AMP-activated protein kinase phosphorylates EMCV, TMEV and SafVleader proteins at different sites

Holly A. Basta 1, Ann C. Palmenberg n

Institute for Molecular Virology and Department of Biochemistry, Robert M. Bock Laboratories, University of Wisconsin–Madison,1525 Linden Dr., Madison, WI 53706, USA

a r t i c l e i n f o

Article history:Received 6 May 2014Returned to author for revisions7 June 2014Accepted 17 June 2014

Keywords:CardiovirusLeader proteinPhosphorylationAMPK

a b s t r a c t

Cardioviruses of the Encephalomyocarditis virus (EMCV) and Theilovirus species encode small, amino-terminal proteins called Leaders (L). Phosphorylation of the EMCV L (LE) at two distinct sites by CK2 andSyk kinases is important for virus-induced Nup phosphorylation and nucleocytoplasmic traffickinginhibition. Despite similar biological activities, the LE phosphorylation sites are not conserved in theTheiloviruses, Saffold virus (LS, SafV) or Theiler's murine encephalitis virus (LT, TMEV) sequences eventhough these proteins also become phosphorylated in cells and cell-free extracts. Site predictionalgorithms, combined with panels of site-specific protein mutations now identify analogous, but nothomologous phosphorylation sites in the Ser/Thr and Theilo protein domains of LT and LS, respectively.In both cases, recombinant AMP-activated kinase (AMPK) was reactive with the proteins at these sites,and also with LE, modifying the same residue recognized by CK2.

& 2014 Elsevier Inc. All rights reserved.

Introduction

Encephalomyocarditis virus (e.g. EMCV) and Theiloviruses (e.g.Vilyusik virus, Theiler's murine encephalitis viruses [TMEV], andSaffold viruses [SafV]) are Cardiovirus species in the Picornaviridaefamily. All isolates from this genus have small, Leader proteins(L) encoded at the amino terminus of their viral polyproteins.These highly acidic peptides of 67- (EMCV, LE), 71- (SafV, LS) or 76-(TMEV, LT) amino acids (aa) carry out important anti-host func-tions. For example, the LE and LT proteins have been attributedwith pro- or anti-apoptotic functions, depending on the viral strainand cell culture system (Arslan et al., 2012; Stavrou et al., 2011;Romanova et al., 2009; Okuwa et al., 2010; Ghadge et al., 1998;Himeda et al., 2005; Carocci et al., 2011). For TMEV (DA), LTexpression in a virus context can inhibit stress granule formationin the cytoplasm, thereby reducing the turnover of RNA (Borgheseand Michiels, 2011). It is not clear whether all cardioviruses shareall these same pathways, or if these particular effects are justobserved downstream consequences of the major L function, thecommon catastrophic inhibition of active cellular nucleo-cytoplasmic trafficking (Lidsky et al., 2006; Bardina et al., 2009;Porter and Palmenberg, 2009; Porter et al., 2010; Delhaye et al.,

2004). The introduction of LE, LT or LS into cells by viral orrecombinant means uniformly triggers massive phosphorylationof Phe/Gly-containing nuclear pore proteins (Nups) (Porter andPalmenberg, 2009; Ricour et al., 2009; Basta et al., 2014). Themodified Nups are refractive to cargo-carrying karyopherins(importins or exportins), halting almost immediately the normalactive nuclear-cytoplasmic transport system for proteins andRNAs, to a mere trickle allowed by diffusion-only. This uniquecardiovirus phenomenon requires a poorly understood mechanisminvolving the binding of L to RanGTPase, followed by activationand misdirection of a particular cohort of mitogen-activatedprotein kinases (MAPKs) (Porter et al., 2010). For EMCV, the beststudied system, MAPKs ERK1/2 and p38, as well as their down-stream substrates, MAPKAP-2 (MK2) and RSK, become potentlyactivated in an LE-dependent manner (Porter et al., 2010). Chemi-cal inhibition of ERK1/2 and p38 pathways abolishes LE-directedNup phosphorylation and protects the cells (Porter et al., 2010).Likewise, LE mutations which prevent RanGTPase interactions orunfold the protein are unable to trigger Nup phosphorylation(Bacot-Davis and Palmenberg, 2013).

