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Differential Phosphorylation of RNA Polymerase III and the Initiation Factor TFIIIB in Saccharomyces cerevisiae.

Lee J, Moir RD, Willis IM - PLoS ONE (2015)

Bottom Line: However, numerous lines of evidence suggest greater complexity in the regulatory network including the phosphoregulation of other pol III components.A relatively high stoichiometry of phosphorylation was observed for several of these proteins and the Rpc82 subunit of the polymerase and the Bdp1 subunit of TFIIIB were found to be differentially phosphorylated.Alanine substitutions at the four phosphosites cause hyper-repression of transcription indicating that phosphorylation of Bdp1 opposes Maf1-mediated repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
The production of ribosomes and tRNAs for protein synthesis has a high energetic cost and is under tight transcriptional control to ensure that the level of RNA synthesis is balanced with nutrient availability and the prevailing environmental conditions. In the RNA polymerase (pol) III system in yeast, nutrients and stress affect transcription through a bifurcated signaling pathway in which protein kinase A (PKA) and TORC1 activity directly or indirectly, through downstream kinases, alter the phosphorylation state and function of the Maf1 repressor and Rpc53, a TFIIF-like subunit of the polymerase. However, numerous lines of evidence suggest greater complexity in the regulatory network including the phosphoregulation of other pol III components. To address this issue, we systematically examined all 17 subunits of pol III along with the three subunits of the initiation factor TFIIIB for evidence of differential phosphorylation in response to inhibition of TORC1. A relatively high stoichiometry of phosphorylation was observed for several of these proteins and the Rpc82 subunit of the polymerase and the Bdp1 subunit of TFIIIB were found to be differentially phosphorylated. Bdp1 is phosphorylated on four major sites during exponential growth and the protein is variably dephosphorylated under conditions that inhibit tRNA gene transcription. PKA, the TORC1-regulated kinase Sch9 and protein kinase CK2 are all implicated in the phosphorylation of Bdp1. Alanine substitutions at the four phosphosites cause hyper-repression of transcription indicating that phosphorylation of Bdp1 opposes Maf1-mediated repression. The new findings suggest an integrated regulatory model for signaling events controlling pol III transcription.

No MeSH data available.


Related in: MedlinePlus

Bdp1 is phosphorylated by PKA, Sch9 and CK2 kinases.A. Wild-type and analog-sensitive pka-as, sch9-as and pka-as sch9-as strains that contained Bdp1HA were grown to mid-log phase and treated with 0.1 μM 1 NM-PP1 or drug vehicle (DMSO) for 1 hour before extract preparation. Bdp1HA was resolved in Phos-tag gels and detected by immunoblotting. B. Bdp1 is phosphorylated in vitro by PKA and CK2. Recombinant wild type (WT) and alanine-substituted Bdp1-6His 2SA (S164A and S178A) and 4SA (S49A, S164A, S178A and S586A) proteins were labeled in vitro by murine PKA (left panels) and human CK2 (right panels). The extent of phosphorylation (top panels) normalized to input protein detected by immunoblotting (lower panels) is reported under each lane. C. Bdp1 is phosphorylated in vitro by yeast Sch9. Recombinant wild-type (WT) and alanine-substituted Bdp1-6His 2SA and 4SA proteins were labeled in vitro with yeast-purified wild-type (left panels) and kinase-dead (KD, right panels) GST-Sch9. Phosphorimage (top panel) shows autophosphorylation of the catalytically active Sch9 and labeling of rBdp1. The extent of phosphorylation (normalized to input rBdp1, middle panel) is reported under each lane. GST-Sch9 proteins, detected by western, are shown in the lower panel.
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pone.0127225.g004: Bdp1 is phosphorylated by PKA, Sch9 and CK2 kinases.A. Wild-type and analog-sensitive pka-as, sch9-as and pka-as sch9-as strains that contained Bdp1HA were grown to mid-log phase and treated with 0.1 μM 1 NM-PP1 or drug vehicle (DMSO) for 1 hour before extract preparation. Bdp1HA was resolved in Phos-tag gels and detected by immunoblotting. B. Bdp1 is phosphorylated in vitro by PKA and CK2. Recombinant wild type (WT) and alanine-substituted Bdp1-6His 2SA (S164A and S178A) and 4SA (S49A, S164A, S178A and S586A) proteins were labeled in vitro by murine PKA (left panels) and human CK2 (right panels). The extent of phosphorylation (top panels) normalized to input protein detected by immunoblotting (lower panels) is reported under each lane. C. Bdp1 is phosphorylated in vitro by yeast Sch9. Recombinant wild-type (WT) and alanine-substituted Bdp1-6His 2SA and 4SA proteins were labeled in vitro with yeast-purified wild-type (left panels) and kinase-dead (KD, right panels) GST-Sch9. Phosphorimage (top panel) shows autophosphorylation of the catalytically active Sch9 and labeling of rBdp1. The extent of phosphorylation (normalized to input rBdp1, middle panel) is reported under each lane. GST-Sch9 proteins, detected by western, are shown in the lower panel.

