14-3-3 protein interacts with and affects the structure of rgs domain of regulator of g protein...

Journal of Structural Biology 170 (2010) 451–461

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Journal of Structural Biology

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14-3-3 protein interacts with and affects the structure of RGS domain of regulatorof G protein signaling 3 (RGS3)

Lenka Rezabkova a,b, Evzen Boura a, Petr Herman c, Jaroslav Vecer c, Lenka Bourova b, Miroslav Sulc d,e,Petr Svoboda b, Veronika Obsilova b, Tomas Obsil a,b,*

a Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republicb Institute of Physiology, Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republicc Faculty of Mathematics and Physics, Institute of Physics, Charles University in Prague, 12116 Prague, Czech Republicd Department of Biochemistry, Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republice Institute of Microbiology, Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history:Received 2 November 2009Received in revised form 12 March 2010Accepted 22 March 2010Available online 27 March 2010

Keywords:14-3-3 proteinRGS domainRGS3PhosphorylationFluorescenceStructure

1047-8477/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.jsb.2010.03.009

Abbreviations: 1,5-IAEDANS, 5-((((2-iodoacetyl)lene-1-sulfonic acid; AEDANS, 5-(((acetylamino)ethyfonic acid; 5-IAF, 5-iodoacetamidofluorescein; FRETtransfer; GAPs, GTPase-activating proteins; LED, lightmatrix assisted laser desorption ionization – timechannel plate photomultiplier tube; MEM, maximuphosphorylated regulator of G protein signaling 3; Pregulator of G protein signaling; RGS3, unphosphorysignaling 3; SVD, singular-value-decomposition.

* Corresponding author at: Department of Physicalistry, Faculty of Science, Charles University in PrRepublic. Fax: +420 224919752.

E-mail address: [email protected] (T. Obsil).

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) for thea-subunit of heterotrimeric G proteins. Several RGS proteins have been found to interact with 14-3-3 pro-teins. The 14-3-3 protein binding inhibits the GAP function of RGS proteins presumably by blocking theirinteraction with Ga subunit. Since RGS proteins interact with Ga subunits through their RGS domains, it isreasonable to assume that the 14-3-3 protein can either sterically occlude the Ga interaction surface ofRGS domain and/or change its structure. In this work, we investigated whether the 14-3-3 protein bind-ing affects the structure of RGS3 using the time-resolved tryptophan fluorescence spectroscopy. Two sin-gle-tryptophan mutants of RGS3 were used to study conformational changes of RGS3 molecule. Ourmeasurements revealed that the 14-3-3 protein binding induces structural changes in both the N-termi-nal part and the C-terminal RGS domain of phosphorylated RGS3 molecule. Experiments with the isolatedRGS domain of RGS3 suggest that this domain alone can, to some extent, interact with the 14-3-3 proteinin a phosphorylation-independent manner. In addition, a crystal structure of the RGS domain of RGS3 wassolved at 2.3 Å resolution. The data obtained from the resolution of the structure of the RGS domain sug-gest that the 14-3-3 protein-induced conformational change affects the region within the Ga-interactingportion of the RGS domain. This can explain the inhibitory effect of the 14-3-3 protein on GAP activity ofRGS3.

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1. Introduction have been identified. Some RGS proteins consist of little more that

Regulator of G protein signaling (RGS) proteins share a highlyconserved �125-amino-acid large domain that was first identifiedby its ability to negatively regulate G protein coupled receptor(GPCR) signaling (Hepler, 1999; Ross and Wilkie, 2000). To date,more than 25 proteins containing RGS or RGS homology domains

ll rights reserved.

amino)ethyl)amino)naphtha-l)amino) naphthalene-1-sul-, Förster resonance energy

-emitting diode; MALDI-TOF,of flight; MCP-PMT, micro

m entropy method; pRGS3,SD, post source decay; RGS,lated regulator of G protein

and Macromolecular Chem-ague, 12843 Prague, Czech

the RGS domain (e.g. RGS1, RGS2, RGS4) while others posses longN-terminal or C-terminal extensions (e.g. RGS3, RGS7, RGS9) thatusually contain additional protein–protein interaction motifs anddomains (Ishii and Kurachi, 2003). RGS proteins function asGTPase-activating proteins (GAPs) for the a-subunit of heterotri-meric G proteins. They bind specifically to the GTP-bound formsof Ga and significantly stimulate their GTPase activity by stabiliz-ing the transition state (Tesmer et al., 1997). In addition, someRGS proteins seem to play additional functions based on eithertheir ability to interact with proteins other than Ga or their nuclearlocalization (Abramow-Newerly et al., 2006b; De Vries and GistFarquhar, 1999).

