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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 is present in various tissues including testis.A. Validation of PPP1R11 antibody was performed as follows: recombinant PPP1R11 and testis protein extracts were separated by SDS-PAGE followed by western blot analysis with affinity purified rabbit polyclonal anti-PPP1R11. Size markers (left) were derived from β-Galactosidase (116-kDa), Phosphorylase b (97.4-kDa), Albumin (66-kDa), Glutamic dehydrogenase (55-kDa), Ovalbumin (45-kDa), Glyceraldehyde-3-phosphate dehydrogenase (36-kDa), Carbonic anhydrase (29-kDa), and Soybean trypsin inhibitor (20-kDa). B. Soluble protein extracts from testis and somatic tissues were separated by SDS-PAGE followed by western blot analysis with the validated affinity purified PPP1R11 antibody, and the same blot was stripped and reprobed with anti-actin used as a loading control.
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pone-0004861-g001: PPP1R11 is present in various tissues including testis.A. Validation of PPP1R11 antibody was performed as follows: recombinant PPP1R11 and testis protein extracts were separated by SDS-PAGE followed by western blot analysis with affinity purified rabbit polyclonal anti-PPP1R11. Size markers (left) were derived from β-Galactosidase (116-kDa), Phosphorylase b (97.4-kDa), Albumin (66-kDa), Glutamic dehydrogenase (55-kDa), Ovalbumin (45-kDa), Glyceraldehyde-3-phosphate dehydrogenase (36-kDa), Carbonic anhydrase (29-kDa), and Soybean trypsin inhibitor (20-kDa). B. Soluble protein extracts from testis and somatic tissues were separated by SDS-PAGE followed by western blot analysis with the validated affinity purified PPP1R11 antibody, and the same blot was stripped and reprobed with anti-actin used as a loading control.

Mentions: A previous study documented the presence of ppp1r11 mRNA in testis, and showed that the steady state level of this message is clearly higher in testis than in other tissues [32]. We confirmed, by northern blot analysis of mRNAs isolated from testis and numerous other tissues, that ppp1r11 testicular mRNA is significantly more abundant than it is in other tissues (data not shown). To determine if the steady state levels of PPP1R11 protein in various tissues reflect these mRNA results, we prepared an affinity-purified polyclonal antibody (see Materials and Methods), and demonstrated that it specifically recognized both recombinant PPP1R11 and endogenous testicular PPP1R11 (Fig. 1A). Subsequent western blots of protein extracts from testis and various somatic tissues probed with our PPP1R11 antibody, established the existence of a single immunoreactive protein at 27-kDa at significantly higher levels in testis than in other tissues (Fig. 1B).


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 is present in various tissues including testis.A. Validation of PPP1R11 antibody was performed as follows: recombinant PPP1R11 and testis protein extracts were separated by SDS-PAGE followed by western blot analysis with affinity purified rabbit polyclonal anti-PPP1R11. Size markers (left) were derived from β-Galactosidase (116-kDa), Phosphorylase b (97.4-kDa), Albumin (66-kDa), Glutamic dehydrogenase (55-kDa), Ovalbumin (45-kDa), Glyceraldehyde-3-phosphate dehydrogenase (36-kDa), Carbonic anhydrase (29-kDa), and Soybean trypsin inhibitor (20-kDa). B. Soluble protein extracts from testis and somatic tissues were separated by SDS-PAGE followed by western blot analysis with the validated affinity purified PPP1R11 antibody, and the same blot was stripped and reprobed with anti-actin used as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654099&req=5

pone-0004861-g001: PPP1R11 is present in various tissues including testis.A. Validation of PPP1R11 antibody was performed as follows: recombinant PPP1R11 and testis protein extracts were separated by SDS-PAGE followed by western blot analysis with affinity purified rabbit polyclonal anti-PPP1R11. Size markers (left) were derived from β-Galactosidase (116-kDa), Phosphorylase b (97.4-kDa), Albumin (66-kDa), Glutamic dehydrogenase (55-kDa), Ovalbumin (45-kDa), Glyceraldehyde-3-phosphate dehydrogenase (36-kDa), Carbonic anhydrase (29-kDa), and Soybean trypsin inhibitor (20-kDa). B. Soluble protein extracts from testis and somatic tissues were separated by SDS-PAGE followed by western blot analysis with the validated affinity purified PPP1R11 antibody, and the same blot was stripped and reprobed with anti-actin used as a loading control.
Mentions: A previous study documented the presence of ppp1r11 mRNA in testis, and showed that the steady state level of this message is clearly higher in testis than in other tissues [32]. We confirmed, by northern blot analysis of mRNAs isolated from testis and numerous other tissues, that ppp1r11 testicular mRNA is significantly more abundant than it is in other tissues (data not shown). To determine if the steady state levels of PPP1R11 protein in various tissues reflect these mRNA results, we prepared an affinity-purified polyclonal antibody (see Materials and Methods), and demonstrated that it specifically recognized both recombinant PPP1R11 and endogenous testicular PPP1R11 (Fig. 1A). Subsequent western blots of protein extracts from testis and various somatic tissues probed with our PPP1R11 antibody, established the existence of a single immunoreactive protein at 27-kDa at significantly higher levels in testis than in other tissues (Fig. 1B).

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