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ERK signaling pathway
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Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signal-regulated kinase and activates MAPK/ERK signaling pathway Hyungdong Kim a, Jun Hyeong Jang a, Yul Eum Song a, b, Taegun Seo a, * a b
Department of Life Science, Dongguk University-Seoul, Goyang, 10326, South Korea Department of Infectious Disease, St. Jude Children’s Research Hospital, Memphis, TN, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2019 Accepted 5 November 2019 Available online xxx
Open reading frame 36 (ORF36) of Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a serine/ threonine-type viral protein kinase (vPK). Previous studies have examined the functions of KSHV vPK; however, its role in the activation of extracellular signal-regulated kinase (ERK1/2) has not yet been described to date. Using HEK 293 cell lines, we performed a human phospho-kinase array analysis to screen for MAPK signaling pathways kinases that are activated by KSHV vPK. In addition, we investigated the regulator protein phosphorylation of up/downstream ERK1/2 pathway; nuclear translocation of phosphorylated ERK1/2; and regulation of transcription factor, inflammatory cytokine, and pro-/antiapoptotic factor by KSHV vPK transfection. Here, we demonstrated that KSHV vPK activates ERK1/2 signaling pathway and plays an important role in the activation of MAPK/ERK signaling pathway. © 2019 Elsevier Inc. All rights reserved.
Keywords: KSHV Viral protein kinase ORF36 MAPK/ERK signaling pathway ERK
1. Introduction Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway plays a critical role in cellular signal cascade and intercellular responses. ERK1/2 has been the major signaling cascade trigger for MAPK pathway, and numerous studies have shown that ERK1/2 is involved in the transduction of diverse classes of extracellular signals (e.g., growth factors, hormones) to the nucleus [1,2]. In the MAPK/ERK signaling cascade, ERK is activated by the upstream Ras/Raf/Mek pathway [3,4]. Activated ERK1/ 2 (p-ERK1/2) translocates to the nucleus and activates diverse transcription factors (TFs), pro-inflammatory cytokines (ICs), growth factors (GFs), and pro-/anti-apoptotic factors [3,5,6]. Activated ERK1/2, in turn, regulates the expression of various intracellular factors [7,8] and controls various cellular processes (e.g., cellular proliferation, differentiation, and growth).
Abbreviations: KSHV, Kaposi’s sarcoma-associated herpesvirus; ORF, open reading frame; vPK, viral protein kinase; MAPK, Mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; TFs, transcription factors; ICs, pro-inflammatory cytokines; GFs, growth factors. * Corresponding author. Department of Life Science, Dongguk University-Seoul, 32 Dongguk-ro, Ilsandong-gu, Goyang, 10326, South Korea. E-mail address: [email protected] (T. Seo).
Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is an oncogenic double-stranded DNA virus of the gammaherpesvirus subfamily [9]. KSHV is the etiologic agent for several human cancers, including multicentric Castleman’s disease, primary effusion lymphoma (PEL), and Kaposi’s sarcoma (KS) [10,11]. Moreover, reactivated KSHV stimulates multiple MAPK pathways [12,13]. The viral protein kinase (vPK) produced by herpesvirus is a promising therapeutic target because of its ability to enhance host cellular protein synthesis and viral lytic replications [14]. KSHV vPK, encoded by ORF 36, belongs to the KSHV late gene [15] and is auto-phosphorylated on serine/threonine residues. Previous studies have shown that autophosphorylation of KSHV vPK blocks the ability of vPK to phosphorylate C-Jun N-terminal kinase (JNK) [15,16]. To understand the mechanisms underlying gammaherpesvirus lytic cycle replication and gammaherpesvirus-mediated tumor genesis, it is necessary to study the relationship between gammaherpesvirus vPK and transduction of signal pathway in host cells. Here, we demonstrate that whether KSHV vPK transduces MAPK/ERK signaling pathway via ERK1/2 activation and translocation of ERK1/2 to cell nucleus and consequently regulates intercellular responses. To address this question, we introduced KSHV vPK (KSHV ORF36), a kinase-dead mutant of KHSV vPK containing alanine instead of lysine at position 108 (KSHV ORF36
https://doi.org/10.1016/j.bbrc.2019.11.038 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: H. Kim et al., Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signalregulated kinase and activates MAPK/ERK signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.11.038
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K108A), or an empty vector into HEK 293, HEK 293T, and BJAB cells. Furthermore, to clarify whether other gammaherpesvirus vPK also activate MAPK/ERK signal pathway and translocate p-ERK1/2 to cell nucleus, we introduced Murine gammaherpesvirus-68 (MHV-68) vPK (MHV-68 ORF36), a kinase-dead mutant of MHV-68 vPK containing glutamine instead of lysine at position 107 (MHV-68 ORF36 K107Q), or an empty vector in the control group. As a member of the gammaherpesvirus subfamily, KSHV and MHV-68 share a significant degree of similarity [17]. MHV-68 is genetically and biologically related to our target KSHV vPK [18]; MHV-68 established latent and lytic infection stage in mice, and previous studies have shown that MHV-68 ORF36 vPK can also activate the MAPK/ERK pathway [19,20]. Experiments conducted in the current study demonstrated that gammaherpesvirus vPK activates MAPK/ERK signaling pathway via phosphorylation of ERK1/2 proteins. Furthermore, KSHV/MHV-68 vPK mediated translocation of p-ERK1/2 into the cell nucleus, which leads to regulation of diverse cellular responses. 2. Materials and methods 2.1. Cell culture and plasmid HEK 293T and HEK 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. EBV and KSHV-negative human Burkitt lymphoma B cells (BJABs) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. An HA-tagged wild-type KSHV vPK (pcDNA3-HA KSHV ORF36), a kinase-dead mutant of KSHV vPK (pcDNA3-HA-KSHV ORF36 K108A), and an empty vector were generated using pcDNA3. The Flag-tagged wild-type MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36), kinase-dead mutant of MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36 K107Q), and empty vector were employed as controls for performing subsequent comparisons. 2.2. Transfection HEK 293 and HEK 293T cells were transfected using calcium phosphate method. The vector plasmid, wild-type KSHV vPK (pcDNA3-HA-KSHV ORF36), kinase-dead mutant of KHSV vPK (pcDNA3-HA-KSHV ORF36 K108A), and MHV-68 vPK (p-FLAG-Tag ORF36) or kinase-dead mutant of MHV-68 vPK (p-FLAG-Tag ORF36 K107Q) were introduced into BJAB cells via electroporation by implementing the following procedure: 5 106 cells were washed twice with serum free RPMI 1640 media and washed twice with ice-cold PBS. Electroporation was performed using Gene Pulser (Bio-Rad Laboratories, Hercules, CA) at 210 V, 955 mF [21,22]. 2.3. Proteome profiling HEK 293 cells were transfected with an empty vector, or KSHV vPK (pcDNA3-HA-KSHV ORF36) using Polyexpress reagent transfection system (Excel gene, Gaithersburg, MD, USA); 48 h post transfection, the cells were washed with phosphate-buffered saline (PBS) and collected. Cell components were extracted using Human phospho-kinase antibody array kit (Proteome Profiler; R&D Systems, Minneapolis, USA) and reacted individually with two membranes (A and B). After an 18-h reaction, the membranes were washed and treated with an antibody cocktail as primary antibody and StreptavidineHRP as secondary antibody according to the manufacturer’s instructions. Detection was performed using an Enhanced Chemiluminescent Substrate (ECL) (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence signals were detected on Kodak X-ray film.