The activity of RGS proteins is tightly regulated through variousmechanisms including the post-translational modifications and theinteraction with other signaling proteins (Fischer et al., 2000; Kimet al., 1999; Roy et al., 2003; Schiff et al., 2000). Certain RGS pro-teins, including RGS3, RGS4, RGS5, RGS7 and RGS16, have beenfound to interact with 14-3-3 proteins (Abramow-Newerly et al.,

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452 L. Rezabkova et al. / Journal of Structural Biology 170 (2010) 451–461

2006a; Benzing et al., 2002, 2000; Niu et al., 2002; Ward and Mil-ligan, 2005). 14-3-3 proteins are a family of acidic regulatory pro-teins that function as molecular scaffolds (Aitken, 2006; Fu et al.,2000; Mackintosh, 2004). Interactions between the 14-3-3 proteinsand their targets are usually controlled by the phosphorylation ofthe binding partner (Muslin et al., 1996). Two optimal 14-3-3 pro-tein binding motifs (R-S-X-pS/pT-X-P and R-X-Y/F-X-pS/pT-X-P)have been identified, although 14-3-3 proteins can also recognizemany sites that do not conform to these optimal motifs includingunphosphorylated sites (Rittinger et al., 1999; Yaffe et al., 1997).Several recent studies showed that the 14-3-3 protein bindinginhibits the GAP function of certain RGS proteins presumably byblocking their interaction with Ga subunit (Abramow-Newerlyet al., 2006a; Benzing et al., 2000; Niu et al., 2002). However, themechanism of this inhibition is still unknown. First studies identi-fied a putative 14-3-3 binding site located within the RGS domainof RGS3 and RGS7 at a conserved SYP motif (Benzing et al., 2002,2000). Other studies, however, identified phosphorylation siteSer264 located at the N-terminus of RGS3 outside of the RGS do-main as a single 14-3-3 binding motif (Niu et al., 2002; Wardand Milligan, 2005).

One mode of the 14-3-3 protein action is the interference withthe protein–protein interaction of the binding partner either bysteric blocking of the binding interface and/or through the modu-lation of the binding partner structure (Bridges and Moorhead,2004; Fu et al., 2000; Hermeking, 2003; van Hemert et al., 2001).To investigate the mechanism of the 14-3-3 protein-dependentinhibition of RGS3 function, we studied whether the 14-3-3f pro-tein binding affects the structure of RGS3 protein using the trypto-phan fluorescence spectroscopy. Two single-tryptophan mutantsof human RGS3 were used to study conformational changes eitherat the N-terminus of RGS3 molecule (Trp295) or within the C-ter-minal RGS domain (Trp424). Our measurements revealed that theinteraction between the 14-3-3 protein and RGS3 phosphorylatedat Ser264 induces significant structural changes in both the N-ter-minal part and the C-terminal RGS domain of RGS3 molecule. Thephosphorylation of Ser264 itself induces significant structuralchanges in the region surrounding nearby located Trp295 but notTrp424 located within the remote RGS domain. Experiments withthe isolated RGS domain of RGS3 suggest that this domain alonecan, to some extent, interact with the 14-3-3 protein in a phos-phorylation-independent manner. In addition, a crystal structureof the RGS domain of RGS3 was solved at 2.3 Å resolution. Thisstructure suggests that the 14-3-3 protein-induced conformationalchange affects the region within the Ga-interacting portion of theRGS domain. This can explain the inhibitory effect of the 14-3-3protein on GAP activity of RGS3.

2. Materials and methods

2.1. Expression, purification, and phosphorylation of RGS3 protein

DNA encoding human RGS3 isoform 1 (residues 229-513) wasligated into pET-15b (Novagen) using the NdeI and BamHI sites.The histidine-tagged protein was expressed by IPTG induction for12 h at 30 �C and purified from Escherichia coli BL21(DE3) cellsusing Chelating Sepharose Fast Flow (Amersham Biosciences)according to the standard protocol. The histidine tag was cleavedby incubation (12 h at 4 �C) with 5 U of thrombin per mg protein.After cleavage, RGS3229–513 was purified by size-exclusion chroma-tography on a Superdex 200 column (Amersham Biosciences) inbuffer containing 20 mM Tris–HCl (pH 7.5), 500 mM NaCl, 1 mMEDTA, 3 mM DTT, 0.01% (w/v) NP-40, and 10% (w/v) glycerol.

Purified RGS3229–513 was phosphorylated by incubation (8 h at6 �C) with 100 U of PKA (Promega) per mg of protein in the pres-

ence of 0.75 mM ATP and 15 mM MgCl2. The reaction was stoppedby the addition of EDTA at final concentration 15 mM. The phos-phorylated protein was re-purified by size-exclusion chromatogra-phy as mentioned above. The completeness of the phosphorylationreaction was checked using the MALDI-TOF mass spectrometry.

The sequence RGS3229–513 contains three tryptophan residueslocated at positions 295, 391 and 424. Mutants of RGS3229–513 con-taining single tryptophan residue (either at positions 295, 391 or424) were created by mutating other two tryptophans to phenylal-anines using the QuikChange approach (Stratagene). All mutationswere confirmed by sequencing. However, only mutants containingTrp295 (RGS3229–513W295) and Trp424 (RGS3229–513W424) weresuccessfully purified. Mutant of RGS3229–513 containing singleTrp391 was unstable and therefore was not used in this study.