Despite having identity variations that can exceed 65%, all cardi-ovirus L proteins are homologs, if not presumed analogs. The LM(EMCV, Mengo) structure as solved by NMR (PDB: 2M7Y) shows aflexible coiled-coil configuration distinguished by an unusual,conserved (genus level) CHCC-based zinc finger (aa 10–22) nearthe amino terminus (Fig. 1). A carboxyl-proximal “acidic” domain(aa 37–52) is also shared, conferring a pI of 3.6–3.8 to each protein.

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Virology

http://dx.doi.org/10.1016/j.virol.2014.06.0260042-6822/& 2014 Elsevier Inc. All rights reserved.

n Corresponding author. Tel.: þ608 262 7519; fax: þ608 262 6690.E-mail address: acpalmen@wisc.edu (A.C. Palmenberg).1 Present address: Rocky Mountian College, Billings, MT, USA.

Virology 462-463 (2014) 236–240

A central linker or hinge region (LE aa 35–40) mediates essentialinteractions with cellular binding partner, RanGTPase, presumablythrough induced-fit contacts (Bacot-Davis and Palmenberg, 2013).Theilovirus L proteins have shorter amino-termini, but they arelonger than LE proteins, because of carboxyl-proximal insertionsthat add 14- or 19-aa respectively to the LS and LT sequences. Thecommon portion of these insertions, the “Theilo domain,” isdiagnostic of that species (BeAn aa 60–73). Only the LT displayadditional, upstream “Ser/Thr-rich” residues (BeAn aa 51–63).The indel domain functions have been partially inferred by geneticmapping with viruses. Point mutations to the LT (DA) Theilodomain reduce viral apoptotic activity, decrease virus persistencein FVB/N mice, and reduce Nup98 phosphorylation with conse-quent decreased inhibition of nucleocytoplasmic trafficking. Theyalso decrease the ability to inhibit IFN-β and RANTES activities,and decrease the ability to inhibit IRF-3 dimerization (Ricour et al.,2009). Such viruses are also unable to inhibit stress granuleformation (Borghese and Michiels, 2011). Equivalent mutationsto the LT Ser/Thr domain (S51A, S53A, and S54A) do not have similareffects (Ricour et al., 2009), although a single amino acid change inthe LT of GDVII virus (P57) and DA virus (S57) correlates withdifferential apoptotic phenotypes (Stavrou et al., 2011), and LTintracellular localization differences (Taniura et al., 2009).

The problem with evaluating such results is that even pointmutations can have unanticipated long-range effects if they un-intentionally disrupt protein structure. Moreover, as has beenshown for LE, directed phosphorylation particular to these regionsplays a very important role in the observed activity (Basta et al.,2014). In cells, LE is sequentially phosphorylated by casein kinase 2(CK2) and spleen tyrosine kinase (Syk) on T47 and Y41, respectively(Basta et al., 2014). The reactions are not obligatory for RanGTPaseinteractions, but they are essential for the subsequent trigger ofMAPK pathways. LT and LS also become phosphorylated, but atdifferent sites than LE, and in reactions that do not require CK2(Basta et al., 2014). Sequence comparisons have suggested S57(Taniura et al., 2009) and/or T63 (Ricour et al., 2009) as possiblesites in LT (DA). We now have experimental validation that LT(BeAn) and LS (SafV-2) and are differentially phosphorylated at S57(Ser/Thr domain) and T58 (Theilo domain), respectively. Bothreactions can be catalyzed by AMP-activated protein kinase(AMPK), an enzyme that can substitute for CK2 in the initialphosphorylation of LE. The identified LS and LT sites have super-imposable structural locales in an extended helical motif, modeledfor the Ser/Thr and Theilo domain insertions.