Mentions: The location of Bdp1 phosphosites within consensus motifs for well-known pro-growth kinases, PKA, Sch9 and CK2, coupled with the dephosphorylation of Bdp1 under repressing conditions, suggested that Bdp1 function might be regulated by these enzymes. Accordingly, reducing the activities of the kinases in vivo should lead to lower levels of Bdp1 phosphorylation. To examine this possibility for PKA and Sch9, Bdp1 was HA-tagged in the analog-sensitive strains (see Fig 1) and the effect of 1NM-PP1 on Bdp1 mobility was assessed using Phos-tag gels. Loss of Sch9 activity by analog treatment of the sch9-as strain increased the relative amount of dephosphorylated Bdp1. Analog treatment of the pka-as and pka/sch9-as strains caused almost complete dephosphorylation of Bdp1 (Fig 4A). Since only one of the identified Bdp1 phosphosites conforms to the PKA consensus, the results suggest that inhibiting PKA activity may have both direct and indirect effects on Bdp1 phosphorylation.


Differential Phosphorylation of RNA Polymerase III and the Initiation Factor TFIIIB in Saccharomyces cerevisiae.

Lee J, Moir RD, Willis IM - PLoS ONE (2015)

Bdp1 is phosphorylated by PKA, Sch9 and CK2 kinases.A. Wild-type and analog-sensitive pka-as, sch9-as and pka-as sch9-as strains that contained Bdp1HA were grown to mid-log phase and treated with 0.1 μM 1 NM-PP1 or drug vehicle (DMSO) for 1 hour before extract preparation. Bdp1HA was resolved in Phos-tag gels and detected by immunoblotting. B. Bdp1 is phosphorylated in vitro by PKA and CK2. Recombinant wild type (WT) and alanine-substituted Bdp1-6His 2SA (S164A and S178A) and 4SA (S49A, S164A, S178A and S586A) proteins were labeled in vitro by murine PKA (left panels) and human CK2 (right panels). The extent of phosphorylation (top panels) normalized to input protein detected by immunoblotting (lower panels) is reported under each lane. C. Bdp1 is phosphorylated in vitro by yeast Sch9. Recombinant wild-type (WT) and alanine-substituted Bdp1-6His 2SA and 4SA proteins were labeled in vitro with yeast-purified wild-type (left panels) and kinase-dead (KD, right panels) GST-Sch9. Phosphorimage (top panel) shows autophosphorylation of the catalytically active Sch9 and labeling of rBdp1. The extent of phosphorylation (normalized to input rBdp1, middle panel) is reported under each lane. GST-Sch9 proteins, detected by western, are shown in the lower panel.
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pone.0127225.g004: Bdp1 is phosphorylated by PKA, Sch9 and CK2 kinases.A. Wild-type and analog-sensitive pka-as, sch9-as and pka-as sch9-as strains that contained Bdp1HA were grown to mid-log phase and treated with 0.1 μM 1 NM-PP1 or drug vehicle (DMSO) for 1 hour before extract preparation. Bdp1HA was resolved in Phos-tag gels and detected by immunoblotting. B. Bdp1 is phosphorylated in vitro by PKA and CK2. Recombinant wild type (WT) and alanine-substituted Bdp1-6His 2SA (S164A and S178A) and 4SA (S49A, S164A, S178A and S586A) proteins were labeled in vitro by murine PKA (left panels) and human CK2 (right panels). The extent of phosphorylation (top panels) normalized to input protein detected by immunoblotting (lower panels) is reported under each lane. C. Bdp1 is phosphorylated in vitro by yeast Sch9. Recombinant wild-type (WT) and alanine-substituted Bdp1-6His 2SA and 4SA proteins were labeled in vitro with yeast-purified wild-type (left panels) and kinase-dead (KD, right panels) GST-Sch9. Phosphorimage (top panel) shows autophosphorylation of the catalytically active Sch9 and labeling of rBdp1. The extent of phosphorylation (normalized to input rBdp1, middle panel) is reported under each lane. GST-Sch9 proteins, detected by western, are shown in the lower panel.
