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Serine 257 phosphorylation regulates association of polyomavirus middle T antigen with 14-3-3 proteins
X Culleré1, P Rose, U Thathamangalam
1Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Abstract:
Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.
Insights
Polyomavirus middle T antigen phosphorylation at serine 257 is key for 14-3-3 protein binding, impacting cell signaling. This site, however, is not essential for fibroblast transformation but affects tumor development.
Area of Science:
- Molecular biology
- Virology
- Cell biology
Background:
- Polyomavirus middle T antigen (MT) is a viral oncoprotein.
- MT interacts with cellular proteins involved in signaling and cell cycle regulation.
- 14-3-3 proteins are known regulators of cellular processes.
Purpose of the Study:
- To identify the specific serine residue phosphorylated in polyomavirus middle T antigen.
- To investigate the role of serine 257 phosphorylation in MT's interaction with 14-3-3 proteins.
- To assess the functional significance of serine 257 phosphorylation in MT-mediated cell transformation and tumor formation.
Main Methods:
- Partial proteolytic mapping and Edman degradation to identify phosphorylation sites.
- Site-directed mutagenesis to create serine 257 mutants (S257A, S257C).
- Isoelectric focusing and immunoprecipitation to analyze MT phosphorylation.
- Cell transformation assays (foci formation, colony formation in agar).
- In vivo tumor induction studies in animal models.
Main Results:
- Serine 257 was identified as a major site of phosphorylation on MT, occurring at near molar stoichiometry.
- Phosphorylation at serine 257 is essential for the association of MT with 14-3-3 proteins.
- Mutant MTs lacking serine 257 phosphorylation (S257A, S257C) retained the ability to transform fibroblasts in vitro.
- A virus expressing S257C MT showed a significant reduction in salivary gland tumor induction.
Conclusions:
- Serine 257 phosphorylation is critical for MT binding to 14-3-3 proteins.
- While not essential for initial cell transformation, serine 257 phosphorylation influences MT's oncogenic potential in vivo, specifically affecting salivary gland tumor formation.
- Further investigation is needed to elucidate the precise mechanism underlying the defect in salivary gland tumor induction for S257C MT.