2.4. Western blot analysis Transfected BJAB and HEK 293T cells were washed in PBS and lysed with RIPA lysis buffer [50 mM Tris-HCl (pH 8), 1% Nonidet P40, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS þ 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Cell lysates were mixed with 5 SDS buffer and boiled at 100 C for 5 min, followed by electrophoresis in 10% gradient SDS-polyacrylamide gel at 120 V for 90 min and transferred onto a nitrocellulose blotting membrane (Amersham-GE healthcare, Little Chalfont, Buckinghamshire, UK) at 300 mA over a 1-h period. The membranes were blocked for 1 h using 5% skimmed milk, followed by appropriate primary antibodies. After washing five times in TBST buffer [TBS containing 0.1% v/v Tween 20, 120 mM NaCl, 20 mM Tris-HCl (pH 7.5)], the membranes were incubated with HRP-conjugated secondary antibody for 1 h. The membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The following antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA): p-Mek1/2 (Ser 217/221) Rabbit, p-ERK (T202/Y204) Rabbit, p-90RSK (Ser380) Rabbit, p-MSK1 (Thr581) Rabbit, and p-SAPK/JNK (Thr183/Tyr185) Rabbit. The secondary antibodies were anti-rabbit IgG, HRP-linked anti-mouse IgG, and HRP-linked antibody from Cell Signaling Technology, Inc. 2.5. Immunofluorescence assay HEK 293T cells were grown on 8-well cell culture slides (SPL Life Science, Seoul, Korea), and each well was respectively transfected with KSHV empty vector, KSHV vPK, kinase-dead mutant of KSHV vPK, MHV-68 empty vector, MHV-68 vPK, or kinase-dead mutant of MHV-68 vPK; 24 h transfection, cells were fixed with 3.7% formaldehyde for 30 min, followed by permeabilization with 1 mL PBS containing 0.2% Triton X-100 (PBST) for 1 h. After washing with icecold PBS three times, cells were blocked for 1 h at room temperature with 1% bovine serum albumin (BSA) prepared in PBS. Subsequently, cells were incubated with anti-p-ERK (p44/42) antibody diluted in 1% BSA-containing PBST for 24 h at 4 C, followed by washing of cells with PBST up to three times and staining with appropriate secondary antibodies diluted in 1% BSA-containing PBST for 1 h. After washing two more times with PBS, fluorescence images of cells were obtained using confocal microscopy (Nikon TE2000, Japan). 2.6. Reverse transcription: PCR and real-time PCR Thirty-six hours after transfection, the total RNA content was extracted with TRIzol (Invitrogen-Life Technologies, Carlsbad, CA) by following the standard procedure to measure mRNA levels of TFs. cDNAs were generated using high-capacity cDNA reverse €rster City, CA, USA) and transcription kits (Applied Biosystems, Fo random primers, mRNA expression was measured using real-time PCR (Rotor Gene, Qiagen, Hilden, Germany). RT-PCR was performed using KAPA SYBR FAST qPCR kit (KAPA Biosystems Inc., Woburn, MA) under the following conditions: pre-denaturation at 95 C for 3 min, followed by 40 repeat cycles of 95 C for 3 s and 60 C for 20 s. The obtained CT values were analyzed using 2-delta CT method, and GAPDH mRNA was used to normalize gene expression. Data are presented as mean ± SD of three independent experiments. 3. Results 3.1. KSHV vPK activates MAPK/ERK signaling pathway To identify whether KSHV vPK can phosphorylate regulator
Please cite this article as: H. Kim et al., Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signalregulated kinase and activates MAPK/ERK signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.11.038
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proteins and to determine which signaling pathway is activated by KSHV vPK transfection, we performed protein phosphorylation screening using human phospho-kinase antibody arrays [23,24]. HEK 293 cells were transfected with expression constructs for HAtagged wild-type KSHV vPK (pcDNA3-HA-KSHV ORF36) or empty vector and analyzed against the array. The result shown that ERK1/ 2 (Fig. 1, box 1), MEK1/2 (Fig. 1, box 2), and RSK1/2 (Fig. 1, box 4) were phosphorylated owing to KSHV vPK transfection as opposed to transfection with the vector control; however, the extent of MSK1/2 (Fig. 1, box 3) and JNK signaling (Fig. 1, box 5) was slightly decreased. Because ERK1/2, MEK1/2, and RSK1/2 are known to be the main signaling cascade regulators in the RAS/RAF/MEK pathway, this result is a key evidence for the activation of MAPK/ ERK pathway and regulation of cellular responses by KSHV vPK.
vPK, kinase-dead mutant KSHV vPK, or vector control, and for comparative analysis, BJAB cells were also electroporated with wild-type MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36), kinase-dead mutant MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36 K107Q), or vector control. As shown in Fig. 2B, Western blot analyses revealed that p-ERK1/2 levels were significantly higher in both KSHV vPK- and MHV-68 vPK-electroporated BJAB cells than those in mock or kinase-dead mutant-electroporated cells, without causing a corresponding change in total ERK levels. Our data suggest that KHSV vPK induces phosphorylation of MEK1/2, 90RSK, and ERK1/2 and activates the MAPK/ERK pathway; additionally, genetically related MHV-69 vPK promotes the phosphorylation of ERK1/2.