2.2. Mass spectrometric analysis of RGS3

Samples were first separated by 12% SDS–PAGE and excised pro-tein bands were digested with trypsin endoprotease (Promega) di-rectly in gel with cysteine modification by iodoacetamide(Sadilkova et al., 2008). Resulting peptide mixtures were extractedby 30% acetonitrile and 0.3% acetic acid and subjected toMALDI-TOF mass spectrometer BIFLEX (Bruker–Daltonics, Bremen,Germany) equipped with a nitrogen laser (337 nm) and gridless de-layed extraction ion source. Ion acceleration voltage was 19 kV andthe reflectron voltage was set to 20 kV. Positive charged spectrawere acquired with a-cyano-4-hydroxy-cinnamic acid as MALDImatrix and calibrated internally using the monoisotopic [M + H]+

ions of RGS3229–513 protein peptides with known sequence. To en-rich phosphorylated peptides from the peptide mixtures the de-scribed procedure was performed (Larsen et al., 2005).

2.3. Crystallization of RGS domain of RGS3, data collection, andstructure determination

RGS domain of RGS3 (residues 384–513) was expressed andpurified according to the same protocol as described above forRGS3229–513. Purified RGS3384–513 in buffer containing 200 mM so-dium citrate (pH 6.5), 100 mM NaCl, 5 mM DTT was concentratedto 10 mg/ml. Crystals grew in 3–4 weeks at 15 �C in hanging dropscomposed of a 1:1 mixture of the protein solution and a well solu-tion consisting of 28% PEG 4000 (w/v), 150 mM magnesium chlo-ride, and 100 mM glycine. Single crystals were transferred into astabilization solution consisting of 28% PEG 4000 (w/v), 150 mMmagnesium chloride, 100 mM glycine, and 30% glycerol (w/v),and the crystals were frozen by rapid immersion in liquid nitrogen.

A complete data set was collected on a MAR345 image platedetector (X-ray Research, Germany) mounted on a Nonius FR591rotating anode source operated at 50 kV/80 mA (Nonius, The Neth-erlands). Data were processed using the MOSFLM (Leslie, 2006).Initial phases were obtained by molecular replacement using thecoordinates of RGS domain of RGS16 (PDB Accession Number2BT2). The model refinement was carried out with REFMAC 5.2(Murshudov et al., 1997). The crystallographic data and refinementstatistic are summarized in Table 4. The structure was refined at2.3 Å resolution and the current model consists of one moleculeof RGS domain of RGS3 and 62 solvent molecules. The atomic coor-dinates and structure factors have been deposited in the RCSB Pro-tein Data Bank and are available under Accession Code 2OJ4.Superimposition of RGS3 with other RGS domains was performedin PyMOL (www.pymol.org). The model of RGS3/Gia1 complexwas build on the bases of the crystal structure of the RGS4/Gia1

complex (Tesmer et al., 1997) using DeepView/Swiss-PDBViewer4.0.1 (Guex and Peitsch, 1997).

http://www.pymol.org

L. Rezabkova et al. / Journal of Structural Biology 170 (2010) 451–461 453

2.4. Expression and purification of 14-3-3 protein

Both human 14-3-3f protein (WT) and its mutant version con-taining no tryptophan residues (mutations Trp59Phe andTrp228Phe, denoted as 14-3-3fnoW) were expressed and purifiedas described previously (Boura et al., 2007).

2.5. 14-3-3 protein binding studies

Steady-state fluorescence measurements were performed on aPerkin–Elmer LS50B fluorescence spectrometer at 22 �C in buffercontaining 20 mM Tris–HCl buffer (pH 7.5), 250 mM NaCl, 1 mMEDTA, 0.01% (v/v) Tergitol NP-40, 3 mM 2-mercaptoethanol, and10% (w/v) glycerol with 250 nM pRGS3229–513 labeled by 1,5-IAE-DANS. Increasing amounts of 14-3-3f labeled by 5-IAF weretitrated into the cuvette. At each 14-3-3f concentration, thesteady-state fluorescence intensity of AEDANS was recorded(excitation at 336 nm and emission at 490 nm). The fraction ofRGS3 bound was calculated from the formula FB = (Imax � Iobs)/(Imax � Imin), where Imax is the maximum fluorescence intensity inthe absence of 14-3-3f, Iobs is the observed intensity for any 14-3-3f concentration; and Imin is the intensity at saturation. FB wasplotted against the 14-3-3f protein concentration and fitted usingthe Eq. (1) to determine the KD for the 14-3-3f:pRGS3229–513 com-plex formation:

FB ¼ fKD þ ½P1� þ ½P2� �ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðKD þ ½P1� þ ½P2�Þ2 � 4½P1�½P2�

qg=2½P1�;

ð1Þ

where KD is the equilibrium dissociation constant, P1 is theRGS3229–513-AEDANS concentration, and P2 is the 14-3-3f-fluores-cein concentration. Nonlinear data fitting assuming 2:1 M ratio be-tween the 14-3-3f protein and RGS3229–513-AEDANS was performedusing the Origin 8.0 package (Microcal Software Inc.).

