Ralf Ruediger

Disruption of protein phosphatase 2A subunit interaction in human cancers with mutations in the Aα subunit gene

The A subunit of protein phosphatase 2A (PP2A) consists of 15 nonidentical repeats. The catalytic C subunit binds to C-terminal repeats 11–15 and regulatory B subunits bind to N-terminal repeats 1–10. Recently, four cancer-associated mutants of the Aα subunit have been described: Glu64→Asp in lung carcinoma, Glu64→Gly in breast carcinoma, Arg418→Trp in melanoma, and Δ171–589 in breast carcinoma. Based on our model of PP2A, we predicted that Glu64→Asp and Glu64→Gly might be defective in B subunit binding, whereas Arg418→Trp and Δ171–589 might bind neither B nor C subunits. We generated these mutants by site-directed mutagenesis and assayed their ability to associate with different forms of B subunits (B, B′, B′′) or with the catalytic C subunit. The results demonstrate that all mutants are defective in binding either B or B and C subunits. Specifically, the N-terminal mutants, Glu64→Asp and Glu64→Gly, are defective in B′ but normal in B, B′′, and C subunit binding, whereas the C-terminal mutants Arg418→Trp and Δ171–589 bind none of the B subunits nor the C subunit. The implications of these findings with regard to the potential role of PP2A as a tumor suppressor are discussed.

Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.

Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.

Alterations in protein phosphatase 2A subunit interaction in human carcinomas of the lung and colon with mutations in the Aβ subunit gene

Protein phosphatase 2A (PP2A) consists of three subunits, A, B and C. The A and B subunits have regulatory functions while C is the catalytic subunit. PP2A core enzyme is composed of subunits A and C, and the holoenzyme of subunits A, B and C. All subunits exist as multiple isoforms or splice variants. The A subunit exists as two isoforms, Aα and Aβ. Here we report about the properties of eight Aβ mutants, which were found in human lung and colon cancer. These mutants were reconstructed by site-directed mutagenesis and assayed for their ability to bind B and C subunits. Two mutants showed decreased binding of PR72, a member of the B′′ family of B subunits, but normal C subunit binding; two mutants exhibited decreased binding of the C subunit and of B′′/PR72; and one mutant showed increased binding of both the C subunit andB′′/PR72. Of three mutants that behaved like the wild-type Aβ subunit, one is a polymorphic variant and another one is altered outside the binding region for B and C subunits. Importantly, we also found that the wild-type Aα and Aβ isoforms, although 85% identical, are remarkably different in their ability to bind B and C subunits. Our findings may have important implications in regard to the role of PP2A as a tumor suppressor.

Characterization of the Aalpha and Abeta subunit isoforms of protein phosphatase 2A: differences in expression, subunit interaction, and evolution.

Protein phosphatase 2A (PP2A) is very versatile owing to a large number of regulatory subunits and its ability to interact with numerous other proteins. The regulatory A subunit exists as two closely related isoforms designated Aalpha and Abeta. Mutations have been found in both isoforms in a variety of human cancers. Although Aalpha has been intensely studied, little is known about Abeta. We generated Abeta-specific antibodies and determined the cell cycle expression, subcellular distribution, and metabolic stability of Abeta in comparison with Aalpha. Both forms were expressed at constant levels throughout the cell cycle, but Aalpha was expressed at a much higher level than Abeta. Both forms were found predominantly in the cytoplasm, and both had a half-life of approx. 10 h. However, Aalpha and Abeta differed substantially in their expression patterns in normal tissues and in tumour cell lines. Whereas Aalpha was expressed at similarly high levels in all tissues and cell lines, Abeta expression varied greatly. In addition, in vivo studies with epitope-tagged Aalpha and Abeta subunits demonstrated that Abeta is a markedly weaker binder of regulatory B and catalytic C subunits than Aalpha. Construction of phylogenetic trees revealed that the conservation of Aalpha during the evolution of mammals is extraordinarily high in comparison with both Abeta and cytochrome c, suggesting that Aalpha is involved in more protein-protein interactions than Abeta. We also measured the binding of polyoma virus middle tumour antigen and simian virus 40 (SV40) small tumour antigen to Aalpha and Abeta. Whereas both isoforms bound polyoma virus middle tumour antigen equally well, only Aalpha bound SV40 small tumour antigen.

Constant expression and activity of protein phosphatase 2A in synchronized cells

The levels of the A, B, and C subunits of protein phosphatase 2A in extracts from synchronized embryonic bovine tracheal cells were determined by immunoblotting with subunit-specific antibodies. A constant amount of each subunit was found in resting cells as well as in growing cells from all stages of the cell cycle. The phosphatase activity of protein phosphatase 2A was also constant. A quantitative comparison showed that the A and C subunits were present in similar amounts, whereas the B subunit was present at a significantly lower level. Together, the A, B, and C subunits represented approximately 0.2% of the total cellular protein.