3.2. KSHV and MHV-68 vPK phosphorylates ERK1/2 and activates MAPK/ERK pathway
Previous studies have reported that activated ERK1/2 translocates to the nucleus [26,27]. To compare the ability of KSHV vPK and MHV-68 vPK- mediated phosphorylated ERK1/2 with normal p-ERK1/2, we performed immunofluorescence analyses. To monitor the subcellular localization of p-ERK1/2 in cells, we introduced wild-type vPK, kinase-dead mutant vPK or empty vector of KSHV vPK or MHV-68, and incubated transfected cells with anti-p-ERK1/2 and FITC-labeled primary and secondary antibodies, respectively. As shown in Fig. 3, confocal microscopy revealed the presence of p-ERK1/2 in the nucleus of HEK 293T cells transfected with wild-type KSHV vPK or MHV-68 vPK (Fig. 3 E and N) but not in empty vector-transfected cells (Fig. 3 B and K) or cells transfected with kinase-dead KSHV mutant or MHV-68 vPK mutants (Fig. 3H and Q). This is demonstrated more clearly in the merged images of FITC- (anti-p-ERK1/2) and DAPI-stained cell nuclei (Fig. 3C, F, I, L, O and R), which show an overlap of p-ERK and nuclear signals in the cells transfected with wild-type KSHV vPK or MHV-68 vPK (Fig. 3 F and O, indicated by white arrow), but not in the cells transfected with the corresponding kinase-dead mutants [KSHV vPK-K108A and MHV-68 vPK-K107Q, respectively (Fig. 3 I and R)] or vector control (Fig. 3C and L); thus, these findings strengthen the results of the assay conducted using human phospho-kinase antibody array kit (Fig. 1) and western blot analysis (Fig. 2). Collectively, these results suggest that nuclear ERK1/2 in cells which were either transfected with KSHV vPK or MHV-68 vPK gets phosphorylated and p-ERK1/2 is translocated into the nucleus.
Next, using western blot analyses, we confirmed that KSHV vPK activated the up- and down-stream of the ERK1/2 signaling pathway (ERK1/2, MEK 1/2, MSK1/2, and RSK1/2). Fig. 2A demonstrates a dramatic increase in the activation of the MEK pathway, as evidenced by elevated baseline levels of phosphorylated MEK1/2 (p-MEK), the immediate upstream ERK-activating kinases. In contrast, phosphorylation of SAPK/JNK, members of another MAPK family, was not affected by KSHV vPK. To further confirm ERK1/2 pathway activation, we examined the phosphorylation status of the two immediate downstream targets of activated ERK1/2, namely MSK1 and RSK1, by measuring phosphorylated MSK1 (p-MSK1) (Thr581) and phosphorylated 90RSK (p-90RSK) (Ser380) levels via Western blot assays. Both p-MSK1 (Thr581) and p-90RSK (Ser380) levels were increased (Fig. 2A). Collectively, these results indicate that activation of ERK1/2 by KSHV vPK is associated with the activation of the upstream effector, MEK1/2, and downstream effectors, MSK1 and 90RSK. To extend this analysis to other related gammaherpesvirus vPKs, we subsequently investigated whether ERK1/ 2 was also activated by vPK from Murine gammaherpesvirus 68 (MHV-68). MHV-68, as a member of gamma-2-herpesvirus [18,19], had a close genetic and biologic relationship with KSHV; furthermore, vPK of MHV-68 (MHV-68 ORF 36) shares homology with that of KSHV [25]. BJAB cells were electroporated with wild-type KSHV
3.3. Gammaherpesvirus vPK induces p-ERK1/2 translocation to the nucleus
3.4. vPK mediated activated ERK1/2 regulates the expression of diverse intracellular factor
Fig. 1. KSHV vPK activates MAPK/ERK pathway in HEK 293 cells. Whole-cell lysates were analyzed using a human phospho-kinase antibody array to detect the status of phosphorylation in HEK 293 cells which were transfected with (A) empty vector or (B) wild-type KSHV vPK (pcDNA3-HA KSHV ORF 36). Boxes from 1 to 5 represent activated proteins isolated using the human phospho-kinase antibody array, and circle represents positive control.
A previous study reported that activated ERK1/2 regulates the expression of various TF genes [11,12,28]. Owing to the findings of phosphokinase array assay, western blotting, and immunofluorescence analysis that wild-type vPK from KSHV and MHV-68 mediate the activation of the ERK1/2, we hypothesized that ERK1/2 phosphorylation by gammaherpesvirus vPK can induce diverse intracellular response in wild-type vPK-transfected cells. To determine the influence of wild-type vPK-mediated regulation of ERK1/2 gene expression, we measured changes in the mRNA expression level of TFs, pro-inflammatory cytokine (IC), and pro-/anti-apoptotic factors, which have previously been reported to be affected by ERK1/2 activation [29e32], by real-time PCR. Changes in mRNA expression levels of vPK (KHSV and MHV-68 vPK) were compared with mock or kinase-dead mutants (KSHV vPK K108A, MHV-68 vPK K107Q). As shown in Fig. 4, mRNA expression level of ATF2, ATF3, C-Jun, C-Myc, IL-8, and Bcl-2 were higher in wild-type vPK-transfected cells than in mock- or kinase-dead mutant vPK-transfected cells. RT-PCR results show that mRNA expression is regulated by ERK1/2 activation; these results suggest that the wild-type vPK of KSHV and MHV-68
Please cite this article as: H. Kim et al., Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signalregulated kinase and activates MAPK/ERK signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.11.038
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Fig. 2. KSHV vPK and MHV-68 vPK induce ERK phosphorylation and activate MAPK/ERK signal pathway. (A) HEK 293T cells were transfected with KSHV vPK (pcDNA3-HA KSHV ORF 36), KSHV vPK-dead mutant (pcDNA3-HA KSHV ORF36 K108A), and empty vector. The activity of MEK1/2, ERK1/2, MSK1, 90RSK, and SAPK/JNK were analyzed via Western blotting, which was performed using phospho-specific antibody; HA-tagged mock. b-actin served as a loading control. The results shown are representative of those from three independent experiments. (B) The empty vector for KSHV vPK, wild-type KSHV vPK (pcDNA3-HA KSHV ORF36), kinase-dead mutant KSHV vPK (pcDNA3-HA KSHV ORF36-K108A), empty vector, wild-type MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36), or kinase-dead mutant MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36-K107Q) were transfected via electroporation individually into BJAB cells. Western blot was analyzed using p-ERK antibody, and b-actin served as a loading control. The results shown are representative of those from three independent experiments.