2.6. Time-resolved fluorescence measurements

Fluorescence experiments were measured on a time-correlatedsingle photon counting instrument with a pulsed UV-LED excita-tion (PicoQuant, PLS 295-10) and a cooled MCP-PMT (Hamamatsu,R3809U-50) detector. Subnanosecond excitation pulses with a rep-etition rate of 10 MHz and 500 ps half-width were generated at295 nm. The LED emission was cleaned up by a custom made band-pass filter. Fluorescence signal was isolated by a monochromator at355 nm with a slit-width of 15 nm. A color glass filter (Zeiss, UG1)placed in front of the input slit enhanced suppression of scatteredlight. The decays were acquired in 512 channels with a time scaleof 100 ps per channel. The data were typically acquired until a peakcount of 105 was reached. Samples were placed in a thermostaticholder and all experiments were performed at 22 �C in buffer con-taining 20 mM Tris–HCl buffer (pH 7.5), 250 mM NaCl, 1 mM EDTA,0.01% (v/v) Tergitol NP-40, 3 mM 2-mercaptoethanol, and 10% (w/v) glycerol. Concentration of RGS3229–513 (single tryptophan mu-tants) was 10 lM and 14-3-3fnoW concentration was 20 lM.

Fluorescence decays were processed as described previously(Obsilova et al., 2004, 2005). Fluorescence was assumed to decaymultiexponentially according to the formula:

IðtÞ ¼X

i

ai � expð�t=siÞ ð2Þ

where si and ai are fluorescence lifetimes and corresponding ampli-tudes, respectively. The intensity decay I(t) was analyzed by a max-imum entropy method for oversampled data (SVD-MEM) codedaccording to the Bryan (Bryan, 1990). Typically we used 100 life-times in the range from 20 ps to 20 ns, equidistantly spaced in thelogarithmic scale. The program yielded a set of amplitudes ai repre-

senting a lifetime distribution in the decay. The mean lifetime wascalculated as:

smean ¼X

i

fisi ð3Þ

where index i runs over all lifetime components and fi represents anintensity fraction of the corresponding component:

fi ¼ aisi

Xi

aisi

,ð4Þ

The fluorescence anisotropy decays r(t) were obtained from theparallel I||(t) and perpendicular I\(t) decay components. Data wereanalyzed by a method similar to the one published by Brochon(1994) using a program developed at the Institute of Physics,Charles University, Prague, Czech Republic. We used a model inde-pendent SVD-MEM approach that does not set prior limits on theshape of the distribution. The anisotropies were analyzed for a ser-ies of exponentials:

rðtÞ ¼X

i

bi � expð�t=/iÞ ð5Þ

where the amplitudes bi represent a distribution of the correlationtimes /i. The bi are related to the initial anisotropy r0 by theformula:X

bi ¼ r0: ð6Þ

We used 100 correlation times /i equidistantly spaced in thelogarithmic scale and ranging from 50 ps to 200 ns. For multimodaldistributions the mean correlation time associated with the m-thpeak of the distribution was calculated from the formula:

�/m ¼X

k

bm;k � /m;k

Xk

bm;k

,ð7Þ

where index k runs over the nonzero amplitudes of the m-th peak ofthe distribution only. The area of the peak represents the associatedmean amplitude �bm:.

3. Results

3.1. Preparation and characterization of phosphorylated RGS3

The 14-3-3 protein binding inhibits the GAP function of certainRGS proteins, including RGS3, presumably by blocking their inter-action with Ga subunit (Abramow-Newerly et al., 2006a; Benzinget al., 2000; Niu et al., 2002). Since the GAP activity is conferredby an RGS domain, it is reasonable to speculate that the 14-3-3protein binding might block the interaction between the RGS pro-tein and Ga either through the structural modulation of RGS do-main or by the steric blocking of the Ga interaction interface.Main goal of this work was to study whether the 14-3-3 proteinbinding affects the structure of the RGS3 protein, especially, of itsRGS domain.

Since the preparation of milligram quantities of full length hu-man RGS3 isoform 1 (residues 1–519) has been shown to be veryproblematic due to the high instability of the recombinant protein(Ward and Milligan, 2005), we prepared a construct of RGS3 (iso-form 1) consisting of residues 229–513 (Fig. 1). The sequence ofRGS3229–513 covers the region containing phosphorylation site/14-3-3 binding motif (Ser264) and the C-terminal RGS domain(residues 384–513). This protein is fairly soluble and stable to beprepared in sufficient quantity for spectroscopic studies. Althoughthe kinase responsible for phosphorylation of RGS3 at Ser264in vivo is unknown, the work by Niu et al. (2002)) suggested thepossible role of PKA. Our recombinant RGS3229–513 protein can be

Fig. 1. Interaction of RGS3 with the 14-3-3f protein. Schematic representation of primary structure of RGS3229–513 (isoform 1) protein. Black vertical bar denotes location ofthe 14-3-3 protein binding site. Gray ellipses denote the locations of tryptophan residues.


stoichiometrically phosphorylated in vitro by PKA at Ser264. Boththe location of the phosphorylation site and the completeness ofthe reaction were confirmed by the MALDI-TOF mass spectrome-try. In all tested constructs (WT and mutants containing singleTrp residue) the comparison of positive MALDI-TOF mass spectraof digested unphosphorylated and phosphorylated proteins clearlydemonstrates two phosphorylated peptides of pRGS3229–513 pro-tein (Fig. 2). The detected peaks in positive ion mass spectra ofphosphorylated pRGS3229–513 having the protonized mass of1589.7 (m/z) corresponds to phosphorylated peptide RTHSEGSLL-QEPR (sequence 261–273) and the second peptide with sequenceRRTHSEGSLLQEPR (sequence 260–273) having the mass of 1745.7(m/z). On the other hand, unphosporylated RGS3229–513 mass spec-trum provided no peaks with same values of m/z there but two cor-responding signals at m/z 1353.7 (sequence 262–273) and m/z1509.7 (sequence 261–273). The identity and structure of bothphosphorylated peptides were further corroborated by analysis oftheir PSD spectra to authenticate serine Ser264 in all constructsof pRGS3229–513 as a phosphorylated amino acid residue.

