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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–regulated phosphorylation sites in ERK2 and its substrate EppA. (A) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on ERK2 (DDB0191457) and EppA (DDB0233660). Solid lines represent averaged data for class I sites and dashed lines from class III sites from a single experiment. (B) ERK2 T176/Y178 phosphorylation in response to DIF-1. Ax2 cells starved for 5 h in KK2 were treated with 5 mM caffeine (10 min) and then washed out before cells (1 × 107 cells/ml) were treated with 1 μM cAMP for 2 min before addition of 100 nM DIF-1. Samples were collected at the times indicated and then immunoblotted using anti–phospho p44/42 MAPK antibody. Blots were stripped and reprobed with anti-actin antibody as a loading control. Results are representative of at least three independent experiments.
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Figure 7: DIF-1–regulated phosphorylation sites in ERK2 and its substrate EppA. (A) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on ERK2 (DDB0191457) and EppA (DDB0233660). Solid lines represent averaged data for class I sites and dashed lines from class III sites from a single experiment. (B) ERK2 T176/Y178 phosphorylation in response to DIF-1. Ax2 cells starved for 5 h in KK2 were treated with 5 mM caffeine (10 min) and then washed out before cells (1 × 107 cells/ml) were treated with 1 μM cAMP for 2 min before addition of 100 nM DIF-1. Samples were collected at the times indicated and then immunoblotted using anti–phospho p44/42 MAPK antibody. Blots were stripped and reprobed with anti-actin antibody as a loading control. Results are representative of at least three independent experiments.

Mentions: Mitogen-activated protein kinase (MAPK) cascades are conserved signaling pathways that transfer extracellular signals to a range of regulatory pathways. ERK2, one of the two Dictyostelium MAPKs, has a role in cAMP relay and chemotaxis and lies downstream of the cAR1 receptor. ERKs are regulated by phosphorylation of a highly conserved TEY sequence in the activation loop of the kinase. Residues T176 and Y178 are phosphorylated in response to cAMP (Maeda et al., 1996; Kosaka and Pears, 1997). We identified T176 and Y178 in ERK2 as class III phosphorylation sites, and both are rapidly dephosphorylated in response to DIF-1 (Y178, maximally ninefold dephosphorylated at 1 min; Figure 7A). We confirmed these ERK2 phosphorylation changes using a phospho-specific antibody designed to recognize T202 and Y204 of human ERK2. We used caffeine treatment to inhibit endogenous cAMP production (Brenner and Thoms, 1984), which temporarily blocks the fluctuating phosphorylation of ERK2. We stimulated cells with cAMP to cause rapid phosphorylation of ERK2 and then monitored the phosphorylation state of T176/Y178 in response to DIF-1. The late decrease in phosphorylation in control cells seems very likely to be due to slow hydrolysis of cAMP. However, within just 30 s of DIF-1 addition, ERK2 T176/Y178 was completely dephosphorylated before levels returned to baseline within 2–3 min (Figure 7, A and B). These data are consistent with DIF-1 causing a transient accelerated dephosphorylation of ERK2.


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–regulated phosphorylation sites in ERK2 and its substrate EppA. (A) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on ERK2 (DDB0191457) and EppA (DDB0233660). Solid lines represent averaged data for class I sites and dashed lines from class III sites from a single experiment. (B) ERK2 T176/Y178 phosphorylation in response to DIF-1. Ax2 cells starved for 5 h in KK2 were treated with 5 mM caffeine (10 min) and then washed out before cells (1 × 107 cells/ml) were treated with 1 μM cAMP for 2 min before addition of 100 nM DIF-1. Samples were collected at the times indicated and then immunoblotted using anti–phospho p44/42 MAPK antibody. Blots were stripped and reprobed with anti-actin antibody as a loading control. Results are representative of at least three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 7: DIF-1–regulated phosphorylation sites in ERK2 and its substrate EppA. (A) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on ERK2 (DDB0191457) and EppA (DDB0233660). Solid lines represent averaged data for class I sites and dashed lines from class III sites from a single experiment. (B) ERK2 T176/Y178 phosphorylation in response to DIF-1. Ax2 cells starved for 5 h in KK2 were treated with 5 mM caffeine (10 min) and then washed out before cells (1 × 107 cells/ml) were treated with 1 μM cAMP for 2 min before addition of 100 nM DIF-1. Samples were collected at the times indicated and then immunoblotted using anti–phospho p44/42 MAPK antibody. Blots were stripped and reprobed with anti-actin antibody as a loading control. Results are representative of at least three independent experiments.
Mentions: Mitogen-activated protein kinase (MAPK) cascades are conserved signaling pathways that transfer extracellular signals to a range of regulatory pathways. ERK2, one of the two Dictyostelium MAPKs, has a role in cAMP relay and chemotaxis and lies downstream of the cAR1 receptor. ERKs are regulated by phosphorylation of a highly conserved TEY sequence in the activation loop of the kinase. Residues T176 and Y178 are phosphorylated in response to cAMP (Maeda et al., 1996; Kosaka and Pears, 1997). We identified T176 and Y178 in ERK2 as class III phosphorylation sites, and both are rapidly dephosphorylated in response to DIF-1 (Y178, maximally ninefold dephosphorylated at 1 min; Figure 7A). We confirmed these ERK2 phosphorylation changes using a phospho-specific antibody designed to recognize T202 and Y204 of human ERK2. We used caffeine treatment to inhibit endogenous cAMP production (Brenner and Thoms, 1984), which temporarily blocks the fluctuating phosphorylation of ERK2. We stimulated cells with cAMP to cause rapid phosphorylation of ERK2 and then monitored the phosphorylation state of T176/Y178 in response to DIF-1. The late decrease in phosphorylation in control cells seems very likely to be due to slow hydrolysis of cAMP. However, within just 30 s of DIF-1 addition, ERK2 T176/Y178 was completely dephosphorylated before levels returned to baseline within 2–3 min (Figure 7, A and B). These data are consistent with DIF-1 causing a transient accelerated dephosphorylation of ERK2.

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