Fig. 3. Nuclear localization of p-ERK in KSHV/MHV-68 vPK-transfected HEK 293T cells. In HEK 293T cells transfected with mock, wild-type vPK (pcDNA3-HA-KSHV ORF36, pFLAG-Tag MHV-68 ORF36), or kinase-dead mutant (pcDNA3-HA-KSHV vPK ORF36 K108A, p-FLAG-Tag MHV-68 ORF36 K107Q) were subjected to indirect immunofluorescence with anti-p-ERK primary antibody and FITC-conjugated secondary antibody. The nuclei were stained with 4, 6-diamino-2-phenylindole (blue); 24 h after transfection, cells were observed using a confocal laser scanning microscope. DAPI (blue): (A), (D), (G), (J), (M), and (P); FITC (Anti-p-ERK) (green): (B), (E), (H), (K), (N), and (Q); Merge: (C), (F), (I), (L), (O), and (R). The white arrow of (F) and (O) represent the overlapped signal of FITC and DAPI.
activates ERK1/2 and promotes translocation of p-ERK1/2 to the nucleus, which in turn results in the regulation of cellular responses. 4. Discussion The protein kinases play a crucial role in the cell signaling network, and dysregulation of protein kinase activity or presence of
mutant protein kinases have been reported to be a trigger for diverse disease such as diabetes [33], Alzheimer’s disease [34], and various types of cancers [35,36]. Among the various protein kinases, ERK1/2 is known to be a critical regulator of cellular survival, proliferation, migration, anti-apoptosis [37], and pro-apoptosis [38]. In the MAPK/ERK pathway, activation of MEK1 causes phosphorylation of threonine and tyrosine residues of ERK1/2, and p-ERK1/2 subsequently translocates to the nucleus and activates TFs, pro-ICs,
Please cite this article as: H. Kim et al., Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signalregulated kinase and activates MAPK/ERK signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.11.038
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Fig. 4. Change in mRNA level of transcription factors caused by vPK activity. Total cDNA was synthesized via reverse transcription, and mRNA levels of ATF2, ATF3, IL-8, C-Myc, CJun, and Bcl-2 were measured via real-time PCR. The mRNA levels of each transcription factor were compared among empty vector, wild-type vPK or kinase-dead mutant vPK. Results are expressed as mean ± SD. Assays for each transcription factor were performed in triplicate. *P, 0.05; **P, 0.01; ***P, 0.001.
pro-/anti-apoptotic factors, and GFs. Earlier studies have shown the importance of vPK and consistent ERK1/2 pathway activation in KSHV lytic replication or KS lesion developments. The results of previous studies are consistent with the present study, i.e., the expression of ICs and GFs mediates reactivation of KSHV and development of KS lesion [39];, the consistent transcriptional activation of AP-1 response elements is regulated by ERK and JNK pathways during KS lesion development [40]; and Raf/MEK/ERK signaling pathway activation plays a key role in reactivation of KHSV latency [41]. In concordance with the results of other studies, KSHV vPK, which inhibits the Wnt signaling pathway, exerts a suppressive effect on host cell cycle progression, interacts with RNA helicase A, and regulates host gene expression during the lytic cycle [20,42]. However, despite decades of studies, the relationship between KSHV vPK and ERK1/2 activation has still not been fully understood. In our study, we used the human phospho-kinase antibody array kit and performed western blot analysis to examine the regulator proteins phosphorylation, which was induced by KSHV vPK. The phosphorylation of upstream (MEK1) and downstream (90RSK) of ERK1/2 signaling pathway were significantly increased compare to mock or kinase-dead vPK transfected cell line. Contrastingly, the phosphorylation of SAPK/JNK or MSK1 was not affected by KSHV vPK. We suggest that KSHV vPK participates in the regulation of MAPK/ERK signaling pathway via ERK1/2 activation (Fig. 2A). We also monitored the effect of MHV-68 vPK, a genetically related gammaherpesvirus vPK, on BJAB cell line for evaluating the phosphorylating ability of ERK1/2; furthermore, similar results with regard to vPK from two different viruses revealed that gammaherpesvirus vPK has the ability to activate MAPK/ERK pathway (Fig. 2B). A previous study revealed that in the MAPK/ERK signaling pathway, p-ERK1/2 translocates to the nucleus and regulates TFs, ICs, and pro-/anti-apoptotic factors [4,32,43], which lead to cellular proliferation and impact survival and apoptotic functions [38]. To determine whether gammaherpesvirus vPK mediated the translocation of p-ERK1/2 to the nucleus, we performed immunofluorescence analysis (Fig. 3), and the results suggest that KSHV/MHV-
68 vPK-dependent ERK1/2 phosphorylation may represent an initiating event for the activation of MAPK/ERK signaling pathway. We performed a series of RT-PCRs to verify that the translocated pERK1/2 regulates TFs, ICs, and pro-/anti-apoptotic factors and confirmed that p-ERK1/2 regulates diverse TFs including ATF2, ATF3, C-Jun, and C-Myc as well as influences the level of proinflammatory cytokines or pro-/anti-apoptotic factors such as IL8, and Bcl-2. Taken together, the present set of findings suggest that KSHV/MHV-68 vPK activates MAPK/ERK signaling pathway, translocates p-ERK1/2 to the nucleus, and regulates TFs, GFs, proICs, or pro-/anti-apoptotic factors during KSHV lytic cycle in order to stabilize KSHV replication and KS lesion development. Our study clearly revealed the relationship between KSHV/MHV-68 vPK and MAPK/ERK signaling pathway activation; however, many questions still remain unanswered. Further investigations regarding vPKmediated MAPK/ERK signaling transduction and its effects on activation or dysregulation of GFs or other intercellular factors in host cells are warranted for understanding gammaherpesvirusassociated lytic replication cycle and lesion development. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF2017R1A2B4009448). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.038 References [1] Y.-p. Liu, Y.-n. Tan, Z.-l. Wang, L. Zeng, Z.-x. Lu, L.-l. Li, W. Luo, M. Tang, Y. Cao, Phosphorylation and nuclear translocation of STAT3 regulated by the EpsteinBarr virus latent membrane protein 1 in nasopharyngeal carcinoma, Int. J. Mol. Med. 21 (2008) 153e162. [2] N. Parikh, R.L. Shuck, T.-A. Nguyen, A. Herron, L.A. Donehower, Mouse tissues
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Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signal-regulated kinase and activates MAPK/ERK signaling pathway Hyungdong Kim a, Jun Hyeong Jang a, Yul Eum Song a, b, Taegun Seo a, * a b
Department of Life Science, Dongguk University-Seoul, Goyang, 10326, South Korea Department of Infectious Disease, St. Jude Children’s Research Hospital, Memphis, TN, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2019 Accepted 5 November 2019 Available online xxx
Open reading frame 36 (ORF36) of Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a serine/ threonine-type viral protein kinase (vPK). Previous studies have examined the functions of KSHV vPK; however, its role in the activation of extracellular signal-regulated kinase (ERK1/2) has not yet been described to date. Using HEK 293 cell lines, we performed a human phospho-kinase array analysis to screen for MAPK signaling pathways kinases that are activated by KSHV vPK. In addition, we investigated the regulator protein phosphorylation of up/downstream ERK1/2 pathway; nuclear translocation of phosphorylated ERK1/2; and regulation of transcription factor, inflammatory cytokine, and pro-/antiapoptotic factor by KSHV vPK transfection. Here, we demonstrated that KSHV vPK activates ERK1/2 signaling pathway and plays an important role in the activation of MAPK/ERK signaling pathway. © 2019 Elsevier Inc. All rights reserved.
