Limits...
The Dictyostelium prestalk inducer differentiation-inducing factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes.

Sugden C, Urbaniak MD, Araki T, Williams JG - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.

Show MeSH
Overview of phosphoproteomic profiling. (A) Experimental workflow for one replicate experiment. Differently SILAC labeled Dictyostelium Ax2 cells were starved for 5 h before treatment with DIF-1. Cells harvested at 1, 5, 8, and 15 min were pooled with untreated cells as triplex experimental samples. (A and B) Samples were trypsin digested and fractionated by SCX, and phosphopeptides were enriched using TiO2; then each sample was analyzed by LC-MS/MS. Data from triplicate experiments were analyzed using MaxQuant to identify and quantify phosphorylation site changes. (B) Summary of phosphorylation sites identified. (C) Heat-map representation of TPM-based clustering of DIF-1–regulated class I phosphorylation sites. Clusters are separated by horizontal lines and labeled. Numbers in parentheses indicate a four-digit binary label for each cluster based on the change in phosphorylation state: 1, phosphorylation; 0, dephosphorylation. Cluster 1 (1111) has only a single site and is not represented. Averaged temporal profiles (n ≥ 2) for each phosphorylation site in the five most populated clusters are presented.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4325849&req=5

Figure 1: Overview of phosphoproteomic profiling. (A) Experimental workflow for one replicate experiment. Differently SILAC labeled Dictyostelium Ax2 cells were starved for 5 h before treatment with DIF-1. Cells harvested at 1, 5, 8, and 15 min were pooled with untreated cells as triplex experimental samples. (A and B) Samples were trypsin digested and fractionated by SCX, and phosphopeptides were enriched using TiO2; then each sample was analyzed by LC-MS/MS. Data from triplicate experiments were analyzed using MaxQuant to identify and quantify phosphorylation site changes. (B) Summary of phosphorylation sites identified. (C) Heat-map representation of TPM-based clustering of DIF-1–regulated class I phosphorylation sites. Clusters are separated by horizontal lines and labeled. Numbers in parentheses indicate a four-digit binary label for each cluster based on the change in phosphorylation state: 1, phosphorylation; 0, dephosphorylation. Cluster 1 (1111) has only a single site and is not represented. Averaged temporal profiles (n ≥ 2) for each phosphorylation site in the five most populated clusters are presented.

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).


The Dictyostelium prestalk inducer differentiation-inducing factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes.

Sugden C, Urbaniak MD, Araki T, Williams JG - Mol. Biol. Cell (2014)

Overview of phosphoproteomic profiling. (A) Experimental workflow for one replicate experiment. Differently SILAC labeled Dictyostelium Ax2 cells were starved for 5 h before treatment with DIF-1. Cells harvested at 1, 5, 8, and 15 min were pooled with untreated cells as triplex experimental samples. (A and B) Samples were trypsin digested and fractionated by SCX, and phosphopeptides were enriched using TiO2; then each sample was analyzed by LC-MS/MS. Data from triplicate experiments were analyzed using MaxQuant to identify and quantify phosphorylation site changes. (B) Summary of phosphorylation sites identified. (C) Heat-map representation of TPM-based clustering of DIF-1–regulated class I phosphorylation sites. Clusters are separated by horizontal lines and labeled. Numbers in parentheses indicate a four-digit binary label for each cluster based on the change in phosphorylation state: 1, phosphorylation; 0, dephosphorylation. Cluster 1 (1111) has only a single site and is not represented. Averaged temporal profiles (n ≥ 2) for each phosphorylation site in the five most populated clusters are presented.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325849&req=5

Figure 1: Overview of phosphoproteomic profiling. (A) Experimental workflow for one replicate experiment. Differently SILAC labeled Dictyostelium Ax2 cells were starved for 5 h before treatment with DIF-1. Cells harvested at 1, 5, 8, and 15 min were pooled with untreated cells as triplex experimental samples. (A and B) Samples were trypsin digested and fractionated by SCX, and phosphopeptides were enriched using TiO2; then each sample was analyzed by LC-MS/MS. Data from triplicate experiments were analyzed using MaxQuant to identify and quantify phosphorylation site changes. (B) Summary of phosphorylation sites identified. (C) Heat-map representation of TPM-based clustering of DIF-1–regulated class I phosphorylation sites. Clusters are separated by horizontal lines and labeled. Numbers in parentheses indicate a four-digit binary label for each cluster based on the change in phosphorylation state: 1, phosphorylation; 0, dephosphorylation. Cluster 1 (1111) has only a single site and is not represented. Averaged temporal profiles (n ≥ 2) for each phosphorylation site in the five most populated clusters are presented.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.

Show MeSH