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PP1gamma2 and PPP1R11 are parts of a multimeric complex in developing testicular germ cells in which their steady state levels are reciprocally related.

Cheng L, Pilder S, Nairn AC, Ramdas S, Vijayaraghavan S - PLoS ONE (2009)

Bottom Line: A biochemical approach was employed to learn how expression versus deficiency of PP1gamma2, the predominant PP1 isoform in male germ cells, affects spermatogenesis.Methods used in this study include column chromatography, western blot and northern blot analyses, GST pull-down assays, immunoprecipitation, non-denaturing gel electrophoresis, phosphatase enzyme assays, protein sequencing, and immunohistochemistry.However, in PP1alpha- spleen, where PP1gamma2 normally is not expressed, PPP1R11 levels remained unchanged.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Kent State University, Kent, Ohio, United States of America.

ABSTRACT
Mice lacking the protein phosphatase 1 gamma isoforms, PP1gamma1 and PP1gamma2, are male-sterile due to defective germ cell morphogenesis and apoptosis. However, this deficiency causes no obvious abnormality in other tissues. A biochemical approach was employed to learn how expression versus deficiency of PP1gamma2, the predominant PP1 isoform in male germ cells, affects spermatogenesis. Methods used in this study include column chromatography, western blot and northern blot analyses, GST pull-down assays, immunoprecipitation, non-denaturing gel electrophoresis, phosphatase enzyme assays, protein sequencing, and immunohistochemistry. We report for the first time that in wild-type testis, PP1gamma2 forms an inactive complex with actin, protein phosphatase 1 regulatory subunit 7 (PPP1R7), and protein phosphatase 1 regulatory subunit 11 (PPP1R11), the latter, a potent PP1 inhibitor. Interestingly, PPP1R11 protein, but not its mRNA level, falls significantly in PP1gamma- testis where mature sperm are virtually absent. Conversely, both mature sperm numbers and the PPP1R11 level increase substantially in PP1gamma- testis expressing transgenic PP1gamma2. PPP1R11 also appears to be ubiquitinated in PP1gamma- testis. The levels of PP1gamma2 and PPP1R11 were increased in phenotypically normal PP1alpha- testis. However, in PP1alpha- spleen, where PP1gamma2 normally is not expressed, PPP1R11 levels remained unchanged. Our data clearly show a direct reciprocal relationship between the levels of the protein phosphatase isoform PP1gamma2 and its regulator PPP1R11, and suggest that complex formation between these polypeptides in testis may prevent proteolysis of PPP1R11 and thus, germ cell apoptosis.

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Sds22, PPP1R11, and PP1γ2 are bound to each other in crude testis protein extracts.A. The recombinant proteins GST-PPP1R11 and control GST were incubated with testis cell lysates in the presence of Glutathione-Sepharose beads. The eluted proteins and testis extracts alone were resolved by SDS-PAGE and subjected to western blot analysis with anti-Sds22 and anti-PP1γ2 antibodies. B. Testis protein extracts and buffer controls were incubated with anti-PP1γ2, anti-PPP1R11, or preimmune serum immobilized on Protein G-Sepharose-4 beads, as indicated at the top of the figure. The immunoprecipitates were separated by SDS-PAGE and immunoblotted for the proteins indicated at the bottom of the figure. (ND: not done).
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pone-0004861-g002: Sds22, PPP1R11, and PP1γ2 are bound to each other in crude testis protein extracts.A. The recombinant proteins GST-PPP1R11 and control GST were incubated with testis cell lysates in the presence of Glutathione-Sepharose beads. The eluted proteins and testis extracts alone were resolved by SDS-PAGE and subjected to western blot analysis with anti-Sds22 and anti-PP1γ2 antibodies. B. Testis protein extracts and buffer controls were incubated with anti-PP1γ2, anti-PPP1R11, or preimmune serum immobilized on Protein G-Sepharose-4 beads, as indicated at the top of the figure. The immunoprecipitates were separated by SDS-PAGE and immunoblotted for the proteins indicated at the bottom of the figure. (ND: not done).

Mentions: The protein PPP1R11 has been reported to co-localize with PP1γ1 in nucleoli and with PP1α in centrosomes in somatic cells, and has been co-precipitated with PP1γ1 and PP1α but not PP1β in HEK cells [28]. However, it was not known whether PPP1R11 binds PP1γ2 in testicular germ cells. To determine if PPP1R11 is capable of binding to PP1γ2 in vitro, we made GST-tagged recombinant PPP1R11 protein in bacteria, immobilized it on glutathione Sepharose 4B beads, and incubated it with mouse testis extracts. After extensive washing, bound proteins released by reduced glutathione were analyzed on western blots probed with anti-PP1γ2 and anti-Sds22 antibodies. Figure 2A demonstrates that PP1γ2 from testis extracts was bound to GST-PPP1R11 but not to GST alone. Not surprisingly, Sds22 was also identified as a potential PPP1R11 and/or PP1γ2 binding protein in testis extracts (Fig. 2A).