3.2. RGS3 and the 14-3-3 protein form a stable complex

Formation of the complex between the 14-3-3f protein andRGS3 was monitored and quantified using the fluorescence bindingassay based on the steady-state Förster resonance energy transfer(FRET). The titration of the 14-3-3f protein to the phosphorylatedpRGS3229–513 yielded the apparent equilibrium dissociation con-stant (KD) of 220 ± 10 nM assuming the 2:1 M ratio between 14-3-3f and phosphorylated pRGS3229–513 (Fig. 3). The stoichiometryof the complex was estimated using the size-exclusion chromatog-raphy experiments (data not shown). The titration of the 14-3-3fprotein to the unphosphorylated RGS3229–513 revealed low affinityinteraction. Similar titration performed with the isolated RGS do-main of the RGS3 protein (residues 384–513) also revealed weakbut not negligible interaction between the two components. Bind-ing data of both the unphosphorylated RGS3 and the isolated RGSdomain could not be reasonably fitted to Eq. (1) in order to obtainthe KD.

3.3. Preparation of single tryptophan mutants of RGS3 for fluorescencemeasurements

To investigate the effect of the 14-3-3f protein binding on thestructure of pRGS3229–513, the time-resolved tryptophan fluores-cence intensity and anisotropy decay measurements of single tryp-tophan residues located at two different positions within the RGS3molecule were performed. Mutant denoted as RGS3229–513W295contains single tryptophan residue Trp295 located in the vicinityof the phosphorylation site/14-3-3 binding motif (Ser264) whilemutant denoted as RGS3229–513W424 contains single tryptophanresidue Trp424 located within the C-terminal RGS domain. Mutantof RGS3229–513 containing single Trp391 was unstable and there-fore was not used in this study. Circular dichroism spectra showedno significant differences between RGS3229–513WT and tryptophanmutants (data not shown). Completeness of the phosphorylation

was confirmed by the MALDI-TOF mass spectrometry as describedabove. The human 14-3-3fnoW protein mutant missing all Trp res-idues (mutations Trp59 to Phe59 and Trp228 to Phe228) was usedin all tryptophan fluorescence measurements. We have previouslyshown that these two mutations have no effect on the bindingproperties of the 14-3-3f protein (Obsilova et al., 2004, 2008).

3.4. Interaction between the N-terminal part of RGS3229–513 and the14-3-3f protein

The indole is an environmentally sensitive fluorophore thatreflects variations in both polarity of its microenvironment andthrough-space Trp interactions (Vivian and Callis, 2001). Photo-physics of the indole moiety is rather complex and Trp exhibitsbiexponential decay even in an isotropic aqueous solution (Petrichet al., 1983). Situation in proteins becomes further complicated.Due to a dynamic nature of the protein structure and relatedconformational heterogeneity, even single Trp proteins frequentlyexhibit complicated nonexponential decays and a detailed inter-pretation of particular decay components is often impossible(Bajzer and Prendergast, 1993; Chen and Barkley, 1998). In thiswork we therefore used a mean fluorescent lifetime (smean) as arobust qualitative indicator of Trp microenvironment changescaused by external perturbations.

Results of the fluorescence measurements of theRGS3229–513W295 mutant reflect both the effect of PKA-inducedphosphorylation of Ser264 itself and the 14-3-3fnoW protein bind-ing on the RGS3 structure surrounding residue Trp295. We foundthat both the phosphorylation of Ser264 itself and the binding ofthe 14-3-3fnoW protein induce an increase of the smean. Simulta-neously, lifetime-independent fluorescence anisotropy decays doc-ument suppression of Trp295 segmental motion both upon thephosphorylation of RGS3 itself and upon the binding of the 14-3-3fnoW protein. This is well evidenced by a decrease of sum ofamplitudes ð�b1 þ �b2Þ associated with the fast motions of Trp295and by an increase in the correlation time �/2 (Table 1, Fig. 4). Since�b3 ¼ r0 � ð�b1 þ �b2Þ (see Eq. (6)), we can judge the local rigidizationof the RGS3 structure from the increase of �b3. The effect well dem-onstrates itself in Fig. 4 by significantly elevated anisotropies atlong times after excitation. These data show that both the phos-phorylation of Ser264 itself and the 14-3-3fnoW protein bindingaffect the structure of the N-terminal region of RGS3 molecule.Our data also indicate that the removal of two naturally occurringTrp residues from the RGS domain (to create RGS3229–513W295mutant) causes protein aggregation that results in extremely longcorrelation times �/3 (>100 ns) that reflect overall rotational diffu-sion of the large aggregates. This aggregation disallowed us to re-solve changes in �/3 caused by interaction of RGS3229–513W295with the 14-3-3fnoW protein.