Keywords: KSHV Viral protein kinase ORF36 MAPK/ERK signaling pathway ERK
1. Introduction Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway plays a critical role in cellular signal cascade and intercellular responses. ERK1/2 has been the major signaling cascade trigger for MAPK pathway, and numerous studies have shown that ERK1/2 is involved in the transduction of diverse classes of extracellular signals (e.g., growth factors, hormones) to the nucleus [1,2]. In the MAPK/ERK signaling cascade, ERK is activated by the upstream Ras/Raf/Mek pathway [3,4]. Activated ERK1/ 2 (p-ERK1/2) translocates to the nucleus and activates diverse transcription factors (TFs), pro-inflammatory cytokines (ICs), growth factors (GFs), and pro-/anti-apoptotic factors [3,5,6]. Activated ERK1/2, in turn, regulates the expression of various intracellular factors [7,8] and controls various cellular processes (e.g., cellular proliferation, differentiation, and growth).
Abbreviations: KSHV, Kaposi’s sarcoma-associated herpesvirus; ORF, open reading frame; vPK, viral protein kinase; MAPK, Mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; TFs, transcription factors; ICs, pro-inflammatory cytokines; GFs, growth factors. * Corresponding author. Department of Life Science, Dongguk University-Seoul, 32 Dongguk-ro, Ilsandong-gu, Goyang, 10326, South Korea. E-mail address: [email protected] (T. Seo).
Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is an oncogenic double-stranded DNA virus of the gammaherpesvirus subfamily [9]. KSHV is the etiologic agent for several human cancers, including multicentric Castleman’s disease, primary effusion lymphoma (PEL), and Kaposi’s sarcoma (KS) [10,11]. Moreover, reactivated KSHV stimulates multiple MAPK pathways [12,13]. The viral protein kinase (vPK) produced by herpesvirus is a promising therapeutic target because of its ability to enhance host cellular protein synthesis and viral lytic replications [14]. KSHV vPK, encoded by ORF 36, belongs to the KSHV late gene [15] and is auto-phosphorylated on serine/threonine residues. Previous studies have shown that autophosphorylation of KSHV vPK blocks the ability of vPK to phosphorylate C-Jun N-terminal kinase (JNK) [15,16]. To understand the mechanisms underlying gammaherpesvirus lytic cycle replication and gammaherpesvirus-mediated tumor genesis, it is necessary to study the relationship between gammaherpesvirus vPK and transduction of signal pathway in host cells. Here, we demonstrate that whether KSHV vPK transduces MAPK/ERK signaling pathway via ERK1/2 activation and translocation of ERK1/2 to cell nucleus and consequently regulates intercellular responses. To address this question, we introduced KSHV vPK (KSHV ORF36), a kinase-dead mutant of KHSV vPK containing alanine instead of lysine at position 108 (KSHV ORF36
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Please cite this article as: H. Kim et al., Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signalregulated kinase and activates MAPK/ERK signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.11.038
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H. Kim et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
K108A), or an empty vector into HEK 293, HEK 293T, and BJAB cells. Furthermore, to clarify whether other gammaherpesvirus vPK also activate MAPK/ERK signal pathway and translocate p-ERK1/2 to cell nucleus, we introduced Murine gammaherpesvirus-68 (MHV-68) vPK (MHV-68 ORF36), a kinase-dead mutant of MHV-68 vPK containing glutamine instead of lysine at position 107 (MHV-68 ORF36 K107Q), or an empty vector in the control group. As a member of the gammaherpesvirus subfamily, KSHV and MHV-68 share a significant degree of similarity [17]. MHV-68 is genetically and biologically related to our target KSHV vPK [18]; MHV-68 established latent and lytic infection stage in mice, and previous studies have shown that MHV-68 ORF36 vPK can also activate the MAPK/ERK pathway [19,20]. Experiments conducted in the current study demonstrated that gammaherpesvirus vPK activates MAPK/ERK signaling pathway via phosphorylation of ERK1/2 proteins. Furthermore, KSHV/MHV-68 vPK mediated translocation of p-ERK1/2 into the cell nucleus, which leads to regulation of diverse cellular responses. 2. Materials and methods 2.1. Cell culture and plasmid HEK 293T and HEK 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. EBV and KSHV-negative human Burkitt lymphoma B cells (BJABs) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. An HA-tagged wild-type KSHV vPK (pcDNA3-HA KSHV ORF36), a kinase-dead mutant of KSHV vPK (pcDNA3-HA-KSHV ORF36 K108A), and an empty vector were generated using pcDNA3. The Flag-tagged wild-type MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36), kinase-dead mutant of MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36 K107Q), and empty vector were employed as controls for performing subsequent comparisons. 2.2. Transfection HEK 293 and HEK 293T cells were transfected using calcium phosphate method. The vector plasmid, wild-type KSHV vPK (pcDNA3-HA-KSHV ORF36), kinase-dead mutant of KHSV vPK (pcDNA3-HA-KSHV ORF36 K108A), and MHV-68 vPK (p-FLAG-Tag ORF36) or kinase-dead mutant of MHV-68 vPK (p-FLAG-Tag ORF36 K107Q) were introduced into BJAB cells via electroporation by implementing the following procedure: 5 106 cells were washed twice with serum free RPMI 1640 media and washed twice with ice-cold PBS. Electroporation was performed using Gene Pulser (Bio-Rad Laboratories, Hercules, CA) at 210 V, 955 mF [21,22]. 2.3. Proteome profiling HEK 293 cells were transfected with an empty vector, or KSHV vPK (pcDNA3-HA-KSHV ORF36) using Polyexpress reagent transfection system (Excel gene, Gaithersburg, MD, USA); 48 h post transfection, the cells were washed with phosphate-buffered saline (PBS) and collected. Cell components were extracted using Human phospho-kinase antibody array kit (Proteome Profiler; R&D Systems, Minneapolis, USA) and reacted individually with two membranes (A and B). After an 18-h reaction, the membranes were washed and treated with an antibody cocktail as primary antibody and StreptavidineHRP as secondary antibody according to the manufacturer’s instructions. Detection was performed using an Enhanced Chemiluminescent Substrate (ECL) (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence signals were detected on Kodak X-ray film.