Results and discussion

Phosphorylation site mapping

A dataset of cardiovirus L proteins (Fig. 1) was compiled fromGenBank, aligned by ClustalX (Larkin et al., 2007) and is summar-ized using Weblogo format (Crooks et al., 2004). The LT (BeAn),

LS (SafV-2) and LE (EMCV-R) sequences are those from GenBankfiles, m16020, am922293, and m81861 respectively. Phosphoryla-tion predictions for these proteins used online program suites,including PPSP (set to “high sensitivity” for “all kinases”) (Xue etal., 2006), NetPhosK1.0 (set to “prediction without filtering” (fast),threshold¼0.5) (Blom et al., 2004), NetPhos2.0 (Blom et al., 1999),Phosida (Gnad et al., 2011), DiPHOS (specified predict for viruses)(Iakoucheva et al., 2004), Phosphomotif Finder (Amanchy et al.,2007) and ScanSite (set to “low stringency”) (Obenauer, 2003).

For LE (recombinant or viral), CK2 phosphorylation at T47 isthe primary, obligatory event in cells or in vitro, followed by aSyk reaction at Y41 (Basta et al., 2014). The LT-GST and LS-GSTproteins do not react with CK2 and the required site (T47XXD/E) isnot conserved in this species. Credible alternative predictedphosphorylation sites, common to each Theilo sequence groupincluded 6 Tyr/Thr residues in LS, and 8 Tyr/Thr/Ser residues in LT(filled symbols in Fig. 1). These, and structurally debilitating (C11)mutations in the respective zinc finger domains, were individuallyconstructed into LT- and LS-GST plasmids. Each protein wasexpressed and purified. When added to HeLa cytosol in thepresence of γ-32P-ATP, all of the LT-GST sequences were labeled,except C11A, Y7L and S57A (Fig. 2A). Some proteins, especially thosewith phosphomimetic glutamate mutations (S51D, T53D, and T59D),actually registered more strongly than wild-type (wt) when thesignals were normalized (densitometry) to the unmodified par-ental fusion protein band (αGST, top band). When this type ofdiversity was observed with the LE-GST proteins (Basta et al., 2014)it was indicative of interdependent phosphorylation events, one ofwhich (at T47) was an obligatory precursor for the other (at Y41).

The LS-GST proteins (Fig. 2B) had more variable responses.The wt protein was only weakly labeled in HeLa extracts (Basta etal., 2014), and all members of this panel incorporated less label thanany reactive LT-GST. The membranes required more than twice theexposure to even visualize the positive bands. Relative to eachother, the C11A, Y7L, T36A and Y49F sequences were labeled 2–3

I I I I

I II

I I I II I

II I I

Fig. 1. Cardiovirus Leader sequences. WebLogo depiction summarizes knownvariation in LE, LS and LT proteins. Ser, Thr, and Tyr sites predicted by any onlinephosphorylation algorithm are highlighted ( ). Sites selected for mutagenesis( ) are a subset and include also zinc-finger disruption mutations ( ). Mappedphosphorylation sites from this study (LS, LT) and others (LE) are in orange.

32P

32P

GST

Norm

Norm

P-AMPK

CK2

Tubulin

100

105100

100100

17727 48

Norm

Norm

Norm

~ng/reaction

HeLa

BHK-21

rAMPK

50

LT-GST

GST

GST

LS-GST

wtC11A

Y7LY25F

Y34F

S51D

T 53D

S57A

T 59D

T 63A

wtC11A

Y34F

T 36A

T 40A

Y49F

T 58A

T 7A

GST

0 100

33100 7939 51 5790

10112955130166941153417

670

Fig. 2. LX phosphorylation in cytosol. LT-GST (A) or LS-GST (B) proteins wereincubated with HeLa cytosol and [γ-32P]-ATP. After GST extraction and SDS-PAGE,the proteins were detected by autoradiography (32P), or Western analysis (αGST).After densitometry, the signals were normalized to the GST control (“0”) and wt(“100”) samples. The uppermost αGST band is the fusion protein. (C) Equivalentcytosol samples (HeLa, BHK) to A and B were probed byWestern analysis for AMPK,CK2 and tubulin signals, relative to a standardized sample of rAMPK.

