Polyoma and SV40 proteins differentially regulate PP2A to activate distinct cellular signaling pathways involved in growth control

Abstract

Binding of Src family kinases to membrane-associated polyoma virus middle T-antigen (PyMT) can result in the phosphorylation of PyMT tyrosine 250, which serves as a docking site for the binding of Shc and subsequent activation of the Raf-MEK-ERK (MAP) kinase cascade. In a screen for PyMT variants that could not activate the ARF tumor suppressor, we isolated a cytoplasmic nontransforming mutant (MTA) that encoded a C-terminal truncated form of the PyMT protein. Surprisingly, MTA was able to strongly activate the MAP kinase pathway in the absence of Src family kinase and Shc binding. Interestingly, the polyoma small T-antigen (PyST), which shares with MTA both partial amino acid sequence homology and cellular location, also activates the MAP kinase cascade. Activation of the MAP kinase cascade by both MTA and PyST has been demonstrated to be PP2A-dependent. Neither MTA nor PyST activate the phosphorylation of AKT. The SV40 small T-antigen, which is similar to PyST in containing a J domain and in binding to the PP2A AC dimer, does not activate the MAP kinase cascade, but does stimulate phosphorylation of AKT in a PP2A-dependent manner. These findings highlight a novel role of PP2A in stimulating the MAP kinase cascade and indicate that the similar polyoma and SV40 small T-antigens influence PP2A to activate discrete cellular signaling pathways involved in growth control.

Fig. 1.

PyMT Shc and PI3-kinase signaling. A diagrammatic representation of Shc and PI3-kinase (PI3K) pathways stimulated by membrane-bound PyMT is shown. Association of Src family kinases with membrane-bound PyMT can result in the phosphorylation of PyMT tyrosines 250 (Y250) and 315 (Y315). Phosphorylation of PyMT Y250 can serve as a docking site for Shc, which recruits the Grb2 adaptor protein, which binds the Sos guanine nucleotide exchange factor resulting in the activation of Ras. Ras activation can lead to the activation of the Raf-Map kinase cascade, resulting in the phosphorylation of MEK and ERK. Phosphorylation of PyMT tyrosine 315 (Y315) results in the binding of the PI3-kinase regulatory p85 subunit and subsequent activation of PI3-kinase activity resulting in the phosphorylation of a number of cellular targets including AKT.

Fig. 2.

PyMT binding sites. A diagrammatic representation of the membrane localized PyMT wild type (Top), the cytoplasmic MTA (Middle), and the nuclear MT11 (Bottom) proteins are shown. The location of the binding sites for Hsc70 (J domain), PP2A A and C subunits (PP2A domain), Src family kinases, 14-3-3, Shc, PI3-kinase, and PLCγ, as well as the position of the membrane localization signal on the 421-aa PyMT wild-type protein, are shown. PyMT shares its N-terminal 191 aa (shown in gray) with PyST, which contains the J and PP2A domains. The MTA frameshift protein contains the first 337 aa of PyMT attached to 6 aa from a small ORF of the 3′ untranslated region of the PyST (stippled) at its C terminus (8). The MT11 frameshift protein contains the N-terminal 338 aa of PyMT attached to 85 aa of PyLT (containing nuclear localization sequence) (diagonal crosshatch) and 4 aa of vector sequence (horizontal crosshatch) at its C terminus (8).

Fig. 3.

MTA and MT11 have reduced MT-associated in vitro kinase activity. (Left) Wild-type PyMT proteins from PyE REF52 cells and PyMT DNp53REF52 cells, and the mutant PyMT proteins from MT11 and MTA REF52 cells after immunoprecipitation with the 762 antibody and Western blotting with the PyC antibody. (Right) Autoradiogram of the same PyMT immunoprecipitated species shown in Left, after incubation in the presence of γ-ATP to detect MT-associated kinase activity. The wild-type PyMT from PyE REF52 and PyMT DNp53REF52 cells contain an associated kinase activity, which is able to phosphorylate the PyMT protein, whereas little kinase activity (2–5%) is found associated with the mutant proteins from the MT11 and MTA cells.

