Figure 1. The scaffold Bem1 directly stimulates Cdc24 GEF activity in a reversible manner via PAK-dependent phosphorylation.
(A) Fluorescence intensity change associated with the nucleotide exchange of GDP-Cdc42 for mant-GTP Cdc42. Fluorescence was measured after the addition of GDP-Cdc42 (9 µM) to reactions containing Mant-GTP (100 nM) and GMP-PNP (100 µM) in the absence (blue curve) and presence (red curve) of Bem1 (5 µM) and Cdc24 (60 nM). (B) Observed kinetic rate constants were obtained by fitting trace data to a single exponential equation. Error bars show SD and confidence where *p<0.05 and ***p<0.001. (C) In vitro kinase reactions in which the indicated proteins were incubated with Cdc24-6xHis. At the times indicated, samples were removed and analysed by SDS-PAGE and Western blotting using anti-His antibody to detect the electrophoretic mobility shift of Cdc24-6xHis. (D) A Western blot showing the phosphorylation of Cdc24 in the samples used for subsequent GEF assays. (E) The observed kinetic rate constants of Cdc24 GEF activity obtained by fitting trace data to a single exponential equation. The reactions indicated on the right had additional Bem1 added to 5 µM.
DOI: http://dx.doi.org/10.7554/eLife.25257.002
Figure 1—figure supplement 1. The interaction of Cdc42 with mant- nucleotides and the dependence of scaffold stimulation on the PB1 domain.
