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. 2019 Oct 4;294(40):14546-14561.
doi: 10.1074/jbc.RA119.009601. Epub 2019 Aug 1.

Casein kinase-2-mediated phosphorylation increases the SUMO-dependent activity of the cytomegalovirus transactivator IE2

Vasvi Tripathi  1 , Kiran Sankar Chatterjee  1 , Ranabir Das  2
Affiliations

Casein kinase-2-mediated phosphorylation increases the SUMO-dependent activity of the cytomegalovirus transactivator IE2

Vasvi Tripathi et al. J Biol Chem. .

Abstract

Many viral factors manipulate the host post-translational modification (PTM) machinery for efficient viral replication. In particular, phosphorylation and SUMOylation can distinctly regulate the activity of the human cytomegalovirus (HCMV) transactivator immediate early 2 (IE2). However, the molecular mechanism of this process is unknown. Using various structural, biochemical, and cell-based approaches, here we uncovered that IE2 exploits a cross-talk between phosphorylation and SUMOylation. A scan for small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) revealed two SIMs in IE2, and a real-time SUMOylation assay indicated that the N-terminal SIM (IE2-SIM1) enhances IE2 SUMOylation up to 4-fold. Kinetic analysis and structural studies disclosed that IE2 is a SUMO cis-E3 ligase. We also found that two putative casein kinase 2 (CK2) sites adjacent to IE2-SIM1 are phosphorylated in vitro and in cells. The phosphorylation drastically increased IE2-SUMO affinity, IE2 SUMOylation, and cis-E3 activity of IE2. Additional salt bridges between the phosphoserines and SUMO accounted for the increased IE2-SUMO affinity. Phosphorylation also enhanced the SUMO-dependent transactivation activity and auto-repression activity of IE2. Together, our findings highlight a novel mechanism whereby SUMOylation and phosphorylation of the viral cis-E3 ligase and transactivator protein IE2 work in tandem to enable transcriptional regulation of viral gene.