2.4. Western blot analysis Transfected BJAB and HEK 293T cells were washed in PBS and lysed with RIPA lysis buffer [50 mM Tris-HCl (pH 8), 1% Nonidet P40, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS þ 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Cell lysates were mixed with 5 SDS buffer and boiled at 100 C for 5 min, followed by electrophoresis in 10% gradient SDS-polyacrylamide gel at 120 V for 90 min and transferred onto a nitrocellulose blotting membrane (Amersham-GE healthcare, Little Chalfont, Buckinghamshire, UK) at 300 mA over a 1-h period. The membranes were blocked for 1 h using 5% skimmed milk, followed by appropriate primary antibodies. After washing five times in TBST buffer [TBS containing 0.1% v/v Tween 20, 120 mM NaCl, 20 mM Tris-HCl (pH 7.5)], the membranes were incubated with HRP-conjugated secondary antibody for 1 h. The membranes were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The following antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA): p-Mek1/2 (Ser 217/221) Rabbit, p-ERK (T202/Y204) Rabbit, p-90RSK (Ser380) Rabbit, p-MSK1 (Thr581) Rabbit, and p-SAPK/JNK (Thr183/Tyr185) Rabbit. The secondary antibodies were anti-rabbit IgG, HRP-linked anti-mouse IgG, and HRP-linked antibody from Cell Signaling Technology, Inc. 2.5. Immunofluorescence assay HEK 293T cells were grown on 8-well cell culture slides (SPL Life Science, Seoul, Korea), and each well was respectively transfected with KSHV empty vector, KSHV vPK, kinase-dead mutant of KSHV vPK, MHV-68 empty vector, MHV-68 vPK, or kinase-dead mutant of MHV-68 vPK; 24 h transfection, cells were fixed with 3.7% formaldehyde for 30 min, followed by permeabilization with 1 mL PBS containing 0.2% Triton X-100 (PBST) for 1 h. After washing with icecold PBS three times, cells were blocked for 1 h at room temperature with 1% bovine serum albumin (BSA) prepared in PBS. Subsequently, cells were incubated with anti-p-ERK (p44/42) antibody diluted in 1% BSA-containing PBST for 24 h at 4 C, followed by washing of cells with PBST up to three times and staining with appropriate secondary antibodies diluted in 1% BSA-containing PBST for 1 h. After washing two more times with PBS, fluorescence images of cells were obtained using confocal microscopy (Nikon TE2000, Japan). 2.6. Reverse transcription: PCR and real-time PCR Thirty-six hours after transfection, the total RNA content was extracted with TRIzol (Invitrogen-Life Technologies, Carlsbad, CA) by following the standard procedure to measure mRNA levels of TFs. cDNAs were generated using high-capacity cDNA reverse €rster City, CA, USA) and transcription kits (Applied Biosystems, Fo random primers, mRNA expression was measured using real-time PCR (Rotor Gene, Qiagen, Hilden, Germany). RT-PCR was performed using KAPA SYBR FAST qPCR kit (KAPA Biosystems Inc., Woburn, MA) under the following conditions: pre-denaturation at 95 C for 3 min, followed by 40 repeat cycles of 95 C for 3 s and 60 C for 20 s. The obtained CT values were analyzed using 2-delta CT method, and GAPDH mRNA was used to normalize gene expression. Data are presented as mean ± SD of three independent experiments. 3. Results 3.1. KSHV vPK activates MAPK/ERK signaling pathway To identify whether KSHV vPK can phosphorylate regulator
Please cite this article as: H. Kim et al., Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signalregulated kinase and activates MAPK/ERK signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.11.038
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proteins and to determine which signaling pathway is activated by KSHV vPK transfection, we performed protein phosphorylation screening using human phospho-kinase antibody arrays [23,24]. HEK 293 cells were transfected with expression constructs for HAtagged wild-type KSHV vPK (pcDNA3-HA-KSHV ORF36) or empty vector and analyzed against the array. The result shown that ERK1/ 2 (Fig. 1, box 1), MEK1/2 (Fig. 1, box 2), and RSK1/2 (Fig. 1, box 4) were phosphorylated owing to KSHV vPK transfection as opposed to transfection with the vector control; however, the extent of MSK1/2 (Fig. 1, box 3) and JNK signaling (Fig. 1, box 5) was slightly decreased. Because ERK1/2, MEK1/2, and RSK1/2 are known to be the main signaling cascade regulators in the RAS/RAF/MEK pathway, this result is a key evidence for the activation of MAPK/ ERK pathway and regulation of cellular responses by KSHV vPK.
vPK, kinase-dead mutant KSHV vPK, or vector control, and for comparative analysis, BJAB cells were also electroporated with wild-type MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36), kinase-dead mutant MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36 K107Q), or vector control. As shown in Fig. 2B, Western blot analyses revealed that p-ERK1/2 levels were significantly higher in both KSHV vPK- and MHV-68 vPK-electroporated BJAB cells than those in mock or kinase-dead mutant-electroporated cells, without causing a corresponding change in total ERK levels. Our data suggest that KHSV vPK induces phosphorylation of MEK1/2, 90RSK, and ERK1/2 and activates the MAPK/ERK pathway; additionally, genetically related MHV-69 vPK promotes the phosphorylation of ERK1/2.