H.A. Basta, A.C. Palmenberg / Virology 462-463 (2014) 236–240 237

times less efficiently than wt. The T40A and T58A had reduced labelincorporation by about 25%. The most active LS-GST sequences werewt and Y34F.

LX-GST phosphorylation by rAMPK

For LE, disruption of the zinc finger domain at C19 unfolds theprotein, as measured by single-proton NMR, and renders itinactive as substrate for reactions with cytosol or recombinantkinases (Bacot-Davis and Palmenberg, 2013). The C11 in LT or LS isthe structural analog to LE C19 with presumably similar functions.Indeed, neither Theilovirus protein with this mutation wasstrongly labeled in Hela cytosol. Given the inability to discriminatebetween structural and phosphorylation phenotypes at Y7, thislocation was noted, but not pursued further.

Instead, the S57 site of LT-GST, the only other potent mutation forthis protein, was suggested by the algorithms as a potential targetfor AMP-kinase, a ubiquitous sensor of cellular energy homeostasis,and an enzyme common to all organisms from yeast to mammals.AMPK is involved in the replication cycles of many viruses (forreview, see Mankouri and Harris (2011)). Recombinant AMPK wasactive with each of the wt LX-GST proteins but not with GST control(Fig. 3A). Surprisingly, LE-GST reacted even more strongly thanLT-GST or LS-GST, an unexpected finding since neither of themapped LE-GST sites (Y41, T47) had been previously assigned to thisenzyme (Basta et al., 2014). Parallel reactions with mutant proteinsconfirmed that AMPK was selecting the same LE site (T47) as thepreviously mapped CK2 activity (Fig. 3B).

For LT-GST, mutation at S57, but not T63, also abolished AMPKreactivity (Fig. 3D). The LT-dependent apoptotic phenotypes(Stavrou et al., 2011), and LT intracellular localization differences(Taniura et al., 2009) previously assigned to the S57 (DA) and P57(GDVII) sequences are now logically explained by the phosphoryla-tion status of this residue. We do not yet know if GDVII or otherviruses of this subtype use an alternative primary site instead. Therewas no evidence in cytosol experiments (Hela or BKH, not shown)that BeAn LT-T63 was a viable, reactive site (Fig. 2A), and certainly itwas unreactive with rAMPK (Fig. 3D). With the LS-GST protein,mutation at T58 but not T63 also prevented rAMPK reactions(Fig. 3C). The selection of this subset of proteins for testing wasbased on the site-prediction algorithms, as these were the only LXsites that fit the enzyme profile.

AMPK in cells

With standard reaction conditions, 50 ng of rAMPK put anaverage of 1 phosphate onto each substrate LE-GST protein (not

shown). The cytosol reactions with the same amount of substrateusually required longer exposures, suggesting a lower saturation oflabel. When tested side-by-side for antibody-band signals, relativeto rAMPK, a sample of HeLa cell lysate registered about 27 ng ofenzyme, as present in similar reactions (Fig. 3). BHK extracts gavea higher concentration, so there is variability in the availability ofAMPK in these cell types. This means that native AMPK is probablynot a dominant, high concentration enzyme in HeLa cells. Thealternative possibility that LS-GST (and LT-GST, LE-GST) is perhaps anon-ideal substrate is not supported by the incorporation data.The listed specific activity for rAMPK on its preferred acetyl-CoAcarboxylase substrate is 737 nmol/min/mg, a value that shouldlabel 2 μg of LS-GST (�62 pmol) within 3–5 min, as was observedin the actual reactions.

