Positive feedback induces switch between distributive and processive phosphorylation of Hog1

Area of Science:

• Cellular Biology
• Biochemistry
• Systems Biology

Background:

• Mitogen-activated protein kinase (MAPK) pathways are essential for cellular decision-making, often exhibiting ultrasensitivity.
• The in vivo phosphorylation mechanism and activation dynamics of MAP kinases remain incompletely understood.
• Previous theoretical analyses suggested distributive mechanisms generate ultrasensitivity.

Purpose of the Study:

• To characterize the in vivo regulation and activation dynamics of the MAP kinase Hog1 in Saccharomyces cerevisiae.
• To elucidate the phosphorylation mechanism of Hog1 and its contribution to pathway ultrasensitivity.
• To investigate the role of a positive feedback loop involving the MAP kinase-kinase Pbs2.

Main Methods:

• Topologically different ordinary differential equation (ODE) models were employed.
• Models were parameterized using multimodal activation data.
• Mutagenesis studies (S248A, S248E) and in vitro binding assays were performed.

Main Results:

• The best-fitting model demonstrated a switch between distributive and processive phosphorylation.
• A positive feedback loop involving Hog1 phosphorylation of Pbs2 on S248 was identified.
• Mutant analysis and in vitro data confirmed the role of Pbs2-S248 phosphorylation in regulating Hog1 affinity and feedback.
• Simulations indicated this mixed mechanism enhances stimulus sensitivity and robustness.

Conclusions:

• Hog1 activation involves a mixed distributive-processive phosphorylation mechanism regulated by positive feedback.
• Direct phosphorylation of Pbs2 by Hog1 on S248 is a key regulatory event.
• This complex activation strategy is vital for robust cellular responses to stimuli.