Fig. 4.

Interaction of PyMT proteins with Shc and PP2A. Wild-type and mutant PyMT proteins were immunoprecipitated (IP) from PyMT DNp53REF52 (MT REF52) and MTA and MT11 REF52 cells with the 762 anti-Py antibody, gel fractionated, and probed with different antibodies. (Top) Various IPs probed with PyC anti-PY antibody and the locations of the different PyMT proteins. (Middle) Fractionated IP proteins probed with anti-Shc antibody. The location of the 66kd, 52kd, and 42kd Shc species are indicated. (Bottom) Fractionated IP proteins probed with a mixture of antibodies against the PP2A A and C subunits. The location of the PP2A 65kd A and 35kd C subunits are indicated. The location of the nonspecific cross-reactivity to the antibody heavy chain (Ab) used in the immunoprecipitation is indicated in Middle and Bottom.

Fig. 5.

MTA PyMT activates the MAP kinase pathway in a PP2A-dependent manner. (A) ERK and MEK, but not AKT, phosphorylation is stimulated in MTA REF52 cells. Gel fractionated extracts derived from REF52, DNp53REF52, PyMT transformed DNp53REF52, MT11, and MTA REF52 cells were probed with phospho-specific antibodies to AKT, ERK, and MEK. The total amount of ERK and MEK in the different extracts is shown. (B) Effect of inactivation of the PyMT Shc or PP2A-binding sites on the stimulation of phospho-ERK in MTA-expressing cells. REF52 cells were infected with retroviruses containing either the wild-type MTA sequence (MTA WT) or MTA sequence mutated in the PP2A binding domain (MTA PP2A-) or mutated in the Shc binding tyrosine 250 (MTA Y250F). ERK phosphorylation was measured in extracts from each infected cell population using phospho-specific antibodies after gel fractionation (Upper). The filter was reprobed with a PyMT antibody (Lower) to show expression levels of the different MTA proteins. (C) Phosphorylation of the Raf1 serine 388 activating site is stimulated in MTA cells. Phosphorylation of Raf1 at the activating serine 338 site (P-S338) was measured by using a phospho-specific antibody after gel fractionation of Raf1 immunoprecipitates from extracts of MTA, MT11, DNp53REF52, DNp53REF52 cells treated with EGF and PyMT transformed DNp53REF52 cells. In Lower, total Raf1 in each sample is shown as a loading control.

Fig. 6.

PyST activates the MAP kinase pathway whereas SVST activates the PI3K pathway. (A) PyST stimulates ERK phosphorylation in a PP2A-dependent manner. REF52 cells were infected with retroviruses containing the MTA sequence or PyST wild type, PyST mutated in the PP2A (PyST PP2A-) or J (PyST J Domain-) domain, or no insert (empty). Extracts from the infected cells were gel fractionated and assessed for phosphorylated AKT (P-AKT) or phosphorylated ERK (P-ERK) using phospho-specific antibodies. The amount of total ERK in each extract is shown. (B) PyST stimulates PP2A-dependent ERK phosphorylation, whereas SVST stimulates PP2A-dependent AKT phosphorylation. REF52 cells were infected with retroviruses containing either wild-type PyST or wild-type SVST or PyST or SVST mutated in either the PP2A or J domain or empty vector. Extracts of the infected cells were gel fractionated and probed with phospho-specific antibodies as (A) above. The amount of total ERK in each extract is shown. (C) PyST stimulates ERK phosphorylation and SVST stimulates AKT phosphorylation in both rat and human cells. Gel fractionated extracts from either rat REF52 cells or human BJ fibroblasts infected with retroviruses containing either no insert (empty) or PyST or SVST were probed with phospho-specific antibodies against ERK and AKT.