Signaling from polyomavirus middle T and small T defines different roles for protein phosphatase 2A

Abstract

Polyomavirus causes a broad spectrum of tumors as the result of the action of its early proteins. This work compares signaling from middle T antigen (MT), the major transforming protein, to that from small T antigen (ST). The abilities of MT mutants to promote cell cycle progression in serum-starved NIH 3T3 cells were compared. Transformation-defective mutants lacking association with SHC or with phosphatidylinositol 3-kinase (PI3-K) retained the ability to induce DNA synthesis as measured by bromodeoxyuridine incorporation. Only when both interactions were lost in the Y250F/Y315F double mutant was MT inactive. ST promoted cell cycle progression in a manner dependent on its binding of protein phosphatase 2A (PP2A). Since the Y250F/Y315F MT mutant was wild type for PP2A binding yet unable to promote cell cycle progression, while ST was capable of promoting cell cycle progression, these experiments revealed a functional difference in MT and ST signaling via PP2A. Assays testing the abilities of MT and ST to induce the c-fos promoter and to activate c-jun kinase led to the same conclusion. ST, but not Y250F/Y315F MT, was able to activate the c-fos promoter through its interaction with PP2A. In contrast, MT, but not ST, was able to activate c-jun kinase by virtue of its interaction with PP2A.

FIG. 1

Schematic diagram of MT and its known associated proteins. The shaded area at the N terminus represents the 191 amino acids shared with the 195-amino-acid ST. The shaded area near the C terminus represents the membrane attachment site.

FIG. 2

BrdU incorporation assays with wild-type MT, insAL107/del205-214, and NG59. Growing ATCC NIH 3T3 cells were transiently transfected with CMV–β-galactosidase and 0.625 μg of CMV-neoBam (control) or 0.625, 0.125, or 0.025 μg of CMV-MT (WT) or 0.625 μg of insAL107/del205-214 or NG59, placed in 0.2% CS-DMEM at 5 h posttransfection, and labeled with BrdU for 8 h starting at 40 h after transfection. Transfected cells were detected by staining for β-galactosidase; BrdU incorporation was measured by immunostaining. % BrdU, percentage of transfected cells (β-galactosidase positive) also staining for BrdU. Error bars represent standard deviations of the means.

FIG. 3

(A) BrdU incorporation assays with wild-type MT, del205-214, insAL107, Y250F, Y315F, Y250F/Y315F, and ST. By using the same protocol described in the legend for Fig. 2, growing NIH 3T3 cells were transiently transfected with CMV–β-galactosidase and with 0.625 μg of either CMV-neo (control), CMV-MT (WT), del205-214, insAL107, Y250F, Y315F, Y250F/Y315F, ST, or STinsAL107, placed into 0.2% serum, and labeled with BrdU. Error bars represent standard errors of the means. (B) Extracts of cells transfected with equal amounts of wild-type or mutant MT DNAs, cells transfected with 1/5 the amount of wild-type MT, or cells transfected with wild-type or mutant STs were resolved in SDS-PAGE gels. The gels were then analyzed using Western blotting with the antibody PN116.

FIG. 4

³⁵S-labeled MT immunoprecipitates from insAL107-, wild type-, and Y250F/Y315F-transfected cells were resolved by IEF and SDS-PAGE. The positions of heat shock 70 (hs 70), the 63- and 36-kDa subunits of PP2A, and a fragment of MT lacking the C terminus are shown.

FIG. 5

Wild-type MT, insAL107, Y250F, Y315F, Y250F/Y315F, Py1387T, insAL107/Py1387T, ST, and STins107AL in the activation of the fos promoter in starved cells. Growing NIH 3T3 cells were transfected with pfos-CAT reporter plasmid and either CMV-neo (control), 5, 1, or 0.2 μg of CMV-MT (WT), or 5 μg of insAL107, Y250F, Y315F, Y250F/Y315F, Py1387T, insAL107/Py1387T, ST, or STins107AL. After transfection cells were placed into 0.2% CS-DMEM. CAT assays were performed as described in Materials and Methods. Error bars represent standard deviations of the means.

FIG. 6

MT activates jnk. (Top) Jnk activation. 293T cells were transfected with FLAG-JNK1 and control or MT as indicated. At 48 h posttransfection, JNK1 was harvested with anti-FLAG antibody. JNK expression was determined with an anti-FLAG blot. JNK activity was assayed by using GST–c-JUN and [γ-³²P]ATP as described in Materials and Methods. (Bottom) Comparison of the abilities of wild type, insAL107 MT, Y250F/Y315F MT, and ST to activate jnk in 293T cells. Specific activities (S.A.) were determined by dividing activity determined by PhosphorImager by the level of JNK determined by densitometry of an anti-FLAG blot.