT transcription factor activation

“…A majority of these rhythmic as well as arrhythmic stimuli lead to oscillations in second messengers (e.g. calcium, cAMP, PKA, MAPK) 14–18 . Among these, the effect of frequency modulation of calcium oscillations on downstream transcription factor activation has been extensively studied using calcium-clamped cells 19,20 .…”

Visualization of the spatial and temporal dynamics of MAPK signaling using fluorescence imaging techniques

“…Theoretical studies of MAPK dynamics have predicted the existence of oscillatory kinase activation [99,100]. This hypothesis was proven by studies that focused on feedback regulation of MAPKs in mammalian and yeast cells [101][102][103][104][105]108]. Nakayama and colleagues investigated FGF-induced oscillatory Hes1 expression and ERK activation and found that a negative feedback phosphorylation of the guanine nucleotide exchange factor Sos (which activates Ras) by ERK [106] is required for this oscillation [101].…”

“…Importantly, oscillatory MAPK dynamics have been difficult to address without single-cell measurement because averaging cell population responses will negate stochastic or asynchronous oscillation occurring in individual cells. Surprisingly, oscillatory activation of MAPK has also been found in the yeast Fus3 pathway [102], suggesting that some conserved regulatory mechanism may underlie the oscillatory dynamics of MAPK signaling.…”