Mentions: The location of Bdp1 phosphosites within consensus motifs for well-known pro-growth kinases, PKA, Sch9 and CK2, coupled with the dephosphorylation of Bdp1 under repressing conditions, suggested that Bdp1 function might be regulated by these enzymes. Accordingly, reducing the activities of the kinases in vivo should lead to lower levels of Bdp1 phosphorylation. To examine this possibility for PKA and Sch9, Bdp1 was HA-tagged in the analog-sensitive strains (see Fig 1) and the effect of 1NM-PP1 on Bdp1 mobility was assessed using Phos-tag gels. Loss of Sch9 activity by analog treatment of the sch9-as strain increased the relative amount of dephosphorylated Bdp1. Analog treatment of the pka-as and pka/sch9-as strains caused almost complete dephosphorylation of Bdp1 (Fig 4A). Since only one of the identified Bdp1 phosphosites conforms to the PKA consensus, the results suggest that inhibiting PKA activity may have both direct and indirect effects on Bdp1 phosphorylation.

Bottom Line: However, numerous lines of evidence suggest greater complexity in the regulatory network including the phosphoregulation of other pol III components.A relatively high stoichiometry of phosphorylation was observed for several of these proteins and the Rpc82 subunit of the polymerase and the Bdp1 subunit of TFIIIB were found to be differentially phosphorylated.Alanine substitutions at the four phosphosites cause hyper-repression of transcription indicating that phosphorylation of Bdp1 opposes Maf1-mediated repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America.

ABSTRACT
The production of ribosomes and tRNAs for protein synthesis has a high energetic cost and is under tight transcriptional control to ensure that the level of RNA synthesis is balanced with nutrient availability and the prevailing environmental conditions. In the RNA polymerase (pol) III system in yeast, nutrients and stress affect transcription through a bifurcated signaling pathway in which protein kinase A (PKA) and TORC1 activity directly or indirectly, through downstream kinases, alter the phosphorylation state and function of the Maf1 repressor and Rpc53, a TFIIF-like subunit of the polymerase. However, numerous lines of evidence suggest greater complexity in the regulatory network including the phosphoregulation of other pol III components. To address this issue, we systematically examined all 17 subunits of pol III along with the three subunits of the initiation factor TFIIIB for evidence of differential phosphorylation in response to inhibition of TORC1. A relatively high stoichiometry of phosphorylation was observed for several of these proteins and the Rpc82 subunit of the polymerase and the Bdp1 subunit of TFIIIB were found to be differentially phosphorylated. Bdp1 is phosphorylated on four major sites during exponential growth and the protein is variably dephosphorylated under conditions that inhibit tRNA gene transcription. PKA, the TORC1-regulated kinase Sch9 and protein kinase CK2 are all implicated in the phosphorylation of Bdp1. Alanine substitutions at the four phosphosites cause hyper-repression of transcription indicating that phosphorylation of Bdp1 opposes Maf1-mediated repression. The new findings suggest an integrated regulatory model for signaling events controlling pol III transcription.

No MeSH data available.


Related in: MedlinePlus