Keywords: SUMO-interacting motif (SIM); enzyme kinetics; host–pathogen interaction; nuclear magnetic resonance (NMR); phosphorylation; post-translational modification (PTM); sumoylation; transcription co-activator; viral protein; viral transcription.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Interactions between IE2–SIM1 and SUMO. A, schematic of the domains in IE2. The three predicted SIMs and the two SUMOylation site Lys-175 and Lys-180 are shown. Yellow circles with S denote SUMO. The transactivation domains are shown as blue boxes with TAD. The fragment 5B, serine-rich region, and DBA-binding domain are shown. B, overlay of the 15N-edited HSQC spectra of free 15N-SUMO1 (red) with different stoichiometric ratios of IE2–SIM1 as given in the top left-hand side of the spectra. C, two regions of the spectra are expanded to show a shift of SUMO1 resonances during titration. D, CSPs in SUMO1 upon binding to IE2–SIM1. The CSPs between the free and the bound form are calculated as CSP = ((δHfree − δHbound)2 + (δNfree − δNbound)/5)2)1/2, where δH and δN are the chemical shift of the amide hydrogen and nitrogen, respectively. The yellow and red dashed lines indicate 1× S.D. and 2× S.D., respectively. The secondary structure alignment of SUMO1 against its sequence is provided above the plot. The residues with CSPs significantly above the dashed lines are present at the interface of the SUMO1/IE2–SIM1 complex. E, significant CSPs are mapped onto the SUMO1 structure. The residues with CSP above the yellow and red lines are colored in yellow and red, respectively. F, CSPs in SUMO2 upon binding to IE2–SIM1. G, significant CSPs mapped on the SUMO2 structure. The residues with CSP above yellow and orange lines are colored in yellow and orange, respectively.
Figure 2.
Figure 2.
CK2 phosphorylates IE2. A, phosphorylation of IE2 detected in cells by mass spectrometry-based proteomics. All vertical black lines denote detected phosphorylation sites in IE2. Green and red vertical lines denote detected phosphorylation sites in IE2 that are predicted MAPK and CK2 sites, respectively. The region around SIM1 is expanded to show that only two serines immediately adjacent to SIM1 are phosphorylated. B, schematic of IE2–SIM1 and IE2-ppSIM1. C, IE2–SIM1 was incubated with CK2 and γ-ATP, run on SDS-polyacrylamide gel, and analyzed using autoradiography. CP is the control peptide that is a known substrate of CK2. In-CK2 in the last lane is inactivated CK2. The higher molecular weight band corresponds to CK2, which auto-phosphorylates itself. This band is not observed in the heat-inactivated lane. D, mass spectra of IE2–SIM1, and IE2–SIM1 incubated with CK2 (IE2-ppSIM1). The spectra of synthesized IE2–ppSIM1 is given below as a reference. The lines with an asterisk are coming from impurities.
Figure 3.
Figure 3.
Molecular basis of the enhanced interaction between SUMO and IE2-ppSIM1. A, selected strips from the 13C,15N-filtered (F1), 13C,15N-edited (F2), and NOESY HSQC spectra depicting intermolecular NOEs between 13C-bonded protons of 13C,15N-labeled SUMO1 and unlabeled IE2-ppSIM1. 13C and 1H assignment of SUMO1 atoms are given on the right and left of the strips, respectively. The protons of IE2–ppSIM1 that show NOEs to SUMO1 are assigned. B and D highlights the hydrophobic interactions in the SUMO1/IE2–ppSIM1 and SUMO2/IE2–ppSIM1 complexes, respectively. The SUMO1/2 surface is colored white, except the hydrophobic patches are colored green. The IE2–ppSIM1 backbone is shown as an orange ribbon. The side chains of central hydrophobic residues CIVI are shown as yellow spheres. C and E shows the hydrogen bonds between phosphorylated side chains of IE2–SIM1 with SUMO1 and SUMO2, respectively. The hydrogen bonds are shown as black lines. The two phosphoserines and the residues in SUMO1/2 that form hydrogen bonds are shown. Nitrogen atoms are colored in blue; oxygen atoms are colored in red; and phosphorus atoms are colored in yellow.
Figure 4.
Figure 4.
In vitro SUMOylation of IE2–NTD. A, FITC fluorophore-labeled IE2–NTD used as a substrate in SUMOylation assays. The substrate lysines and SIM1 are shown. B, products of the SUMOylation reaction with SUMO1 and IE2–NTD as the substrate is resolved on the SDS-polyacrylamide gel and imaged with a filter at 519 nm corresponding to FITC fluorescence. Bands of free IE2–NTD or conjugated with one, two, or multiple (n) SUMO1s are marked. The time points are given at the top of the gel. C, same as B except that IE2–NTDm (CIVI to AAAA) was used as the substrate. D and E, fraction of free IE2–NTD is plotted against time for SUMOylation reactions using SUMO1 and SUMO2, respectively. F, experimental design to monitor SUMOylation of IE2–NTD in real time. G, change of fluorescence anisotropy with time for IE2–NTD and IE2–NTDm in a SUMOylation reaction using SUMO1. H, same as in G using SUMO2 instead of SUMO1.
Figure 5.
Figure 5.
IE2–SIM1 enhances SUMOylation of IE2. A, kinetic data for SUMOylation of IE2–NTD and IE2–NTDm. B and C are the calculated Km and Vmax values, respectively. D, IE2 and SIM mutant IE2 (smIE2) SUMOylation detected in HEK293T cells. E, fraction of IE2∼SUMO over total IE2 is quantified from D and plotted.
Figure 6.
Figure 6.
Mechanism of cis-SUMO-E3 ligase activity of IE2. The three possible mechanisms are shown in A–C. Cys-93 is the catalytic cysteine in UBC9, which is conjugated to Gly-97 in SUMO1. The critical residue His-20 for noncovalent UBC9–SUMO interaction is shown. The critical residue Lys-14 for covalent UBC9–SUMO interaction is also shown. D, CSPs observed in UBC9 upon titration with IE2–NTD. E, CSPs in SUMO1 within the UBC9∼SUMO1 conjugate, upon titration with IE2–NTD. F, 10 lowest energy model structures of the UBC9∼SUMO1/IE2–NTD complex. UBC9, SUMO1, and IE2–NTD are color-coded as in A. The active site Cys-93 is colored purple; Gly-97 of SUMO1 is colored yellow, and Lys-180 is colored blue. G, same as in F for the SUMO1/UBC9∼SUMO1/IE2–NTD, where SUMO1 is noncovalently bound to UBC9. H, same as in F for the SUMO1–UBC9∼SUMO1/IE2–NTD complex, where SUMO1–UBC9 denotes the SUMO1 covalently linked to Lys-14 of UBC9. I, lowest energy structure of the UBC9∼SUMO1/IE2–NTD complex. J, close-up of the active site shows that Glu-178 forms a salt bridge with Lys-101, and Lys-180 attacks the active site. The inset is shown the complete structure, where the zoomed region is marked with a box. K, gel-shift assay; L, fluorescent anisotropy assay monitored the rate of SUMOylation using either WT, K14R, or H20D UBC9.
Figure 7.
Figure 7.
Phosphorylation enhanced SUMOylation of IE2–NTD. A, schematic of IE2–NTD and the phosphorylated IE2–NTD. B, SUMOylation of IE2–NTD and IE2–ppNTD against time. The SUMOylation reaction using either IE2–NTD or IE2–ppNTD was run for different times, resolved on the SDS-polyacrylamide gel, and imaged with a filter at 519 nm corresponding to FITC fluorescence. The time of reaction is given at the top. M stands for the marker. C, IE2–NTD∼SUMO conjugate was quantified and plotted against time for IE2–NTD and IE2–ppNTD. D, real-time fluorescence anisotropy measurement of IE2–NTD and IE2–ppNTD SUMOylation. E, SUMOylation of IE2 and phosphorylation mutant pmIE2 observed in HEK293T cells. Cell lysates 48 h post-transfection were separated on SDS-PAGE and blotted with anti-HA. F, ratio of conjugated and total IE2 is quantified from E and plotted against the time of transfection. G, Michaelis-Menten curves for IE2–NTD and IE2–ppNTD. H and I are the calculated Km and Vmax, respectively.
Figure 8.
Figure 8.
Functional implications of phosphorylation-induced enhanced SIM–SUMO interaction and SUMOylation. A, luciferase transactivation assays were performed in HEK293T cells 36 h post-transfection with or without IE2 or mutants of IE2 and SUMO1. The relative luciferase activity is plotted against the IE2 or its mutants. B same is repeated without transfection of SUMO1. C, luciferase auto-repression activity was monitored using IE2 and its mutants. C denotes the control where IE2 was not transfected. D, model of phosphorylation-induced enhanced IE2 transactivation/auto-repression activity. IE2 is colored in light blue, and the DNA-binding domain (DBD) is colored in dark blue. IE2 binds to the TBP, which binds to the promoter. Phosphorylation increases the interaction between IE2–SIM1 and SUMOylated transcription factors (e.g. TAF12) to enhance the transactivation activity. IE2 can directly bind to the cis-regulatory sequences (crs) for auto-repression via the DBD. Phosphorylation can enhance SUMOylation of IE2 to increase its association with the chromatin modifiers like the HDAC/HMT/CoREST complex and increase auto-repression. HDAC, histone deacetylase, HMT, histone methyltransferase.
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