Structure prediction

The Theilo and Ser/Thr domains of LS-GST and LT-GST containthe respective T58 and S57 mutation-mapped AMPK phosphoryla-tion sites for these proteins (Fig. 1). These sites are not homologsbecause the alignments place the LS S57 within the context of theTheilo domain, paired vertically with LT T63, a non-phosphorylatedsite. Unique to the Theilovirus proteins, however, the sequenceswithin both inserted domains uniformly show strong, conservedpatterns of 3–4 amino acid periodicities (e.g. Ser/Thr, Asp/Gly,Leu/Val). Such patterns typically identify strong α-helical regions.When queried with the Lasergene Protean Suite, every algorithmconcurred on this configuration for the short (�4 turn, LS), orlonger (�7 turn, LT) insertions. If this model is true, as depicted inFig. 4, the central helical portions, scaled to the determined LMstructure, would place both Theilovirus AMPK sites virtually ontop of each other, a proximity dictated by the respective helicalfolds. The Ser/Thr domain (e.g. LT) then would behave physicallyjust like an extended Theilo domain (e.g. LS), with a consequentlydisplaced AMPK site. The LT residue T63, the sequence homologto LS T58 was not phosphorylated by AMPK. That activity isapparently shifted upstream, by 2 turns of the putative helix, tothe structural analog, S57. Possibly, the adjacent proximity tothe upstream acidic domain plays a role in this site selection.

GST

L E-GST

L S-GST

L T-GST

GST

wt

T 47A

wt

wt

T 36A

T 58A

S57A

T 63A

32P

Norm

Silver stain

LE-GST LS-GST LT-GST

100 1020 100 76 64 23 100 100 38 120170

αGST

- LX-GST

Fig. 3. Phosphorylation of LX-GST by rAMPK. Recombinant GST and LX-GST proteinswere reacted with rAMPK (50 ng) and [γ-32P]-ATP as in Methods. After pulldownand protein fractionation, gel bands were detected by phosphoimaging (32P) andsilver stain, or after transfer to membranes, by Western analyses (αGST). Thephosphorylation signals as measured by densitometry were normalized (norm) tothe top band (full-length fusion protein) of LE-GST (A, B) LS-GST (C), or LT-GST (D)αGST signal. Other GST-reactive bands in these panels are from inappropriate LXtranslational start sites during bacterial protein expression.

N’

C’

LT

LS

S57

T58

zinc finger

acidic domain

Theilo domain

LM

Ser/Thr + Theilodomains

Fig. 4. Structure models. An LM configuration as determined by NMR (PDB: 2M7Y)shows the location of Theilovirus Ser/Thr and Theilo domain insertions, modeledhere as helices. Within these contexts, the mapped LT and LS AMPK sites occupyanalogous locations relative to the start of each helix.

H.A. Basta, A.C. Palmenberg / Virology 462-463 (2014) 236–240238

The LE-GST primary site, recognized by CK2 or AMPK, is just on theother side of this acidic patch, again suggesting an analogous if nothomologous function.

Conclusions

Why do cardiovirus LX use different phosphorylation siteswhen the end goal of nucleocytoplasmic trafficking inhibition isthe same? Perhaps phosphorylation variability could just be falloutof cell-type preference by EMCV, Saffold, and TMEV, i.e. eachvirus type has adapted to use a kinase abundant in their cell typeof choice. Alternatively, the site selection and required kinase(s) could represent distinct regulatory methods. LE from highlyvirulent EMCV is phosphorylated by the highly expressed andconstitutively active CK2 and also at the same site, by AMPK, anenzyme which is less abundant in some cell types. The Theilo-viruses, however, are not as robust in their host range, and at leastfor the sites mapped here seem to rely on AMPK and not CK2,possibly slowing the viral lifecycle in a way that is helpful for theestablishment of persistence. The LT site for AMPK, S57, is a knowndeterminant distinguishing between persistent demyelinatingstrains of TMEV (DA, BeAn) and neurovirulent strains (GDVII).Recombinant conversion of DA S57 to P57 results in a GDVIIphenotype (high titer, large plaques) in BHK-21 cells, while theopposite conversion (GDVII P57 to S57) gives a DA phenotype (lowtiter, small plaques) (Takano-Maruyama et al., 2006). Apoptoticphenotypes are also localized to this residue (Stavrou et al., 2011).RanGTPase binding is ubiquitous to all cardiovirus L proteins, butthe phosphorylation status of the LX apparently does not con-tribute to this step (Bacot-Davis and Palmenberg, 2013). Therefore,LX phosphorylation must be required for the subsequent ternary(or quaternary?) complexes that actually carry out Nup phosphor-ylation. Differential partner selection after Ran binding is logicalexperimental directions predicted by these findings.