3.5. The structure of RGS domain of RGS3 is changed upon the bindingof the 14-3-3f protein

To investigate the possible structural changes of RGSdomain upon the 14-3-3f protein binding, we used mutant

Fig. 2. MALDI-TOF mass spectra of trypsinized phosphorylated pRGS3229–513 protein (A) and unphosphorylated RGS3229–513 protein (B).


RGS3229–513W424 containing single tryptophan residue Trp424 lo-cated within the C-terminal RGS domain. As could be expected,analysis of the fluorescence data for RGS3229–513W424 revealedthat the PKA-induced phosphorylation of Ser264 itself has no effecton the fluorescence properties of relatively distant Trp424. How-

ever, binding of the 14-3-3fnoW significantly reduced smean ofTrp424 in both phosphorylated and unphosphorylated RGS3229–

513W424, the reduction being much larger for the higher affinityphosphorylated form (decrease from 5.7 to 4.5 ns) (Table 2, Figs.5 and 6). These observations suggest a binding-induced change

Fig. 3. FRET-based binding assay used to estimate the apparent equilibriumdissociation constant of the pRGS3229–513:14-3-3f complex (j). The KD of220 ± 10 nM for binding of the phosphorylated pRGS3229–513 to the 14-3-3f proteinwas estimated by fitting the resulting curve using Eq. (1). The unphosphorylatedRGS3229–513 (d) and the isolated RGS domain of RGS3 (N) bind the 14-3-3f proteinwith significantly lower affinity. Data obtained for unphosphorylated RGS3 and theisolated RGS domain could not be reasonably fitted using Eq. (1) to yield the KD.

Fig. 4. Time-resolved tryptophan fluorescence anisotropy decays of the RGS3229–

513W295. (A) The effect of the 14-3-3 protein on tryptophan fluorescence ofunphosphorylated RGS3229–513W295 mutant. Lines represent the best fit. (B) Theeffect of the phosphorylation itself and the 14-3-3 protein on tryptophanfluorescence of phosphorylated pRGS3229–513W295 mutant. Lines represent thebest fit.


in the microenvironment of Trp424. The lifetime-independentfluorescence anisotropy decays of Trp424 show that the bindingof the 14-3-3fnoW protein suppresses the fast Trp424 movementassociated with the correlation time �/1 (amplitude �b1 decreases)and causes appearance of a slower segmental motion characterizedby the rotational correlation time �/2 in the nanosecond range. Theamplitude �b3 associated with the overall protein rotation does notessentially change. Samples containing RGS3229–513W424 do notshow any signs of aggregation and the rotational correlation time�/3 correctly reflects the expected size of RGS3 protein (�32 kDa).The increase in �/3 upon the 14-3-3fnoW protein binding (from22 ns to 70 ns) reflects an increased molecular-mass of the formed14-3-3fnoW/pRGS3 complex. Binding-induced change in the char-acter of the Trp424 motion is illustrated by the rotational correla-tion time distributions in Fig. 6. Importantly, our data also suggestthat the unphosphorylated RGS3229–513W424 interacts with the14-3-3fnoW protein. This conclusion is based on the fact thatchanges in smean, �b1; �b2; and �/3 of the unphosphorylated proteinresemble the changes observed for the phosphorylated pRGS3229–

513W424. Such phosphorylation-independent interaction was alsoobserved in the case of RGS3229–513W295 mutant (Table 1). Thesedata are in a good agreement with the low affinity interactionobserved for the unphosphorylated RGS3229–513 WT using theFRET-based binding assay (Fig. 3).

Table 1Kinetic parameters of the RGS3229–513W295 mutant.

Protein smean (ns)a �b1

RGS3229–513W295 3.5 0.028RGS3229–513W295 + 14-3-3fnoW 4.2 0.037pRGS3229–513W295 3.7 0.045pRGS3229–513W295 + 14-3-3fnoW 4.0 0.028

a Standard deviation is better than 0.1 ns.

3.6. 14-3-3f protein interacts with the isolated RGS domain of RGS3

The time-resolved fluorescence measurements of the singletryptophan RGS3 mutants suggest that the 14-3-3fnoW proteincan weakly interact with the unphosphorylated RGS3. In addition,the FRET-base binding assay indicated low affinity interaction be-tween the 14-3-3f protein and both the unphosphorylated RGS3and the isolated RGS domain of RGS3 (Fig. 3). Therefore, it is en-tirely possible that the 14-3-3f protein might directly interact withthe RGS domain itself in a phosphorylation-independent manner.To test this possibility, we decided to study the interaction be-tween the 14-3-3fnoW protein and the isolated RGS domain in de-tail. The results of these measurements are listed in Table 3. For thefree isolated domain the longest correlation time �/3 of 12 ns corre-sponds to the overall rotation of the protein fragment (15 kD). Inthe presence of the 14-3-3fnoW protein a new long component

�/1 (ns) �b2�/2 (ns) �b3

�/3 (ns)

<0.2 0.098 0.6 0.074 >100<0.2 0.054 1.5 0.109 >100<0.2 0.062 1.4 0.093 >100<0.2 0.053 1.3 0.119 >100

Table 2Kinetic parameters of the RGS3229–513W424 mutant.