3.2. KSHV and MHV-68 vPK phosphorylates ERK1/2 and activates MAPK/ERK pathway
Previous studies have reported that activated ERK1/2 translocates to the nucleus [26,27]. To compare the ability of KSHV vPK and MHV-68 vPK- mediated phosphorylated ERK1/2 with normal p-ERK1/2, we performed immunofluorescence analyses. To monitor the subcellular localization of p-ERK1/2 in cells, we introduced wild-type vPK, kinase-dead mutant vPK or empty vector of KSHV vPK or MHV-68, and incubated transfected cells with anti-p-ERK1/2 and FITC-labeled primary and secondary antibodies, respectively. As shown in Fig. 3, confocal microscopy revealed the presence of p-ERK1/2 in the nucleus of HEK 293T cells transfected with wild-type KSHV vPK or MHV-68 vPK (Fig. 3 E and N) but not in empty vector-transfected cells (Fig. 3 B and K) or cells transfected with kinase-dead KSHV mutant or MHV-68 vPK mutants (Fig. 3H and Q). This is demonstrated more clearly in the merged images of FITC- (anti-p-ERK1/2) and DAPI-stained cell nuclei (Fig. 3C, F, I, L, O and R), which show an overlap of p-ERK and nuclear signals in the cells transfected with wild-type KSHV vPK or MHV-68 vPK (Fig. 3 F and O, indicated by white arrow), but not in the cells transfected with the corresponding kinase-dead mutants [KSHV vPK-K108A and MHV-68 vPK-K107Q, respectively (Fig. 3 I and R)] or vector control (Fig. 3C and L); thus, these findings strengthen the results of the assay conducted using human phospho-kinase antibody array kit (Fig. 1) and western blot analysis (Fig. 2). Collectively, these results suggest that nuclear ERK1/2 in cells which were either transfected with KSHV vPK or MHV-68 vPK gets phosphorylated and p-ERK1/2 is translocated into the nucleus.
Next, using western blot analyses, we confirmed that KSHV vPK activated the up- and down-stream of the ERK1/2 signaling pathway (ERK1/2, MEK 1/2, MSK1/2, and RSK1/2). Fig. 2A demonstrates a dramatic increase in the activation of the MEK pathway, as evidenced by elevated baseline levels of phosphorylated MEK1/2 (p-MEK), the immediate upstream ERK-activating kinases. In contrast, phosphorylation of SAPK/JNK, members of another MAPK family, was not affected by KSHV vPK. To further confirm ERK1/2 pathway activation, we examined the phosphorylation status of the two immediate downstream targets of activated ERK1/2, namely MSK1 and RSK1, by measuring phosphorylated MSK1 (p-MSK1) (Thr581) and phosphorylated 90RSK (p-90RSK) (Ser380) levels via Western blot assays. Both p-MSK1 (Thr581) and p-90RSK (Ser380) levels were increased (Fig. 2A). Collectively, these results indicate that activation of ERK1/2 by KSHV vPK is associated with the activation of the upstream effector, MEK1/2, and downstream effectors, MSK1 and 90RSK. To extend this analysis to other related gammaherpesvirus vPKs, we subsequently investigated whether ERK1/ 2 was also activated by vPK from Murine gammaherpesvirus 68 (MHV-68). MHV-68, as a member of gamma-2-herpesvirus [18,19], had a close genetic and biologic relationship with KSHV; furthermore, vPK of MHV-68 (MHV-68 ORF 36) shares homology with that of KSHV [25]. BJAB cells were electroporated with wild-type KSHV
3.3. Gammaherpesvirus vPK induces p-ERK1/2 translocation to the nucleus
3.4. vPK mediated activated ERK1/2 regulates the expression of diverse intracellular factor
Fig. 1. KSHV vPK activates MAPK/ERK pathway in HEK 293 cells. Whole-cell lysates were analyzed using a human phospho-kinase antibody array to detect the status of phosphorylation in HEK 293 cells which were transfected with (A) empty vector or (B) wild-type KSHV vPK (pcDNA3-HA KSHV ORF 36). Boxes from 1 to 5 represent activated proteins isolated using the human phospho-kinase antibody array, and circle represents positive control.
A previous study reported that activated ERK1/2 regulates the expression of various TF genes [11,12,28]. Owing to the findings of phosphokinase array assay, western blotting, and immunofluorescence analysis that wild-type vPK from KSHV and MHV-68 mediate the activation of the ERK1/2, we hypothesized that ERK1/2 phosphorylation by gammaherpesvirus vPK can induce diverse intracellular response in wild-type vPK-transfected cells. To determine the influence of wild-type vPK-mediated regulation of ERK1/2 gene expression, we measured changes in the mRNA expression level of TFs, pro-inflammatory cytokine (IC), and pro-/anti-apoptotic factors, which have previously been reported to be affected by ERK1/2 activation [29e32], by real-time PCR. Changes in mRNA expression levels of vPK (KHSV and MHV-68 vPK) were compared with mock or kinase-dead mutants (KSHV vPK K108A, MHV-68 vPK K107Q). As shown in Fig. 4, mRNA expression level of ATF2, ATF3, C-Jun, C-Myc, IL-8, and Bcl-2 were higher in wild-type vPK-transfected cells than in mock- or kinase-dead mutant vPK-transfected cells. RT-PCR results show that mRNA expression is regulated by ERK1/2 activation; these results suggest that the wild-type vPK of KSHV and MHV-68
Please cite this article as: H. Kim et al., Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signalregulated kinase and activates MAPK/ERK signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.11.038
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Fig. 2. KSHV vPK and MHV-68 vPK induce ERK phosphorylation and activate MAPK/ERK signal pathway. (A) HEK 293T cells were transfected with KSHV vPK (pcDNA3-HA KSHV ORF 36), KSHV vPK-dead mutant (pcDNA3-HA KSHV ORF36 K108A), and empty vector. The activity of MEK1/2, ERK1/2, MSK1, 90RSK, and SAPK/JNK were analyzed via Western blotting, which was performed using phospho-specific antibody; HA-tagged mock. b-actin served as a loading control. The results shown are representative of those from three independent experiments. (B) The empty vector for KSHV vPK, wild-type KSHV vPK (pcDNA3-HA KSHV ORF36), kinase-dead mutant KSHV vPK (pcDNA3-HA KSHV ORF36-K108A), empty vector, wild-type MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36), or kinase-dead mutant MHV-68 vPK (p-FLAG-Tag MHV-68 ORF36-K107Q) were transfected via electroporation individually into BJAB cells. Western blot was analyzed using p-ERK antibody, and b-actin served as a loading control. The results shown are representative of those from three independent experiments.