Reduced expression of the Aα subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the Aα and Aβ subunit genes

Protein phosphatase 2A (PP2A) consists of 3 subunits: the catalytic subunit, C, and the regulatory subunits, A and B. The A and C subunits both exist as 2 isoforms (α and β) and the B subunit as multiple forms subdivided into 3 families, B, B′ and B″. It has been reported that the genes encoding the Aα and Aβ subunits are mutated in various human cancers, suggesting that they may function as tumor suppressors. We investigated whether Aα and Aβ mutations occur in human gliomas. Using single strand conformational polymorphism analysis and DNA sequencing, 58 brain tumors were investigated, including 23 glioblastomas, 19 oligodendrogliomas and 16 anaplastic oligodendrogliomas. Only silent mutations were detected in the Aα gene and no mutations in the Aβ gene. However, in 43% of the tumors, the level of Aα was reduced at least 10‐fold. By comparison, the levels of the Bα and Cα subunits were mostly normal. Our data indicate that these tumors contain very low levels of core and holoenzyme and high amounts of unregulated catalytic C subunit.

Human Cancer-Associated Mutations in the Aα Subunit of Protein Phosphatase 2A Increase Lung Cancer Incidence in Aα Knock-In and Knockout Mice

ABSTRACT Strong evidence has indicated that protein phosphatase 2A (PP2A) is a tumor suppressor, but a mouse model for testing the tumor suppressor activity was missing. The most abundant forms of trimeric PP2A holoenzyme consist of the scaffolding Aα subunit, one of several regulatory B subunits, and the catalytic Cα subunit. Aα mutations were discovered in a variety of human carcinomas. All carcinoma-associated mutant Aα subunits are defective in binding the B or B and C subunits. Here we describe two knock-in mice expressing cancer-associated Aα point mutants defective in binding B′ subunits, one knockout mouse expressing truncated Aα defective in B and C subunit binding, and a floxed mouse for generating conditional Aα knockouts. We found that the cancer-associated Aα mutations increased the incidence of cancer by 50 to 60% in lungs of FVB mice treated with benzopyrene, demonstrating that PP2A acts as a tumor suppressor. We show that the effect of Aα mutation on cancer incidence is dependent on the tumor suppressor p53. The finding that the Aα mutation E64D, which was detected in a human lung carcinoma, increases the lung cancer incidence in mice suggests that this mutation also played a role in the development of the carcinoma in which it was discovered.

Mouse model for probing tumor suppressor activity of protein phosphatase 2A in diverse signaling pathways

Evidence that protein phosphatase 2A (PP2A) is a tumor suppressor in humans came from the discovery of mutations in the genes encoding the Aα and Aβ subunits of the PP2A trimeric holoenzymes, Aα-B-C and Aβ-B-C. One point mutation, Aα-E64D, was found in a human lung carcinoma. It renders Aα specifically defective in binding regulatory B’ subunits. Recently, we reported a knock-in mouse expressing Aα-E64D and an Aα knockout mouse. The mutant mice showed a 50–60% increase in the incidence of lung cancer induced by benzopyrene. Importantly, PP2A’s tumor suppressor activity depended on p53. These data provide the first direct evidence that PP2A is a tumor suppressor in mice. In addition, they suggest that PP2A is a tumor suppressor in humans. Here, we report that PP2A functions as a tumor suppressor in mice that develop lung cancer triggered by oncogenic K-ras. We discuss whether PP2A may function as a tumor suppressor in diverse tissues, with emphasis on endometrial and ovarian carcinomas, in which Aα mutations were detected at a high frequency. We propose suitable mouse models for examining whether PP2A functions as tumor suppressor in major growth-stimulatory signaling pathways, and we discuss the prospect of using the PP2A activator FTY720 as a drug against malignancies that are driven by these pathways.

Purification of PP2A Holoenzymes by Sequential Immunoprecipitation with Anti-Peptide Antibodies

Understanding the multiple functions of protein phosphatase 2A (PP2A) rests on elucidating the enzymatic properties of over 50 different possible forms of the PP2A holoenzyme. We describe a procedure for highly purifying each one of these forms. This procedure is based on coexpressing in 293 cells one scaffolding A subunit, one regulatory B subunit, and one catalytic C subunit, each tagged with a different sequence, and purifying the trimeric holoenzyme by three consecutive immunoprecipitations with antibodies against the tags. In a few hours and from a small number of cells, sufficient enzyme can be purified for enzymatic studies. Purification of six different holoenzymes in parallel can easily be accomplished.