Protein smean (ns)a �b1�/1 (ns) �b2

�/2 (ns) �b3�/3 (ns)

RGS3229–513W424 5.8 0.089 <0.1 – – 0.111 17RGS3229–513W424 + 14-3-3fnoW 5.0 0.048 <0.1 0.035 2.0 0.117 30pRGS3229–513W424 5.7 0.077 <0.1 – – 0.123 22pRGS3229–513W424 + 14-3-3fnoW 4.5 0.039 <0.1 0.046 2.2 0.115 70


Fig. 5. Time-resolved tryptophan fluorescence anisotropy decays of the RGS3229–

513W424. (A) The effect of the 14-3-3 protein on tryptophan fluorescence ofunphosphorylated RGS3229–513W424 mutant. Lines represent the best fit. (B) Theeffect of the 14-3-3 protein on tryptophan fluorescence of phosphorylatedpRGS3229–513W424 mutant. Lines represent the best fit. Fig. 6. Distributions of rotational correlation times of pRGS3229–513W424. (A) In the

absence of the 14-3-3fnoW protein. (B) In the presence of the 14-3-3fnoW protein.


with the correlation time �/4 > 50 ns and a significant amplitude�b4 = 0.037 appeared in the time-resolved fluorescence anisotropydecay. The appearance of �/4 suggests that a fraction of the isolatedRGS domain complexed with the 14-3-3fnoW protein. The largevalue of �/4 is related to a slow rotation of the resulting high molec-ular-mass complex. Data from Table 3 also indicate the interactionof the RGS domain with the 14-3-3fnoW protein is rather loose,because a rotational movement of the free domain itself is resolv-able in the anisotropy decay in the presence of 14-3-3fnoW(�/3 = 12 ns). Compared to the RGS domain alone, the presence ofthe 14-3-3fnoW protein causes also an appearance of a new shortcomponent with �/1 = 0.3 ns and �b1 = 0.009 indicating changes inthe character of the fast Trp movements upon the 14-3-3fnoWbinding. Recovered changes in distributions of the rotational corre-lation times are graphically presented in Fig. 7. Taking together, weconclude that the isolated RGS domain of RGS3 can interact withthe 14-3-3f protein in a phosphorylation-independent manner.

3.7. Crystal structure of RGS domain of RGS3

Since we used the time-resolved tryptophan fluorescence spec-troscopy as a main tool to study the 14-3-3f protein-induced con-formational changes of the RGS domain of RGS3, we needed toknow the exact location and the interactions of its two conservedtryptophan residues (Trp391 and Trp424). Therefore, we deter-mined the crystal structure of the RGS domain of RGS3 at 2.3 Å res-olution (Table 4). As expected, the domain forms a bundle of ninea-helices that fold into two subdomains similar to other RGS do-mains (Fig. 8). The terminal and smaller subdomain consists ofhelices a1, a2, a3, a8, and a9, whereas the larger subdomain con-tains helices a4, a5, a6, and a7. The structural comparison of theRGS domain of RGS3 with available structures of the B/R4 family,to where RGS3 belongs (Jean-Baptiste et al., 2006), revealed no dra-matic differences. The highest root mean square deviation

Table 3Kinetic parameters for the isolated RGS domain of RGS3 (RGS3384–513).

Protein smean (ns)a �b1�/1 (ns) �b2

�/2 (ns) �b3�/3 (ns) �b4

�/4 (ns)

RGS3384–513 5.0 – – 0.026 1.9 0.200 12 – –RGS3384–513 + 14-3-3fnoW 4.9 0.009 0.3 0.023 2.1 0.164 12 0.037 >50


Fig. 7. 14-3-3 protein interacts with isolated RGS domain of RGS3. (A) Time-resolved tryptophan fluorescence anisotropy decays of RGS domain of RGS3(RGS3384–513) in the presence and in the absence of 14-3-3fnoW. (B) Distributionsof rotational correlation times of RGS3384–513 in the absence of 14-3-3fnoW. (C)Distributions of rotational correlation times of RGS3384–513 in the presence of 14-3-3fnoW.

Table 4Crystallographic data and refinement statistics.

Space group: C 2 (C 1 2 1); a = 77.24 Å,b = 60.85 Å, c = 57.41 Å, b = 135.44�

Asymmetric unit: One monomer of RGS domain of RGS3

Data CollectionWavelength (Å) 1.54179Resolution (Å) 28–2.3 (2.42–2.3)Rsym

a 0.055 (0.214)Completeness (%) 96.7 (97.3)Unique reflections (N) 8099Redundancy (-fold) 2.5 (2.4)Average (I/r) 16.5 (5.3)

Refinement statisticsResolution (Å) 20–2.3No. of reflections 8085Rwork 0.193Rfree

b 0.250Asymmetric unit content 1047 protein atoms,

62 solvent atoms

Average B-factors (Å2)Protein 29.14Water 35.14

Rms deviations from ideal valuesBonds (Å) 0.023Angles (degrees) 1.89

Numbers in parenthesis are for the highest resolution shell.a Rsym = R|Ihkl � Ihkli|/RIhkli, where Ihkl is the observed intensity and Ihkli is the

statistically weighted average intensity of multiple observations of symmetry-related reflections.

b Free R-value (Rfree) was calculated using 5% of reflections omitted from therefinement.


