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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 on CanA, the catalytic subunit of calcineurin, and its role in the regulation of DimB phosphorylation. (A) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on CanA (DDB0185021). Phosphorylation fold changes are expressed as log2 ratios relative to pretreatment; a log2 ratio of 1 is a twofold increase in phosphorylation, whereas a log2 ratio of −1 represents a twofold decrease in phosphorylation, that is, dephosphorylation. (B) Amino acid sequence 525–559 of CanA. Underlined 529–548 is a putative CaM-binding domain (Catalano and O'Day, 2008). Highlighted and labeled residues are those identified as class I DIF-1 regulated. (C) DimB S590 phosphorylation in response to DIF-1 and gossypol treatment. Ax2 cells starved for 4 h were treated with DIF-1 and/or gossypol and samples collected at the times indicated and then immunoblotted using anti–pS590 DimB and anti–total DimB antibody. (D) Nuclear translocation of DimB in response to DIF-1 and gossypol. Cells developed and treated as in C. Nuclear accumulation of DimB was assayed immunohistochemically at the stated times using anti–total DimB. Ethanol and dimethyl sulfoxide controls (DIF-1 and gossypol, respectively) both showed no nuclear accumulation. Scale bar, 10 μm.
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Figure 3: DIF-1–regulated phosphorylation sites on CanA, the catalytic subunit of calcineurin, and its role in the regulation of DimB phosphorylation. (A) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on CanA (DDB0185021). Phosphorylation fold changes are expressed as log2 ratios relative to pretreatment; a log2 ratio of 1 is a twofold increase in phosphorylation, whereas a log2 ratio of −1 represents a twofold decrease in phosphorylation, that is, dephosphorylation. (B) Amino acid sequence 525–559 of CanA. Underlined 529–548 is a putative CaM-binding domain (Catalano and O'Day, 2008). Highlighted and labeled residues are those identified as class I DIF-1 regulated. (C) DimB S590 phosphorylation in response to DIF-1 and gossypol treatment. Ax2 cells starved for 4 h were treated with DIF-1 and/or gossypol and samples collected at the times indicated and then immunoblotted using anti–pS590 DimB and anti–total DimB antibody. (D) Nuclear translocation of DimB in response to DIF-1 and gossypol. Cells developed and treated as in C. Nuclear accumulation of DimB was assayed immunohistochemically at the stated times using anti–total DimB. Ethanol and dimethyl sulfoxide controls (DIF-1 and gossypol, respectively) both showed no nuclear accumulation. Scale bar, 10 μm.

Mentions: We identify class I phosphorylation sites on several proteins that have a direct involvement with Ca2+, and most show dephosphorylation in response to DIF-1 (Supplemental Figure S2). In addition, we observe transient phosphorylation upon four phosphorylation sites in an 18-residue stretch of CanA, the catalytic subunit of the protein phosphatase calcineurin (Figure 3A). Calcineurin is a Ca2+- and calmodulin (CaM)-dependent protein phosphatase that is well conserved from yeast to mammals and is critical to many cellular processes (Rusnak and Mertz, 2000). In its inactive form, calcineurin is a heterodimer of CanA and its regulatory subunit CanB. Increasing Ca2+ allows CaM to bind, displacing the autoinhibitory domain and forming an active calcineurin heterotrimer. In this way, cellular Ca2+ regulates calcineurin phosphatase activity. Ca2+-dependent binding of CaM to Dictyostelium CanA has been demonstrated and putative CaM-binding domains identified, but, unusually, Ca2+/CaM is not essential for Dictyostelium calcineurin activity, which can also be activated by long-chain fatty acids, including arachidonic acid (Kessen et al., 1999; Catalano and O'Day, 2008).


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 on CanA, the catalytic subunit of calcineurin, and its role in the regulation of DimB phosphorylation. (A) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on CanA (DDB0185021). Phosphorylation fold changes are expressed as log2 ratios relative to pretreatment; a log2 ratio of 1 is a twofold increase in phosphorylation, whereas a log2 ratio of −1 represents a twofold decrease in phosphorylation, that is, dephosphorylation. (B) Amino acid sequence 525–559 of CanA. Underlined 529–548 is a putative CaM-binding domain (Catalano and O'Day, 2008). Highlighted and labeled residues are those identified as class I DIF-1 regulated. (C) DimB S590 phosphorylation in response to DIF-1 and gossypol treatment. Ax2 cells starved for 4 h were treated with DIF-1 and/or gossypol and samples collected at the times indicated and then immunoblotted using anti–pS590 DimB and anti–total DimB antibody. (D) Nuclear translocation of DimB in response to DIF-1 and gossypol. Cells developed and treated as in C. Nuclear accumulation of DimB was assayed immunohistochemically at the stated times using anti–total DimB. Ethanol and dimethyl sulfoxide controls (DIF-1 and gossypol, respectively) both showed no nuclear accumulation. Scale bar, 10 μm.
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Related In: Results  -  Collection

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Figure 3: DIF-1–regulated phosphorylation sites on CanA, the catalytic subunit of calcineurin, and its role in the regulation of DimB phosphorylation. (A) Temporal profile for DIF-1–induced phosphorylation changes in class I sites on CanA (DDB0185021). Phosphorylation fold changes are expressed as log2 ratios relative to pretreatment; a log2 ratio of 1 is a twofold increase in phosphorylation, whereas a log2 ratio of −1 represents a twofold decrease in phosphorylation, that is, dephosphorylation. (B) Amino acid sequence 525–559 of CanA. Underlined 529–548 is a putative CaM-binding domain (Catalano and O'Day, 2008). Highlighted and labeled residues are those identified as class I DIF-1 regulated. (C) DimB S590 phosphorylation in response to DIF-1 and gossypol treatment. Ax2 cells starved for 4 h were treated with DIF-1 and/or gossypol and samples collected at the times indicated and then immunoblotted using anti–pS590 DimB and anti–total DimB antibody. (D) Nuclear translocation of DimB in response to DIF-1 and gossypol. Cells developed and treated as in C. Nuclear accumulation of DimB was assayed immunohistochemically at the stated times using anti–total DimB. Ethanol and dimethyl sulfoxide controls (DIF-1 and gossypol, respectively) both showed no nuclear accumulation. Scale bar, 10 μm.
Mentions: We identify class I phosphorylation sites on several proteins that have a direct involvement with Ca2+, and most show dephosphorylation in response to DIF-1 (Supplemental Figure S2). In addition, we observe transient phosphorylation upon four phosphorylation sites in an 18-residue stretch of CanA, the catalytic subunit of the protein phosphatase calcineurin (Figure 3A). Calcineurin is a Ca2+- and calmodulin (CaM)-dependent protein phosphatase that is well conserved from yeast to mammals and is critical to many cellular processes (Rusnak and Mertz, 2000). In its inactive form, calcineurin is a heterodimer of CanA and its regulatory subunit CanB. Increasing Ca2+ allows CaM to bind, displacing the autoinhibitory domain and forming an active calcineurin heterotrimer. In this way, cellular Ca2+ regulates calcineurin phosphatase activity. Ca2+-dependent binding of CaM to Dictyostelium CanA has been demonstrated and putative CaM-binding domains identified, but, unusually, Ca2+/CaM is not essential for Dictyostelium calcineurin activity, which can also be activated by long-chain fatty acids, including arachidonic acid (Kessen et al., 1999; Catalano and O'Day, 2008).

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