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Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5.

Jeong JY, Johns J, Sinclair C, Park JM, Rossie S - BMC Cell Biol. (2003)

Bottom Line: Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment.In addition, C-terminal truncation did not prevent further activation by lipid.Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA. jyjeong@purdue.edu

ABSTRACT

Background: Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level.

Results: The specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth.

Conclusions: Many characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests that this enzyme is involved in cell growth or its expression is controlled by metabolic or nutritional signals.

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Effect of C-terminal truncation on Ppt1p activity. (A) Phosphatase activity of full-length Ppt1p (□) or the C-terminal truncation mutant, Ppt1p (1–504) (■), was determined in the absence or presence of 250 μM AA using 32P-casein or 32P-MBP as substrate. Data are the average ± S.D. of triplicate samples from a single experiment. Similar values were obtained in three independent experiments performed under the same conditions. (B) C-terminal sequences of Ppt1p and PP5 are aligned to display the truncation sites for Ppt1p (1–504) and PP5 (1–486) [19]. The truncation sites are indicated with the filled triangles. Bold underlined residues are conserved in both Ppt1p and PP5.
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Figure 3: Effect of C-terminal truncation on Ppt1p activity. (A) Phosphatase activity of full-length Ppt1p (□) or the C-terminal truncation mutant, Ppt1p (1–504) (■), was determined in the absence or presence of 250 μM AA using 32P-casein or 32P-MBP as substrate. Data are the average ± S.D. of triplicate samples from a single experiment. Similar values were obtained in three independent experiments performed under the same conditions. (B) C-terminal sequences of Ppt1p and PP5 are aligned to display the truncation sites for Ppt1p (1–504) and PP5 (1–486) [19]. The truncation sites are indicated with the filled triangles. Bold underlined residues are conserved in both Ppt1p and PP5.

Mentions: Removal of 10–13 residues from the C terminus of PP5 results in a highly active enzyme that is only modestly stimulated by lipid [19]. To examine the role of the C terminus in controlling Ppt1p phosphatase activity, a similar truncation mutant, Ppt1p (1–504), was expressed and characterized (Fig. 1A and 3). Compared to full-length enzyme Ppt1p (1–504) exhibited a two-fold increase in control specific activity with both 32P-casein and 32P-MBP. Lipid did not stimulate Ppt1p (1–504) toward 32P-casein, but still increased activity with 32P-MBP 12-fold. In contrast, C-terminal truncation of PP5 activates the enzyme as extensively as lipid treatment, and little further activation is seen with lipid [19]. In the case of PP5, subtilisin digestion removed both the TPR domain and the C terminus [19]. The observation that C-terminal truncation increased Ppt1p activity toward both 32P-casein and 32P-MBP, but subtilisin digestion increased activity only toward 32P-MBP (see Fig. 2C) suggests that the C terminus of Ppt1p remained intact during subtilisin digestion. However, mass analysis of the subtilisin-derived catalytic fragment would be required to confirm this.


Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5.

Jeong JY, Johns J, Sinclair C, Park JM, Rossie S - BMC Cell Biol. (2003)

Effect of C-terminal truncation on Ppt1p activity. (A) Phosphatase activity of full-length Ppt1p (□) or the C-terminal truncation mutant, Ppt1p (1–504) (■), was determined in the absence or presence of 250 μM AA using 32P-casein or 32P-MBP as substrate. Data are the average ± S.D. of triplicate samples from a single experiment. Similar values were obtained in three independent experiments performed under the same conditions. (B) C-terminal sequences of Ppt1p and PP5 are aligned to display the truncation sites for Ppt1p (1–504) and PP5 (1–486) [19]. The truncation sites are indicated with the filled triangles. Bold underlined residues are conserved in both Ppt1p and PP5.
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Related In: Results  -  Collection

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Figure 3: Effect of C-terminal truncation on Ppt1p activity. (A) Phosphatase activity of full-length Ppt1p (□) or the C-terminal truncation mutant, Ppt1p (1–504) (■), was determined in the absence or presence of 250 μM AA using 32P-casein or 32P-MBP as substrate. Data are the average ± S.D. of triplicate samples from a single experiment. Similar values were obtained in three independent experiments performed under the same conditions. (B) C-terminal sequences of Ppt1p and PP5 are aligned to display the truncation sites for Ppt1p (1–504) and PP5 (1–486) [19]. The truncation sites are indicated with the filled triangles. Bold underlined residues are conserved in both Ppt1p and PP5.
Mentions: Removal of 10–13 residues from the C terminus of PP5 results in a highly active enzyme that is only modestly stimulated by lipid [19]. To examine the role of the C terminus in controlling Ppt1p phosphatase activity, a similar truncation mutant, Ppt1p (1–504), was expressed and characterized (Fig. 1A and 3). Compared to full-length enzyme Ppt1p (1–504) exhibited a two-fold increase in control specific activity with both 32P-casein and 32P-MBP. Lipid did not stimulate Ppt1p (1–504) toward 32P-casein, but still increased activity with 32P-MBP 12-fold. In contrast, C-terminal truncation of PP5 activates the enzyme as extensively as lipid treatment, and little further activation is seen with lipid [19]. In the case of PP5, subtilisin digestion removed both the TPR domain and the C terminus [19]. The observation that C-terminal truncation increased Ppt1p activity toward both 32P-casein and 32P-MBP, but subtilisin digestion increased activity only toward 32P-MBP (see Fig. 2C) suggests that the C terminus of Ppt1p remained intact during subtilisin digestion. However, mass analysis of the subtilisin-derived catalytic fragment would be required to confirm this.

Bottom Line: Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment.In addition, C-terminal truncation did not prevent further activation by lipid.Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA. jyjeong@purdue.edu

ABSTRACT

Background: Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level.

Results: The specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth.

Conclusions: Many characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests that this enzyme is involved in cell growth or its expression is controlled by metabolic or nutritional signals.

Show MeSH
Related in: MedlinePlus