PP1gamma2 and PPP1R11 are parts of a multimeric complex in developing testicular germ cells in which their steady state levels are reciprocally related.

Cheng L, Pilder S, Nairn AC, Ramdas S, Vijayaraghavan S - PLoS ONE (2009)

Sds22, PPP1R11, and PP1γ2 are bound to each other in crude testis protein extracts.A. The recombinant proteins GST-PPP1R11 and control GST were incubated with testis cell lysates in the presence of Glutathione-Sepharose beads. The eluted proteins and testis extracts alone were resolved by SDS-PAGE and subjected to western blot analysis with anti-Sds22 and anti-PP1γ2 antibodies. B. Testis protein extracts and buffer controls were incubated with anti-PP1γ2, anti-PPP1R11, or preimmune serum immobilized on Protein G-Sepharose-4 beads, as indicated at the top of the figure. The immunoprecipitates were separated by SDS-PAGE and immunoblotted for the proteins indicated at the bottom of the figure. (ND: not done).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654099&req=5

pone-0004861-g002: Sds22, PPP1R11, and PP1γ2 are bound to each other in crude testis protein extracts.A. The recombinant proteins GST-PPP1R11 and control GST were incubated with testis cell lysates in the presence of Glutathione-Sepharose beads. The eluted proteins and testis extracts alone were resolved by SDS-PAGE and subjected to western blot analysis with anti-Sds22 and anti-PP1γ2 antibodies. B. Testis protein extracts and buffer controls were incubated with anti-PP1γ2, anti-PPP1R11, or preimmune serum immobilized on Protein G-Sepharose-4 beads, as indicated at the top of the figure. The immunoprecipitates were separated by SDS-PAGE and immunoblotted for the proteins indicated at the bottom of the figure. (ND: not done).
Mentions: The protein PPP1R11 has been reported to co-localize with PP1γ1 in nucleoli and with PP1α in centrosomes in somatic cells, and has been co-precipitated with PP1γ1 and PP1α but not PP1β in HEK cells [28]. However, it was not known whether PPP1R11 binds PP1γ2 in testicular germ cells. To determine if PPP1R11 is capable of binding to PP1γ2 in vitro, we made GST-tagged recombinant PPP1R11 protein in bacteria, immobilized it on glutathione Sepharose 4B beads, and incubated it with mouse testis extracts. After extensive washing, bound proteins released by reduced glutathione were analyzed on western blots probed with anti-PP1γ2 and anti-Sds22 antibodies. Figure 2A demonstrates that PP1γ2 from testis extracts was bound to GST-PPP1R11 but not to GST alone. Not surprisingly, Sds22 was also identified as a potential PPP1R11 and/or PP1γ2 binding protein in testis extracts (Fig. 2A).

Bottom Line: A biochemical approach was employed to learn how expression versus deficiency of PP1gamma2, the predominant PP1 isoform in male germ cells, affects spermatogenesis.Methods used in this study include column chromatography, western blot and northern blot analyses, GST pull-down assays, immunoprecipitation, non-denaturing gel electrophoresis, phosphatase enzyme assays, protein sequencing, and immunohistochemistry.However, in PP1alpha- spleen, where PP1gamma2 normally is not expressed, PPP1R11 levels remained unchanged.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Kent State University, Kent, Ohio, United States of America.

ABSTRACT
Mice lacking the protein phosphatase 1 gamma isoforms, PP1gamma1 and PP1gamma2, are male-sterile due to defective germ cell morphogenesis and apoptosis. However, this deficiency causes no obvious abnormality in other tissues. A biochemical approach was employed to learn how expression versus deficiency of PP1gamma2, the predominant PP1 isoform in male germ cells, affects spermatogenesis. Methods used in this study include column chromatography, western blot and northern blot analyses, GST pull-down assays, immunoprecipitation, non-denaturing gel electrophoresis, phosphatase enzyme assays, protein sequencing, and immunohistochemistry. We report for the first time that in wild-type testis, PP1gamma2 forms an inactive complex with actin, protein phosphatase 1 regulatory subunit 7 (PPP1R7), and protein phosphatase 1 regulatory subunit 11 (PPP1R11), the latter, a potent PP1 inhibitor. Interestingly, PPP1R11 protein, but not its mRNA level, falls significantly in PP1gamma- testis where mature sperm are virtually absent. Conversely, both mature sperm numbers and the PPP1R11 level increase substantially in PP1gamma- testis expressing transgenic PP1gamma2. PPP1R11 also appears to be ubiquitinated in PP1gamma- testis. The levels of PP1gamma2 and PPP1R11 were increased in phenotypically normal PP1alpha- testis. However, in PP1alpha- spleen, where PP1gamma2 normally is not expressed, PPP1R11 levels remained unchanged. Our data clearly show a direct reciprocal relationship between the levels of the protein phosphatase isoform PP1gamma2 and its regulator PPP1R11, and suggest that complex formation between these polypeptides in testis may prevent proteolysis of PPP1R11 and thus, germ cell apoptosis.

Show MeSH
Related in: MedlinePlus