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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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PPP1R11, Sds22, and PP1γ2 from co-eluting fractions are reciprocally co-immunoprecipitated, and co-migrate by native PAGE.A. Superose 6 fractions (C4 and C5 from Figure 3C) of testis extracts containing co-eluted PPP1R11, PP1γ2, and Sds22 were incubated with anti-PPP1R11, anti-PP1γ2, anti-Sds22, 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 co-precipitating proteins as indicated below each blot strip (The reason why Sds22 migrates as a doublet is not known). B. C4 and C5 fractions of testis proteins purified by Superose 6 column chromatography were also separated by native PAGE followed by western blot analysis. Triplicated blot strips probed with either anti-PPP1R11, anti-PP1γ2, or anti-Sds22 antibodies, as indicated, demonstrate that PPP1R11, PP1γ2, and Sds22 co-migrate.
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pone-0004861-g004: PPP1R11, Sds22, and PP1γ2 from co-eluting fractions are reciprocally co-immunoprecipitated, and co-migrate by native PAGE.A. Superose 6 fractions (C4 and C5 from Figure 3C) of testis extracts containing co-eluted PPP1R11, PP1γ2, and Sds22 were incubated with anti-PPP1R11, anti-PP1γ2, anti-Sds22, 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 co-precipitating proteins as indicated below each blot strip (The reason why Sds22 migrates as a doublet is not known). B. C4 and C5 fractions of testis proteins purified by Superose 6 column chromatography were also separated by native PAGE followed by western blot analysis. Triplicated blot strips probed with either anti-PPP1R11, anti-PP1γ2, or anti-Sds22 antibodies, as indicated, demonstrate that PPP1R11, PP1γ2, and Sds22 co-migrate.

Mentions: To expand this assessment, we immunoprecipitated these fractions with anti-PPP1R11, anti-PP1γ2, and anti-Sds22 antibodies, and evaluated the immunoprecipitates by SDS-PAGE/western blotting as in Figure 2B. In all cases, antibodies raised against each of the three proteins were able to reciprocally precipitate the other two proteins (Fig. 4A). The fractions containing the co-eluted proteins from the final sizing column were further analyzed by native PAGE/western blotting. Replicate blot strips were separately probed with anti-PPP1R11, anti-PP1γ2, or anti-Sds22 antibodies (Fig. 4B). The results showed that all three proteins co-migrated, thus, offering further evidence that PPP1R11, Sds22, and PP1γ2 are bound to one another in a single complex in testis.


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)

PPP1R11, Sds22, and PP1γ2 from co-eluting fractions are reciprocally co-immunoprecipitated, and co-migrate by native PAGE.A. Superose 6 fractions (C4 and C5 from Figure 3C) of testis extracts containing co-eluted PPP1R11, PP1γ2, and Sds22 were incubated with anti-PPP1R11, anti-PP1γ2, anti-Sds22, 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 co-precipitating proteins as indicated below each blot strip (The reason why Sds22 migrates as a doublet is not known). B. C4 and C5 fractions of testis proteins purified by Superose 6 column chromatography were also separated by native PAGE followed by western blot analysis. Triplicated blot strips probed with either anti-PPP1R11, anti-PP1γ2, or anti-Sds22 antibodies, as indicated, demonstrate that PPP1R11, PP1γ2, and Sds22 co-migrate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654099&req=5

pone-0004861-g004: PPP1R11, Sds22, and PP1γ2 from co-eluting fractions are reciprocally co-immunoprecipitated, and co-migrate by native PAGE.A. Superose 6 fractions (C4 and C5 from Figure 3C) of testis extracts containing co-eluted PPP1R11, PP1γ2, and Sds22 were incubated with anti-PPP1R11, anti-PP1γ2, anti-Sds22, 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 co-precipitating proteins as indicated below each blot strip (The reason why Sds22 migrates as a doublet is not known). B. C4 and C5 fractions of testis proteins purified by Superose 6 column chromatography were also separated by native PAGE followed by western blot analysis. Triplicated blot strips probed with either anti-PPP1R11, anti-PP1γ2, or anti-Sds22 antibodies, as indicated, demonstrate that PPP1R11, PP1γ2, and Sds22 co-migrate.
Mentions: To expand this assessment, we immunoprecipitated these fractions with anti-PPP1R11, anti-PP1γ2, and anti-Sds22 antibodies, and evaluated the immunoprecipitates by SDS-PAGE/western blotting as in Figure 2B. In all cases, antibodies raised against each of the three proteins were able to reciprocally precipitate the other two proteins (Fig. 4A). The fractions containing the co-eluted proteins from the final sizing column were further analyzed by native PAGE/western blotting. Replicate blot strips were separately probed with anti-PPP1R11, anti-PP1γ2, or anti-Sds22 antibodies (Fig. 4B). The results showed that all three proteins co-migrated, thus, offering further evidence that PPP1R11, Sds22, and PP1γ2 are bound to one another in a single complex in testis.

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