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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.

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DIF-1 induced dephosphorylation of PKB homologues and their substrates. (A) Summary of DIF-1–regulated phosphorylation sites in PKBA, PKBR1, and their substrates. (B) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on PKBA, PKBR1, and their substrate SHAPS (DDB0306661). Solid lines represent averaged data for class I sites and dashed lines are from class III sites from a single experiment. (C) Heat-map representation of the temporal phosphorylation changes of class I DIF-1–regulated phosphorylation sites in PKB substrates. Averaged values, n ≥ 2. (D) PKB AL phosphorylation in response to DIF-1. Ax2 cells starved for 5 h were treated ± 100 nM DIF-1. Results are representative of at least three independent experiments. (E) Antagonism between cAMP and DIF-1. Cells were starved as in D and then treated with 1 μM cAMP ± 100 nM DIF-1. Samples collected at the times indicated and then immunoblotted as in D. (F) The response of PKB substrate phosphorylation to DIF-1. Samples from D were immunoblotted with anti-phosphospecific PKB substrate antibody. Blots were stripped and reprobed with anti-actin antibody as a loading control.
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Figure 6: DIF-1 induced dephosphorylation of PKB homologues and their substrates. (A) Summary of DIF-1–regulated phosphorylation sites in PKBA, PKBR1, and their substrates. (B) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on PKBA, PKBR1, and their substrate SHAPS (DDB0306661). Solid lines represent averaged data for class I sites and dashed lines are from class III sites from a single experiment. (C) Heat-map representation of the temporal phosphorylation changes of class I DIF-1–regulated phosphorylation sites in PKB substrates. Averaged values, n ≥ 2. (D) PKB AL phosphorylation in response to DIF-1. Ax2 cells starved for 5 h were treated ± 100 nM DIF-1. Results are representative of at least three independent experiments. (E) Antagonism between cAMP and DIF-1. Cells were starved as in D and then treated with 1 μM cAMP ± 100 nM DIF-1. Samples collected at the times indicated and then immunoblotted as in D. (F) The response of PKB substrate phosphorylation to DIF-1. Samples from D were immunoblotted with anti-phosphospecific PKB substrate antibody. Blots were stripped and reprobed with anti-actin antibody as a loading control.

Mentions: We identified two closely spaced phosphorylation sites on PKBR1 (T442 and S449), both of which are dephosphorylated in response to DIF-1 (Figure 6A). Averaged temporal data for the class I site, S449, shows consistent dephosphorylation after DIF-1 treatment (Figure 6B). Nine PKBA/PKBR1 substrates have been identified (Kamimura et al., 2008; Liao et al., 2010; Tang et al., 2011), and, significantly, we identified class I phosphorylation sites in no fewer than four of these proteins (Figure 6, A and C): GacG, a RhoGAP; GefN, a RasGEF; PakA, an STE20-family, P21-activated protein kinase; and most, strikingly, SHAPS, which undergoes a 19-fold dephosphorylation (log2 ratio, −4.3) after just 1 min of DIF-1 treatment (Figure 6B).


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)

DIF-1 induced dephosphorylation of PKB homologues and their substrates. (A) Summary of DIF-1–regulated phosphorylation sites in PKBA, PKBR1, and their substrates. (B) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on PKBA, PKBR1, and their substrate SHAPS (DDB0306661). Solid lines represent averaged data for class I sites and dashed lines are from class III sites from a single experiment. (C) Heat-map representation of the temporal phosphorylation changes of class I DIF-1–regulated phosphorylation sites in PKB substrates. Averaged values, n ≥ 2. (D) PKB AL phosphorylation in response to DIF-1. Ax2 cells starved for 5 h were treated ± 100 nM DIF-1. Results are representative of at least three independent experiments. (E) Antagonism between cAMP and DIF-1. Cells were starved as in D and then treated with 1 μM cAMP ± 100 nM DIF-1. Samples collected at the times indicated and then immunoblotted as in D. (F) The response of PKB substrate phosphorylation to DIF-1. Samples from D were immunoblotted with anti-phosphospecific PKB substrate antibody. Blots were stripped and reprobed with anti-actin antibody as a loading control.
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Related In: Results  -  Collection

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Figure 6: DIF-1 induced dephosphorylation of PKB homologues and their substrates. (A) Summary of DIF-1–regulated phosphorylation sites in PKBA, PKBR1, and their substrates. (B) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on PKBA, PKBR1, and their substrate SHAPS (DDB0306661). Solid lines represent averaged data for class I sites and dashed lines are from class III sites from a single experiment. (C) Heat-map representation of the temporal phosphorylation changes of class I DIF-1–regulated phosphorylation sites in PKB substrates. Averaged values, n ≥ 2. (D) PKB AL phosphorylation in response to DIF-1. Ax2 cells starved for 5 h were treated ± 100 nM DIF-1. Results are representative of at least three independent experiments. (E) Antagonism between cAMP and DIF-1. Cells were starved as in D and then treated with 1 μM cAMP ± 100 nM DIF-1. Samples collected at the times indicated and then immunoblotted as in D. (F) The response of PKB substrate phosphorylation to DIF-1. Samples from D were immunoblotted with anti-phosphospecific PKB substrate antibody. Blots were stripped and reprobed with anti-actin antibody as a loading control.
Mentions: We identified two closely spaced phosphorylation sites on PKBR1 (T442 and S449), both of which are dephosphorylated in response to DIF-1 (Figure 6A). Averaged temporal data for the class I site, S449, shows consistent dephosphorylation after DIF-1 treatment (Figure 6B). Nine PKBA/PKBR1 substrates have been identified (Kamimura et al., 2008; Liao et al., 2010; Tang et al., 2011), and, significantly, we identified class I phosphorylation sites in no fewer than four of these proteins (Figure 6, A and C): GacG, a RhoGAP; GefN, a RasGEF; PakA, an STE20-family, P21-activated protein kinase; and most, strikingly, SHAPS, which undergoes a 19-fold dephosphorylation (log2 ratio, −4.3) after just 1 min of DIF-1 treatment (Figure 6B).

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