Induction of cyclin D1 by simian virus 40 small tumor antigen - PubMed (original) (raw)

Induction of cyclin D1 by simian virus 40 small tumor antigen

G Watanabe et al. Proc Natl Acad Sci U S A. 1996.

Abstract

Cell-cycle progression is mediated by a co-ordinated interaction between cyclin-dependent kinases and their target proteins including the pRB and E2F/DP-1 complexes. Immunoneutralization and antisense experiments have established that the abundance of cyclin D1, a regulatory subunit of the cyclin-dependent kinases, may be rate-limiting for G1 phase progression of the cell cycle. Simian virus 40 (SV40) small tumor (t) antigen is capable of promoting G1 phase progression and augments substantially the efficiency of SV40 transformation through several distinct domains. In these studies, small t antigen stimulated cyclin D1 promoter activity 7-fold, primarily through an AP-1 binding site at -954 with additional contributions from a CRE site at -57. The cyclin D1 AP-1 and CRE sites were sufficient for activation by small t antigen when linked to an heterologous promoter. Point mutations of small t antigen between residues 97-103 that reduced PP2A binding were partially defective in the induction of the cyclin D1 promoter. These mutations also reduced activation of MEK1 and two distinct members of the mitogen-activated protein kinase family, the ERKs (extracellular signal regulated kinases) and the SAPKs (stress-activated protein kinases), in transfected cells. Dominant negative mutants of either MEK1, ERK or SEK1, reduced small t-dependent induction of the cyclin D1 promoter. SV40 small t induction of the cyclin D1 promoter involves both the ERK and SAPK pathways that together may contribute to the proliferative and transformation enhancing activity of small t antigen.

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Figures

Figure 1

The cyclin D1 promoter is activated by SV40 small t. (A) The −1745CD1LUC reporter was transfected with either the control plasmid pw2t _dl_888 or the small t expression vector. For the dose response, a representative example from three separate experiments is shown for increasing amounts of transfected expression plasmid normalized to the effect of vector alone. For the effect of pw2t (n = 17 separate transfections) or the CMV small t expression plasmid (n = 7), the data are expressed as mean ± SEM. (B) The −1745CD1LUC reporter was transfected with the SV40 small t expression vector in the cell lines indicated. The data are shown expressed as mean ± SEM with the number of experiments shown as N.

Figure 2

The −953 region of the cyclin D1 promoter is required for activation by small t. (A) A series of 5′ promoter deletions of the cyclin D1 promoter were transfected into JEG-3 cells with the small t expression plasmid. The data are the mean fold induction ± SEM by pw2t determined by comparison with cells transfected with the “cassette” _dl8_88. Data is for N separate transfections as indicated in parenthesis. (B) The cyclin D1(AP-1)TKLUC reporter was transfected with small t into JEG-3 cells. Data are normalized for the effect of the pw2t vector (shown as 1). Comparison was made with the effect of small t on the synthetic reporters, CD1(E2F)TKLUC, CD1(CRE)TKLUC (encoding the sequences resembling a putative CRE binding site at −57), and the synthetic AP-1 site reporter p3TPLUX. The data are shown expressed as mean ± SEM.

Figure 3

The domains of small t required for activation of the cyclin D1 promoter. (A) The −1745CD1LUC reporter was cotransfected with expression vectors encoding either wild-type or mutant SV40 small t proteins into JEG-3 cells. The data shown were adjusted for the effect of expression vector cassette lacking the cDNA on −1745CD1LUC reporter activity. The mean data (± SEM) of_n_ separate transfections (in parenthesis) are shown. (Inset) Western blot of JEG-3 cells transfected with the wild-type small t plasmid, the dl888, or the PP2A binding defective mutant t103 probed with the cyclin D1 antibody. (B) As overexpression of ERK was previously shown to augment activity of the cyclin D1 promoter in JEG-3 cells (13), the −1745CD1LUC reporter was cotransfected with the expression vector encoding wild-type p42ERK in conjunction with either the wild-type or mutant small t proteins into JEG-3 cells. The data were normalized to 1 for the effect of p42ERK on the −1745CD1LUC reporter. The mean data (± SEM) of 7 to 8 separate transfections are shown.

Figure 4

SV40 small t stimulates ERK, MEK, and JNK activity in JEG-3 cells. JEG-3 cells were transfected with a plasmid that expressed wild-type small t antigen (pw2t), a small t mutant with reduced PP2A binding (t103), a small t mutant that does not affect PP2A binding (t4345), or no small t antigen (dl_888). Cells were extracted 48 hr after transfection and kinase assays were done on immunoprecipitates obtained with antibodies against ERK (A), MEK1 (B), or JNK1 (C). Incorporation of [γ-32P] into substrates myelin basic protein (A), kinase-inactive ERK (B), and GST c-Jun (C) was quantitated by acid precipitation and liquid scintillation counting as described. The data are expressed as a ratio between small t-induced kinase activity and that of the_dl888 negative control.

Figure 5

MEK, ERK, and SAPK are required for full induction of the cyclin D1 promoter by small t antigen. JEG-3 cells were transfected with either (A) the activating MEK mutants, MEKΔN3, and MEKΔN3 S218E/S222D or (B) wild type small t and the expression plasmids encoding either dominant negative mutants of MEK (MEKC), p42ERK (MAPKi), SEK, or the MKP-1 expression vector encoding a dual specificity Thr/Tyr phosphatase.

Figure 6

Small t augments c-Jun and CREB transactivation in JEG-3 cells. (A) The GAL4-c-Jun construct (1.2 μg) was transfected with the small t expression plasmids (600 ng) and the (UAS)5E1BTATALUC reporter (4.8 μg) in JEG-3 cells. The mean data (± SEM) of n separate transfections are shown. (Comparison is shown normalized for the effect of_dl_888). (B Inset) An electrophoretic mobility-shift assay was performed with the cyclin D1 CRE/ATF site using nuclear extracts from JEG-3 cells, with the addition of specific antibodies as described. The arrow indicates the primary complex formed and the asterisk indicates the supershifted complex formed with the CREB/CREM antibody HM93. (B) The GAL4-CREB construct (1.2 μg) was transfected with the small t expression plasmid (600 ng) and (UAS)5E1BTATALUC (4.8 μg) either alone or with the addition of an expression plasmid for wild-type or mutant protein kinase A catalytic subunit (C).

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Induction of cyclin D1 by simian virus 40 small tumor antigen - PubMed (original) (raw)