Scaffold-mediated gating of Cdc42 signalling flux

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.DOI: http://dx.doi.org/10.7554/eLife.25257.001

A novel interplay between GEFs orchestrates Cdc42 activity during cell polarity and cytokinesis

“…Surprisingly, we report that active Cdc42 is not sufficient to recruit Scd1 in the absence of Gef1. While it has been proposed that Cdc42 establishes positive feedback through the formation of the ternary complex consisting of the Cdc42 effector PAK (p21-activated kinases) and its associated scaffold protein (Scd2 or Bem1) (Butty et al, 2002;Kozubowski et al, 2008), studies in S. pombe and S. cerevisiae suggest that Pak1 kinase activity antagonizes either the Cdc42 scaffold or the GEF, rather than establishing a positive feedback (Das et al, 2012;Gulli et al, 2000;Kuo et al, 2014;Rapali et al, 2017). In support of this antagonistic role of Pak1, we find that more Scd1 accumulates at cell ends and at the division site in the pak1 switch-off mutant.…”

Avidity‐driven polarity establishment via multivalent lipid– GTP ase module interactions

“…At the cell pole in wild-type cells, Cdc42 displays reduced diffusion compared with elsewhere on the plasma membrane, reflecting activation of Cdc42 at the cell pole (Slaughter et al, 2013;Sartorel et al, 2018). Previous work from our laboratory demonstrated that Bem1, which boosts Cdc42 activation (Rapali et al, 2017), and PS, which recruits Cdc42 activators, contribute to the reduced diffusion and nanoclustering of Cdc42 at the pole (Sartorel et al, 2018). We therefore reasoned that the reduced rate of Cdc42 diffusion and its nanoclustering at the pole may be linked to the lipid interactions exhibited by the Bem1 BC motifs.…”

“…We mutated each cluster individually or all 14 residues simultaneously ( Fig 3A). This FRET-based mant-GTP loading assay serves as a sensitive readout for Bem1 function, since Bem1 boosts GEF activity via interactions with Cdc24 and Cdc42 that are distinct from the N-terminal BC motifs (Ito et al, 2001;Yamaguchi et al, 2007;Rapali et al, 2017). Of the three clusters of basic residues, the most N-terminal BC-1 cluster appeared to be the most important ( Fig 3B).…”

“…GST-Bem1, Cdc24-6xHis, and derivative mutants were expressed and purified from Bl21-CodonPlus (DE3) cells, essentially as previously described (Rapali et al, 2017). Briefly, cells were grown in terrific broth at 37°C until an OD600 nm~3 .…”

“…We next verified that the resulting bc-14E mutant protein retained the ability to boost Cdc24 GEF activity in a GEF assay, and had thus not been non-specifically damaged by the charge-swap mutations. This FRET-based mant-GTP loading assay serves as a sensitive readout for Bem1 function, since Bem1 boosts GEF activity via interactions with Cdc24 and Cdc42 that are distinct from the N-terminal BC motifs (Ito et al, 2001;Yamaguchi et al, 2007;Rapali et al, 2017). The bc-14E mutant was chosen for these experiments because it was expressed at similar levels to wild-type Bem1 in vivo, as demonstrated below.…”