The Dictyostelium prestalk inducer differentiation-inducing factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes.
Bottom Line: The results also provide evidence that the Ca(2+)/calmodulin-dependent phosphatase calcineurin plays a role in DIF-1 signaling to the DimB prestalk transcription factor.This accords with studies that suggest an antagonism between the two inducers and also with the rapid dephosphorylation of the cAMP receptor that we observe in response to DIF-1 and with the known inhibitory effect of DIF-1 on chemotaxis to cAMP.All MS data are available via ProteomeXchange with identifier PXD001555.
Affiliation: College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.Show MeSH
Mentions: To identify and quantify phosphorylation changes in the early response to DIF-1, we metabolically labeled Ax2 cells using SILAC (Ong et al., 2002; Sobczyk et al., 2014). Cell populations were labeled to steady state in “light,” “medium,” or “heavy” medium and then starved in shaking culture, mimicking early Dictyostelium development, for 5 h before DIF-1 treatment. Based on knowledge of the phosphorylation changes of STATc and DimB in response to DIF-1 (Fukuzawa, Araki, et al., 2001; Yamada et al., 2013), cells were harvested 1, 5, 8, and 15 min post–DIF-1 treatment to allow a time course of stimulation to be constructed from two overlapping triplex experimental samples, A (0, 1, 8 min) and B (0, 5, 15 min), with each experiment repeated in biological triplicate (Figure 1A).
Affiliation: College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.