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Abstract
Scaffold proteins modulate signalling pathway activity spatially and temporally. In budding yeast, the scaffold Bem1 contributes to polarity axis establishment by regulating the GTPase Cdc42. Although different models have been proposed for Bem1 function, there is little direct evidence for an underlying mechanism. Here, we find that Bem1 directly augments the guanine exchange factor (GEF) activity of Cdc24. Bem1 also increases GEF phosphorylation by the p21-activated kinase (PAK), Cla4. Phosphorylation abrogates the scaffold-dependent stimulation of GEF activity, rendering Cdc24 insensitive to additional Bem1. Thus, Bem1 stimulates GEF activity in a reversible fashion, contributing to signalling flux through Cdc42. The contribution of Bem1 to GTPase dynamics was borne-out by in vivo imaging: active Cdc42 was enriched at the cell pole in hypophosphorylated cdc24 mutants, while hyperphosphorylated cdc24 mutants that were resistant to scaffold stimulation displayed a deficit in active Cdc42 at the pole. These findings illustrate the self-regulatory properties that scaffold proteins confer on signalling pathways.
Highlights
Scaffold proteins are multivalent binding proteins that assemble and regulate the fidelity of signal transduction cascades, conferring properties ranging from signal amplification, specificity, localisation and switch-like behaviour (Langeberg and Scott, 2015)
The C-terminal PB1 domain of Bem1, which interacts with the C-terminal PB1 domain of Cdc24 (Ito et al, 2001), was required for the stimulation of Cdc24 guanine exchange factor (GEF) activity, since a Bem1 mutant lacking this domain failed to augment GEF activity (Figure 1—figure supplement 1D and E)
The PB1 domain of Bem1 alone was not sufficient to increase GEF activity (Figure 1—figure supplement 1D and E). These results demonstrate that Bem1 can directly increase Cdc24 GEF activity towards Cdc42 by interacting with the PB1 domain of Cdc24
Summary
Scaffold proteins are multivalent binding proteins that assemble and regulate the fidelity of signal transduction cascades, conferring properties ranging from signal amplification, specificity, localisation and switch-like behaviour (Langeberg and Scott, 2015). The interaction of the scaffold with the active GTPase and its upstream GEF led to the idea that Bem might constitute part of a positive feedback loop: activated Cdc could bind Bem, recruiting the GEF to activate additional Cdc, building up a local pool of the active GTPase (Butty et al, 2002; Johnson et al, 2011) This autocatalytic positive feedback loop underpins numerous mathematical models that have been developed to understand the mechanisms that establish polarity (Goryachev and Pokhilko, 2006, 2008; Howell et al, 2012; Savage et al, 2012; Jose et al, 2013)
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