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Catalytically inactive protein phosphatase 2A can bind to polyomavirus middle tumor antigen and support complex
1Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
Interaction between the heterodimeric form of protein phosphatase 2A (PP2A) and polyomavirus middle T antigen (MT) is required for the subsequent assembly of a transformation-competent MT complex. To investigate the role of PP2A catalytic activity in MT complex formation, we undertook a mutational analysis of the PP2A 36-kDa catalytic C subunit. Several residues likely to be involved in the dephosphorylation mechanism were identified and mutated. The resultant catalytically inactive C subunit mutants were then analyzed for their ability to associate with a cellular (B subunit) or a viral (MT) B-type subunit. Strikingly, while all of the inactive mutants were severely impaired in their interaction with B subunit, most of these mutants formed complexes with polyomavirus MT. These findings indicate a potential role for these catalytically important residues in complex formation with cellular B subunit, but not in complex formation with MT. Transformation-competent MT is known to associate with, and modulate the activity of, several cellular proteins, including pp60(c-src) family kinases. To determine whether association of MT with an active PP2A A-C heterodimer is necessary for subsequent association with pp60(c-src), catalytically inactive C subunits were examined for their ability to form complexes containing pp60(c-src) in MT-expressing cells. Two catalytically inactive C subunit mutants that efficiently formed complexes with MT also formed complexes that included an active pp60(c-src) kinase, demonstrating that PP2A activity is not essential in cis in MT complexes for subsequent pp60(c-src) association.
Insights
Protein phosphatase 2A (PP2A) catalytic activity is not essential for polyomavirus middle T antigen (MT) complex formation or subsequent pp60(c-src) kinase association. Inactive PP2A mutants bind MT but not the cellular B subunit.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Protein phosphatase 2A (PP2A) is a key regulator of cellular processes.
- Polyomavirus middle T antigen (MT) hijacks cellular machinery for transformation.
- PP2A interaction with MT is crucial for forming transformation-competent complexes.
Purpose of the Study:
- To investigate the role of PP2A catalytic activity in MT complex assembly.
- To determine if PP2A catalytic residues are essential for binding cellular or viral subunits.
- To assess if PP2A activity is required for pp60(c-src) kinase association with MT complexes.
Main Methods:
- Site-directed mutagenesis of the PP2A catalytic C subunit to create inactive mutants.
- Analysis of mutant PP2A subunit interaction with cellular B subunit and polyomavirus MT.
- Examination of complex formation including pp60(c-src) in MT-expressing cells.
Main Results:
- Catalytically inactive PP2A C subunit mutants showed impaired interaction with the cellular B subunit.
- Most inactive PP2A C subunit mutants efficiently formed complexes with polyomavirus MT.
- Two inactive PP2A C subunit mutants formed complexes with MT that included active pp60(c-src) kinase.
Conclusions:
- PP2A catalytic residues are important for binding the cellular B subunit but not MT.
- PP2A catalytic activity is not required in cis for pp60(c-src) association with MT complexes.
- This suggests PP2A's role in MT-driven transformation may involve mechanisms beyond direct catalytic activity within the MT complex.