Fig. 3. Nuclear localization of p-ERK in KSHV/MHV-68 vPK-transfected HEK 293T cells. In HEK 293T cells transfected with mock, wild-type vPK (pcDNA3-HA-KSHV ORF36, pFLAG-Tag MHV-68 ORF36), or kinase-dead mutant (pcDNA3-HA-KSHV vPK ORF36 K108A, p-FLAG-Tag MHV-68 ORF36 K107Q) were subjected to indirect immunofluorescence with anti-p-ERK primary antibody and FITC-conjugated secondary antibody. The nuclei were stained with 4, 6-diamino-2-phenylindole (blue); 24 h after transfection, cells were observed using a confocal laser scanning microscope. DAPI (blue): (A), (D), (G), (J), (M), and (P); FITC (Anti-p-ERK) (green): (B), (E), (H), (K), (N), and (Q); Merge: (C), (F), (I), (L), (O), and (R). The white arrow of (F) and (O) represent the overlapped signal of FITC and DAPI.
activates ERK1/2 and promotes translocation of p-ERK1/2 to the nucleus, which in turn results in the regulation of cellular responses. 4. Discussion The protein kinases play a crucial role in the cell signaling network, and dysregulation of protein kinase activity or presence of
mutant protein kinases have been reported to be a trigger for diverse disease such as diabetes [33], Alzheimer’s disease [34], and various types of cancers [35,36]. Among the various protein kinases, ERK1/2 is known to be a critical regulator of cellular survival, proliferation, migration, anti-apoptosis [37], and pro-apoptosis [38]. In the MAPK/ERK pathway, activation of MEK1 causes phosphorylation of threonine and tyrosine residues of ERK1/2, and p-ERK1/2 subsequently translocates to the nucleus and activates TFs, pro-ICs,
Please cite this article as: H. Kim et al., Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signalregulated kinase and activates MAPK/ERK signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.11.038
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Fig. 4. Change in mRNA level of transcription factors caused by vPK activity. Total cDNA was synthesized via reverse transcription, and mRNA levels of ATF2, ATF3, IL-8, C-Myc, CJun, and Bcl-2 were measured via real-time PCR. The mRNA levels of each transcription factor were compared among empty vector, wild-type vPK or kinase-dead mutant vPK. Results are expressed as mean ± SD. Assays for each transcription factor were performed in triplicate. *P, 0.05; **P, 0.01; ***P, 0.001.
pro-/anti-apoptotic factors, and GFs. Earlier studies have shown the importance of vPK and consistent ERK1/2 pathway activation in KSHV lytic replication or KS lesion developments. The results of previous studies are consistent with the present study, i.e., the expression of ICs and GFs mediates reactivation of KSHV and development of KS lesion [39];, the consistent transcriptional activation of AP-1 response elements is regulated by ERK and JNK pathways during KS lesion development [40]; and Raf/MEK/ERK signaling pathway activation plays a key role in reactivation of KHSV latency [41]. In concordance with the results of other studies, KSHV vPK, which inhibits the Wnt signaling pathway, exerts a suppressive effect on host cell cycle progression, interacts with RNA helicase A, and regulates host gene expression during the lytic cycle [20,42]. However, despite decades of studies, the relationship between KSHV vPK and ERK1/2 activation has still not been fully understood. In our study, we used the human phospho-kinase antibody array kit and performed western blot analysis to examine the regulator proteins phosphorylation, which was induced by KSHV vPK. The phosphorylation of upstream (MEK1) and downstream (90RSK) of ERK1/2 signaling pathway were significantly increased compare to mock or kinase-dead vPK transfected cell line. Contrastingly, the phosphorylation of SAPK/JNK or MSK1 was not affected by KSHV vPK. We suggest that KSHV vPK participates in the regulation of MAPK/ERK signaling pathway via ERK1/2 activation (Fig. 2A). We also monitored the effect of MHV-68 vPK, a genetically related gammaherpesvirus vPK, on BJAB cell line for evaluating the phosphorylating ability of ERK1/2; furthermore, similar results with regard to vPK from two different viruses revealed that gammaherpesvirus vPK has the ability to activate MAPK/ERK pathway (Fig. 2B). A previous study revealed that in the MAPK/ERK signaling pathway, p-ERK1/2 translocates to the nucleus and regulates TFs, ICs, and pro-/anti-apoptotic factors [4,32,43], which lead to cellular proliferation and impact survival and apoptotic functions [38]. To determine whether gammaherpesvirus vPK mediated the translocation of p-ERK1/2 to the nucleus, we performed immunofluorescence analysis (Fig. 3), and the results suggest that KSHV/MHV-
68 vPK-dependent ERK1/2 phosphorylation may represent an initiating event for the activation of MAPK/ERK signaling pathway. We performed a series of RT-PCRs to verify that the translocated pERK1/2 regulates TFs, ICs, and pro-/anti-apoptotic factors and confirmed that p-ERK1/2 regulates diverse TFs including ATF2, ATF3, C-Jun, and C-Myc as well as influences the level of proinflammatory cytokines or pro-/anti-apoptotic factors such as IL8, and Bcl-2. Taken together, the present set of findings suggest that KSHV/MHV-68 vPK activates MAPK/ERK signaling pathway, translocates p-ERK1/2 to the nucleus, and regulates TFs, GFs, proICs, or pro-/anti-apoptotic factors during KSHV lytic cycle in order to stabilize KSHV replication and KS lesion development. Our study clearly revealed the relationship between KSHV/MHV-68 vPK and MAPK/ERK signaling pathway activation; however, many questions still remain unanswered. Further investigations regarding vPKmediated MAPK/ERK signaling transduction and its effects on activation or dysregulation of GFs or other intercellular factors in host cells are warranted for understanding gammaherpesvirusassociated lytic replication cycle and lesion development. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF2017R1A2B4009448). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.038 References [1] Y.-p. Liu, Y.-n. Tan, Z.-l. Wang, L. Zeng, Z.-x. Lu, L.-l. Li, W. Luo, M. Tang, Y. Cao, Phosphorylation and nuclear translocation of STAT3 regulated by the EpsteinBarr virus latent membrane protein 1 in nasopharyngeal carcinoma, Int. J. Mol. Med. 21 (2008) 153e162. [2] N. Parikh, R.L. Shuck, T.-A. Nguyen, A. Herron, L.A. Donehower, Mouse tissues
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Please cite this article as: H. Kim et al., Kaposi’s sarcoma-associated herpesvirus viral protein kinase phosphorylates extracellular signalregulated kinase and activates MAPK/ERK signaling pathway, Biochemical and Biophysical Research Communications, https://doi.org/ 10.1016/j.bbrc.2019.11.038