Activation of Stat3 transcription factor by Herpesvirus saimiri STP-A oncoprotein - PubMed (original) (raw)

Activation of Stat3 transcription factor by Herpesvirus saimiri STP-A oncoprotein

Young-Hwa Chung et al. J Virol. 2004 Jun.

Abstract

The saimiri transforming protein (STP) oncogene of Herpesvirus saimiri subgroup A strain 11 (STP-A11) is not required for viral replication but is required for lymphoid cell immortalization in culture and lymphoma induction in primates. We previously showed that STP-A11 interacts with cellular Src kinase through its SH2 binding motif and that this interaction elicits Src signal transduction. Here we demonstrate that STP-A11 interacts with signal transducer and activator of transcription 3 (Stat3) independently of Src association and that the amino-terminal short proline-rich motif of STP-A11 and the central linker region of Stat3 are necessary for their interaction. STP-A11 formed a triple complex with Src kinase and Stat3 where Src kinase phosphorylated Stat3, resulting in the nuclear localization and transcriptional activation of Stat3. Consequently, the constitutively active Stat3 induced by STP-A11 elicited cellular signal transduction, which ultimately induced cell survival and proliferation upon serum deprivation. Furthermore, this activity was strongly correlated with the induction of Fos, cyclin D1, and Bcl-XL expression. These results demonstrate that STP-A11 independently targets two important cellular signaling molecules, Src and Stat3, and that these proteins cooperate efficiently to induce STP-A11-mediated transformation.

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Figures

FIG. 1.

FIG. 1.

Activation of Stat transcription activity by STP-A11 and Src expression. 293T cells were transfected with a Stat3 luciferase reporter vector (pLuc-TKS3) and an expression vector containing either wt STP-A11 or the Y115A mutant either alone or together with a Src expression vector. At 48 h posttransfection, cells were harvested, and luciferase activity was measured. Transfection efficiency was normalized with the β-galactosidase reporter vector pGK-βgal. Results are averages from three independent experiments. Error bars, standard deviations. Gels at the bottom show the expression of STP-A11, its mutant, and Src in whole-cell lysates.

FIG. 2.

FIG. 2.

Specific interaction of STP-A11 with Stat3. (A) STP-A11 interacts with Stat3 but not with Stat1 or Stat2. At 48 h after transfection with the carboxy-terminal HA-tagged STP-A11 expression vector alone (lanes 1 and 2) or together with the Stat3 expression vector (lane 3), 293T cells were lysed and used for immunoprecipitation (IP) with an anti-HA (αHA)antibody, followed by immunoblotting with an anti-Stat1, anti-Stat2, or anti-Stat3 antibody. To demonstrate the expression of endogenous Stat1 and Stat2 and transfected Stat3, whole-cell lysates (WCL) were used for immunoblotting with an anti-Stat1, anti-Stat2, or anti-Stat3 antibody and an anti-HA antibody. (B) Stat3 interacts with STP-A11 and is phosphorylated at Y705 by Src kinase associated with STP-A11. At 48 h after transfection with various expression vectors, 293T cells were lysed and used for immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti-Stat3 antibody, an anti-Stat3 Y705 phosphospecific antibody, or an anti-Src antibody. Lane 1, vector only; lane 2, Stat3; lane 3, STP-A11; lane 4, Stat3 plus STP-A11; lane 5, Stat3 plus STP-A11 Y115; lane 6, Stat3 plus STP-A11 plus Src; lane 7, Stat3Y705A plus STP-A11 plus Src.

FIG. 3.

FIG. 3.

