Phosphoproteomic Analysis Reveals the Importance of Kinase Regulation During Orbivirus Infection

Mohl, Bjorn-Patrick; Emmott, Edward; Roy, Polly

doi:10.1074/mcp.m117.067355

Cited by 12 publications

(12 citation statements)

References 65 publications

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“…Moreover, our data provide a molecular basis for this activity through the identification of BRAF as a new interactor of BTV-NS3, together with the fact that BRAF silencing impairs BTV-activated MAPK/ERK signaling. Intriguingly, two other studies have shown that BTV does not activate the MAPK/ERK pathway (38,39). However, these findings do not necessarily contradict our current data.…”

Section: Btv-ns3 Inhibits the Induction Of Ifn-␣/␤ Independently Of Msupporting

confidence: 60%

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway

Kundlacz

Pourcelot

Fablet

et al. 2019

J Virol

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Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its nonstructural protein NS3 (BTV-NS3), is able to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eukaryotic translation initiation factor 4E (eIF4E). By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease in the MAPK/ERK activation by BTV, supporting a model wherein BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the alpha/beta interferon response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel a new essential function of NS3 during BTV infection. IMPORTANCE Bluetongue virus (BTV) is responsible of the arthropod-borne disease bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its nonstructural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway, and this activation is BRAF dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.

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Section: Btv-ns3 Inhibits the Induction Of Ifn-␣/␤ Independently Of Msupporting

confidence: 60%

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway

Kundlacz

Pourcelot

Fablet

et al. 2019

J Virol

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“…We measured CK2 activity indirectly by using an antibody against the phospho-CK2 substrate (motif pS/pTDXE) to measure the amount of phosphorylated CK2 substrates. This method is widely used in literature to measure kinase activity, such as activity of the following kinases CK2 [ 16 , 49 , 50 , 51 , 52 ], AMPK [ 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ], AMT/ATR [ 64 , 65 , 66 , 67 , 68 , 69 , 70 ], PKA [ 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 ], and PKC [ 77 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 ]. Both CK2 knockdown and CX-4945 treatment inhibited the proliferation of CCA cells.…”

Section: Discussionmentioning

confidence: 99%

CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism

Lertsuwan

Sawasdichai

et al. 2018

Cancers

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Cholangiocarcinoma is a disease with a poor prognosis and increasing incidence and hence there is a pressing unmet clinical need for new adjuvant treatments. Protein kinase CK2 (previously casein kinase II) is a ubiquitously expressed protein kinase that is up-regulated in multiple cancer cell types. The inhibition of CK2 activity using CX-4945 (Silmitasertib) has been proposed as a novel treatment in multiple disease settings including cholangiocarcinoma. Here, we show that CX-4945 inhibited the proliferation of cholangiocarcinoma cell lines in vitro. Moreover, CX-4945 treatment induced the formation of cytosolic vacuoles in cholangiocarcinoma cell lines and other cancer cell lines. The vacuoles contained extracellular fluid and had neutral pH, features characteristic of methuosis. In contrast, simultaneous knockdown of both the α and α′ catalytic subunits of protein kinase CK2 using small interfering RNA (siRNA) had little or no effect on the proliferation of cholangiocarcinoma cell lines and failed to induce the vacuole formation. Surprisingly, low doses of CX-4945 increased the invasive properties of cholangiocarcinoma cells due to an upregulation of matrix metallopeptidase 7 (MMP-7), while the knockdown of CK2 inhibited cell invasion. Our data suggest that CX-4945 inhibits cell proliferation and induces cell death via CK2-independent pathways. Moreover, the increase in cell invasion brought about by CX-4945 treatment suggests that this drug might increase tumor invasion in clinical settings.

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“…Moreover, SILAC [ 29 ] have overcome some of the phosphoproteomics quantification problems and improved the insights obtained. [ 23,30 ]…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation Time‐Course Study of the Response during Adenovirus Type 2 Infection

et al. 2020

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PTMs such as phosphorylations are usually involved in signal transduction pathways. To investigate the temporal dynamics of phosphoproteome changes upon viral infection, a model system of IMR‐90 cells infected with human adenovirus type 2 (Ad2) is used in a time‐course quantitative analysis combining titanium dioxide (TiO2) particle enrichment and SILAC‐MS. Quantitative data from 1552 phosphorylated sites clustered the highly altered phosphorylated sites to the signaling by rho family GTPases, the actin cytoskeleton signaling, and the cAMP‐dependent protein kinase A signaling pathways. Their activation is especially pronounced at early time post‐infection. Changes of several phosphorylated sites involved in the glycolysis pathway, related to the activation of the Warburg effect, point at virus‐induced energy production. For Ad2 proteins, 32 novel phosphorylation sites are identified and as many as 52 phosphorylated sites on 17 different Ad2 proteins are quantified, most of them at late time post‐infection. Kinase predictions highlighted activation of PKA, CDK1/2, MAPK, and CKII. Overlaps of kinase motif sequences for viral and human proteins are observed, stressing the importance of phosphorylation during Ad2 infection.

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Phosphoproteomic Analysis Reveals the Importance of Kinase Regulation During Orbivirus Infection

Cited by 12 publications

References 65 publications

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway

CX-4945 Induces Methuosis in Cholangiocarcinoma Cell Lines by a CK2-Independent Mechanism

Phosphorylation Time‐Course Study of the Response during Adenovirus Type 2 Infection

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