In Vitro Phosphorylation of the Polyomavirus Major Capsid Protein VP1 on Serine 66 by Casein Kinase II

Maolin Li,Mary K Lyon,Robert L Garcea

doi:10.1074/jbc.270.43.26006

Abstract

Phosphorylation of the polyomavirus major capsid protein VP1 plays a role in virus assembly and may function in virus-cell recognition. Previous mapping of the in vivo phosphorylation sites on VP1 identified phosphorylation of threonine residues Thr-63 and Thr-156 (Li, M., and Garcea, R. L. (1994) J. Virol. 68, 320-327). Phosphoserine was detected in a tryptic phosphopeptide encompassing residues 58-78. Because of consensus casein kinase II (CK II) sites in this peptide, we examined the in vitro phosphorylation of the purified recombinant VP1 protein by CK II. CK II phosphorylated VP1 on serine, and the resulting tryptic phosphopeptide eluted in a 30-31 min high performance liquid chromatography fraction corresponding to residues 58-78. The VP1 tryptic phosphopeptide also co-migrated in two-dimensional peptide analysis with one of the tryptic peptides obtained from VP1 isolated after in vivo 32P labeling of virus-infected cells. A site-directed mutant VP1 protein, Ser-66 to Ala, was phosphorylated poorly by CK II in vitro. As determined by electron microscopy, all of the mutant proteins were isolated in pentameric form similar to the wild-type protein, although the Ala-66 pentamers had a tendency to self-assemble in vitro into tubular as well as capsid-like structures. These findings identify Ser-66 as a site of VP1 phosphorylation in vitro, and suggest that VP1 may serve as a substrate for CK II in vivo.

Highlights

The polyomavirus capsid is composed of 72 pentamers of the major coat protein VP1 (1)
The data presented in this report show that the polyomavirus major capsid protein VP1 is phosphorylated by Casein kinase II (CK II)
The Ala-66 mutation did not affect pentamer formation, changes in the Ser-66 residue may influence the formation of higher order aggregates of VP1. These data demonstrate that the polyomavirus major capsid protein VP1 is an in vitro substrate for phosphorylation by CK II

Summary

Introduction

The polyomavirus capsid is composed of 72 pentamers of the major coat protein VP1 (1). In vivo phosphate labeling of VP1 during host-range nontransforming mutant virus infections showed that phosphorylation of VP1 on both residues Thr-63 and Thr-156 was defective (9). This regulation could be controlled by activation of a cellular kinase, e.g. pp60c-src, or inactivation of a specific phosphatase, e.g. phosphatase 2A, both activities known to be associated with middle T-antigen (12–14). CK II is activated rapidly when cells are treated with certain growth factors such as serum, epidermal growth factor, and insulin-like growth factor (17– 19) These findings raise the possibility that CK II plays an important role in cellular activities related to cell growth and proliferation (20, 21). The data presented in this report show that the polyomavirus major capsid protein VP1 is phosphorylated by CK II

Methods

Results

Conclusion