PP1[gamma]2 and PPP1R11 Are Parts of a Multimeric Complex in Developing Testicular Germ Cells in which their Steady State Levels Are Reciprocally Related

Mice lacking the protein phosphatase 1 gamma isoforms, PP1[gamma]1 and PP1[gamma]2, 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 def...

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Bibliographic Details
Published in:PloS one 2009-03, Vol.4 (3), p.e4861
Main Authors: Cheng, Lina, Pilder, Stephen, Nairn, Angus C, Ramdas, Shandilya, Vijayaraghavan, Srinivasan
Format: Article
Language:English
Subjects:
Apoptosis
Enzymes
Genetic engineering
Immunohistochemistry
Muscle proteins
Phosphatases
Proteolysis
RNA
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Summary:Mice lacking the protein phosphatase 1 gamma isoforms, PP1[gamma]1 and PP1[gamma]2, 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 PP1[gamma]2, 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, PP1[gamma]2 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 PP1[gamma]-null testis where mature sperm are virtually absent. Conversely, both mature sperm numbers and the PPP1R11 level increase substantially in PP1[gamma]-null testis expressing transgenic PP1[gamma]2. PPP1R11 also appears to be ubiquitinated in PP1[gamma]-null testis. The levels of PP1[gamma]2 and PPP1R11 were increased in phenotypically normal PP1[alpha]-null testis. However, in PP1[alpha]-null spleen, where PP1[gamma]2 normally is not expressed, PPP1R11 levels remained unchanged. Our data clearly show a direct reciprocal relationship between the levels of the protein phosphatase isoform PP1[gamma]2 and its regulator PPP1R11, and suggest that complex formation between these polypeptides in testis may prevent proteolysis of PPP1R11 and thus, germ cell apoptosis.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0004861