Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip

Abstract

Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis.

Figure 1

Tip activates SRF independent of MAPK. Jurkat T cells were transiently transfected with expression plasmids coding for the viral oncoprotein Tip of HVS-C488. Empty vector served as a negative control. For inhibition of MAPK signaling, cells were left untreated or treated with U0126 (25 μM), PD0325901 (1 μM) or solvent (DMSO) for 40 h. As positive controls, vector-transfected cell were stimulated with PMA (20 ng/ml) in the absence or presence of the MEK inhibitors for 15 h. (A) Activity of cotransfected pSRE-Luc displayed as mean values and standard deviations of an assay performed in triplicates, which is representative for three independent experiments. Statistical significance for correlated samples, p < 0.05 (*); p > 0.05 (ns). (B) Activity of cotransfected p3D.A-Luc summarized from six independent experiments. Statistical significance for independent samples, p < 0.001 (***); p > 0.05 (ns). (C) Verification of MEK inhibition by detection of ERK1/2 phosphorylation and expression as well as control of Tip expression by immunoblot analyses with whole cell lysates obtained 48 h post transfection

Figure 2

Effects of actin dynamics and the cofactor MAL on SRF activity. (A) p3D.A-Luc activity of Jurkat T cells transiently transfected with expression plasmids coding for actin wt, actinR62D, MAL and MALΔNΔB1 alone or in combination with a Tip expression construct. (B) Representative expression controls carried out by immunoblot analysis with whole cell lysates 48 h post transfection. Detection of Hsp90α/β served as a loading control. (C) p3D.A-Luc activity of vector- and Tip-transfected cells treated with Latrunculin B (1 μM) and Cytochalasin D (1 μM) for 24 h. The graphs in (A) and (C) display the mean values and standard deviation of three independent experiments. Statistical significance, p > 0.05 (ns); p < 0.05 (*); p < 0.01 (**)

Figure 3

Influence of dominant-negative GTPases on SRF activation. Jurkat T cells were transiently transfected with expression plasmids coding for myc-Rac1-T17N (RacT17N), RhoA-T19N (RhoT19N) and H-Ras-S17N (RasS17N) alone or in combination with a Tip expression construct. (A) Activity of cotransfected p3D.A-Luc displayed as mean values and standard deviation of three independent experiments. Statistical significance, p > 0.05 (ns); p < 0.01 (**). (B) Representative expression control carried out by immunoblot analysis. Detection of β-tubulin served as a loading control

Figure 4

The role of Src-kinase interactions of Tip for SRF activation. (A) Jurkat T cells were transiently transfected with expression plasmids coding for wild-type Tip or its mutants TipΔCSKH, TipmSH3B, TipΔCSKHmSH3, TipY114F, TipY127F and TipY155F. Empty vector (pEF1) served as negative control. Activation of cotransfected p3D.A-Luc displayed as mean values and standard deviation of three independent experiments. Statistical significance, p > 0.05 (ns); p < 0.05 (*). (B) Expression control for Tip and its mutants in total lysates obtained from the cells described in (A). (C) Vector and Tip constructs were cotransfected with p3D.A-Luc into Jurkat T cells, and SFK activity was blocked by PP2 treatment (10 μM) for 40 h. The graph summarizes the reporter activities of six independent experiments. Statistical significance, p > 0.05 (ns); p < 0.001 (***). (D) Total cellular protein tyrosine phosphorylation (pY) and Tip expression in the cells described in (C). Detection of Hsp90α/β served as a loading control

Figure 5

SRF activation by TCR engagement and by constitutively active Rac1 or RhoA. (A) Jurkat T cells were transiently transfected with p3D.A-Luc and vector or expression plasmids coding for Tip and stimulated for 14 h in 6-well plates coated with antibodies directed against CD3 and CD28 (αCD3/CD28). Reporter activation is summarized as mean values and standard error of five independent experiments. Statistical significance, p > 0.05 (ns); p < 0.01 (**). (B) Representative immunoblots for the cells used in (A) displaying ERK1/2 phosphorylation and expression as well as Tip expression. (C) Jurkat T cells were transiently transfected with p3D.A-Luc and vector. Cells were treated with PP2 (10 μM, 40 h), PD0325901 (1 μM, 40 h) or Latrunculin B (1 μM, 24 h) and stimulated for 14 h in 6-well plates coated with antibodies directed against CD3 and CD28 (αCD3/CD28). Reporter activation is summarized as mean values and standard deviation of three independent experiments. Statistical significance, p < 0.05 (*); p < 0.01 (**); p < 0.001 (***). (D) Representative immunoblots for the cells described in (C) displaying ERK1/2 phosphorylation and expression. (E) Vector and expression plasmids coding for Tip, myc-Rac1-G12V (RacG12V) or myc-RhoA-Q63L (RhoQ63L) were cotransfected with p3D.A-Luc. The graph summarizes data of five independent experiments. Statistical significance, p < 0.001 (***). (F) Representative immunoblots displaying myc-Rac1-G12V, myc-RhoA-Q63L and Tip expression. Detection of β-tubulin served as a loading control

Figure 6

Model for Tip-induced SRF activation. The membrane-associated viral oncoprotein Tip engages and activates the Src-family kinase Lck. This interaction is required for STAT3 phosphorylation and for MAL:SRF activation. Signaling flux from Tip:Lck to the RhoGTPases Rac remains to be established (dashed line). Rac promotes a decrease in the G-actin/F-actin ratio and thereby enables nuclear translocation of MAL and coactivation of SRF-dependent gene expression. Tip:Lck is not linked to Ras, MEK1/2, or ERK1/2 and subsequent TCF activation