Identification of the regions of Stat3 and STP-A11 necessary for their interaction. (A) The central linker region of Stat3 is sufficient for interacting with STP-A11. Bacterially purified GST or GST fusion proteins containing the amino-terminal region (ΔCST3), the central linker region, or the carboxyl-terminal region (ΔNST3) of Stat3 were mixed with lysates of 293T cells transfected with the HA-tagged STP-A11 expression vector. Polypeptides present in GST beads were used for immunoblotting (IB) with an anti-HA (α-HA) antibody. Similar amounts of each GST fusion protein were used in this assay (data not shown). ND, N-terminal domain; TAD, transcriptional activation domain. (B) The amino-terminal region of STP-A11 is necessary for interacting with Stat3. Bacterially purified GST or GST fusion proteins containing the amino-terminal regions of STP-A11 were mixed with lysates of 293T cells transfected with the Stat3 expression vector. Polypeptides present in GST beads were used for immunoblotting with an anti-Stat3 antibody. Similar amounts of each GST fusion protein were used in this assay (data not shown). TM, transmembrane domain. (C) The amino-terminal proline-rich motif of STP-A11 is necessary for interacting with Stat3. At 48 h after transfection with a Stat3 expression vector together with an expression vector containing the HA-tagged wt STP-A11 or its mutant, 293T cells were lysed and used for immunoprecipitation with an anti-HA antibody, followed by immunoblotting with an anti-Stat3 antibody.

FIG. 4.

FIG. 4.

Effect of the interaction of STP-A11 with Stat3 and Src on the efficient activation of Stat3 transcriptional activity. 293T cells were transfected with a Stat3 luciferase reporter vector (pLuc-TKS3) and an expression vector containing wt STP-A11 or the Y115A, D1, D2, D3, or Y115A/D1 mutant in the presence or absence of the Src expression vector. At 48 h posttransfection, cells were harvested for measurement of luciferase activity. Transfection efficiency was normalized with the β-galactosidase reporter vector pGK-βgal. Results are averages from three independent experiments. Error bars, standard deviations. Gels at the bottom show expression of STP-A11, its mutants, and Src in whole-cell lysates.

FIG. 5.

FIG. 5.

Nuclear localization of Stat3 upon STP-A11 and Src expression. Cos-1 cells were transfected with a Stat3 expression vector alone or together with pBud expression vectors carrying c-Src and/or wt or mutant STP-A11. At 48 h posttransfection, cells were fixed and reacted with anti-Stat3 (red), anti-STP-A11 (green), and/or anti-Src (green) antibodies, followed by incubation with an Alexa-488-conjugated anti-mouse immunoglobulin G antibody and/or an Alexa-568-conjugated anti-rabbit immunoglobulin G antibody. Immunofluorescence was examined with a Leica confocal microscope. Cells were visualized with Nomarski optics.

FIG. 6.

FIG. 6.

Contribution of STP-A11-induced Stat3 activation to enhanced cell growth. (A) Expression of wt STP-A and its mutants in stable NIH 3T3 cells. Lysates from NIH 3T3-puro (Vec), NIH 3T3-wt STP-A11 (WT), NIH 3T3-STP-A11 Y115A (Y115), and NIH 3T3-STP-A11 D1 (D1) cells were prepared and blotted with an anti-AU1 antibody for detection of STP-A. Tubulin was used for loading equivalent amount of proteins from each sample. (B) Cell growth rate upon serum deprivation. NIH 3T3-puro, NIH 3T3-wt STP-A11, NIH 3T3-STP-A11 Y115A, and NIH 3T3-STP-A11 D1 cells were incubated with 0.1, 0.2, or 1% serum. Cell proliferation rates of these NIH 3T3 cells were measured by a modified MTT assay. Results are averages from three independent experiments. Error bars, standard deviations. (C) Y705 phosphorylation of Stat3 and enhanced expression of Fos, cyclin D1, and Bcl-XL upon STP-A11 expression. Equivalent amounts of polypeptides from NIH 3T3-puro, NIH 3T3-wt STP-A11, NIH 3T3-STP-A11 Y115A, and NIH 3T3-STP-A11 D1 cells were used for immunoblotting with an anti-Stat3 antibody, an anti-Stat3 Y705 phosphospecific antibody, an anti-Fos antibody, an anti-cyclin D1 antibody, or an anti-Bcl-XL antibody.

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