Materials and methods

Plasmids and proteins

Bacterial plasmids pLE-GST, pLS-GST and pLT-GST and selectpoint mutations were prepared as described (Basta et al., 2014)using site-directed mutagenesis via PCR overlap extension meth-ods (Ho et al., 1989). We thank Dr. Howard Lipton for the generousgift of SafV-2 and TMEV (BeAn) cDNA starting materials. Thematched panels linked the LX gene(s) upstream, in-frame with aglutathione S-transferase gene (GST) in a context that alloweddirect cDNA transfection, transcription (via a CMV promoter) andexpression in eukaryotic cells. LX-GST fusion proteins wereexpressed in bacteria and purified on Glutathione Sepharose HighPerformance Column (GE Healthcare Life Sciences) as described(Cornilescu et al., 2008). Proteins prepared this way can havesmaller αGST-reactive bands in addition the full-length fusion,because of alternative bacterial translation start sites in the LXgenes (Basta et al., 2014).

Phosphorylation assays

HeLa or BHK cytosol was prepared via dounce homogenization(Watters and Palmenberg, 2011). Phosphorylation reactions (80 μltotal, with 30 μl cytosol, 2 μl 10 mM ATP, 2 μg LX-GST, 0.75 μl[γ-32P]-ATP) were in GST binding buffer (50 mM HEPES, 150 mMNaCl, 0.5% NP40, pH 7.4) for 45 min (37 1C). Glutathione sepharose4B beads (10 μl per sample, GE Healthcare Life Sciences) were thenadded, followed by agitation (4 1C, 2 h). The beads were collectedby centrifugation (500g), washed with GST buffer (4� ) and then

boiled in SDS gel loading buffer. Protein fractionation was by SDS-PAGE (8% or 10%) with band visualization by phosphorimaging(Typhoon 9200 Variable Mode Imager, GE Healthcare), silver stain,or western analysis.

rAMPK (α1, β1, γ1) was obtained commercially (SignalChem).Specific activity information was from the technical data sheet.Reactions were similar to those described (Basta et al., 2014).Purified LX-GST in buffer (10 μl, 2 μl 0.5 mM AMP, 1 μl 50 ng/μlAMPK, 2 μg LX-GST, 25 mM MOPS pH 7.2, 25 mM MgCl2, 5 mMEGTA, 2 mM EDTA, 0.25 mM DTT) was allowed to react with 2 μlAMPK assay cocktail (0.25 mM ATP, 0.167 μCi/μl [γ-32P]-ATP) for15 min, 30 1C. The LX-GST protein was extracted with glutathionebeads, washed and then fractionated by SDS-PAGE. Phosphoryla-tion signals were quantified by densitometry using TotalLab Quant(Nonlinear Dynamics Ltd., Newcastle, UK), normalizing to inputprotein (from Western analysis or silver stain).

Western analyses

Protein bands were electrotransferred to polyvinylidenedifluoride membranes and treated with primary and secondaryantibodies as described (Basta et al., 2014). The primary antibodiesincluded: αGST (murine mAb, 71087, Novagen), αCK2 (rabbit pAb,06-873, Millipore), αtubulin (murine mAb, T4026, Sigma-Aldrich),and αP-AMPK 23A3 (rabbit mAb, 2603, Cell Signaling).

Acknowledgments

This work was supported by NIH grant AI017331 to ACP.We thank Dr. Adam Swick and Dr. John Yin for BHK-21 cells, andDr. Howard Lipton for the generous gift of SafV-2 and TMEV(BeAn) cDNAs.

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