(r.m.s.d.) was observed for the superimposition with the RGS do-main of RGS18 (1.97 Å over 126 Ca atoms) (PDB Accession Code2OWI), while the smallest r.m.s.d. was calculated for the superim-position with the RGS domain of RGS8 (0.65 Å over 126 Ca atoms)(PDB Accession Code 2IHD). Fig. 8A shows the superimposition ofRGS domains of RGS3, RGS8 and RGS18. While the RGS domainsof RGS3 and RGS8 are almost identical, significant differences inthe packing of helices, especially in the larger subdomain (mainlyhelices a6 and a7), can be observed between RGS domains ofRGS3 and RGS18.

The crystal structure of RGS4 bound to Gia1 (Tesmer et al., 1997)revealed that the Ga interaction surface of the RGS domain isformed by the loop between helices a3 and a4, the loop betweenhelices a5 and a6, and the residues from the end of helix a7 andthe beginning of helix a8. On the basis of the structural similaritybetween the RGS domains of RGS4 and RGS3, we have constructeda model of the complex between the RGS3 and Gia1 (Fig. 8B). Whilethe tryptophan Trp391 is located at the N-terminus of the RGS do-main of RGS3 within the helix a1 where it interacts with residuesfrom helices a2, a3, and a9, the tryptophan Trp424 is located inthe center of the domain within the helix a4 and its side-chaininteracts with residues from the helix a2 and the C-terminal endof helix a7. Considering the close proximity of Trp424 to the Gainteraction interface, this tryptophan residue should probe wellthe conformational changes within the interaction interface of

Fig. 8. The crystal structure of RGS domain of RGS3. (A) The superimposition of RGS domains of RGS3 (shown in brown), RGS8 (shown in green) and RGS18 (shown in blue).While the RGS domains of RGS3 and RGS8 are almost identical, significant differences in the packing of helices, especially in the larger subdomain (mainly helices a6 and a7),can be observed between the RGS domains of RGS3 and RGS18. (B) The model of the complex between RGS3 and Gia1 based on the crystal structure of RGS4 bound to Gia1

(Tesmer et al., 1997). The RGS domain of RGS3 contains two tryptophan residues Trp391 and Trp424 (shown in green). Residues located in the close proximity of Trp424 areshown as sticks. The Gia1 interaction surface of RGS domain of RGS3 is formed by the loop between helices a3 and a4, the loop between helices a5 and a6, and the residuesfrom the end of helix a7 and the beginning of helix a8. The major Gia1 binding sites for the RGS domain (switch segments I–III) are shown in red.


the RGS domain of RGS3. Results of both the time-resolved fluores-cence intensity and anisotropy decays of Trp424 (Table 2, Figs. 5and 6) revealed that the 14-3-3fnoW protein binding does changethe structure of the RGS domain of RGS3 in the region surroundingthis tryptophan residue. Therefore, it is reasonable to speculatethat such structural change in the close vicinity of the Ga interac-tion surface could be responsible for the inhibition of RGS3 interac-tion with Ga protein.

4. Discussion

The 14-3-3 proteins are a family of conserved regulatory mole-cules involved in many biologically important processes, includingcell cycle regulation, apoptosis, control of gene transcription, andmetabolism control (Aitken, 2006; Fu et al., 2000; Hermekingand Benzinger, 2006). Three main modes of the 14-3-3 protein ac-

tion were defined (Bridges and Moorhead, 2004): (i) the 14-3-3protein changes the conformation of its binding partner; (ii) the14-3-3 protein physically occludes sequence specific or structuralfeatures of its binding partner and interferes with its protein–pro-tein interactions; and (iii) the 14-3-3 protein functions as a scaffoldmolecule to anchor proteins within close proximity of one another.

The interaction between the 14-3-3 proteins and two RGS pro-teins, RGS3 and RGS7, was initially described by Benzing et al.(2002 and 2000). It has been suggested that the putative 14-3-3binding motif lies within their RGS domains (in the case of RGS7at Ser434). Latter reports, however, revealed that the 14-3-3 bind-ing motif of RGS3 is not located within its RGS domain but withinthe N-terminal region at Ser264 (Niu et al., 2002; Ward and Milli-gan, 2005). It is now generally accepted that the 14-3-3 proteinsnegatively modulate the function of several RGS proteins and inhi-bit their GAP activity presumably by blocking their interactions


with G proteins (Abramow-Newerly et al., 2006a; Benzing et al.,2000; Niu et al., 2002). Since RGS proteins interact with Ga sub-units through their RGS domains, it is reasonable to assume thatthe 14-3-3 protein can either sterically occlude the Ga interactionsurface of RGS domain and/or change its structure. To investigatethe mechanism of the 14-3-3 protein-dependent inhibition ofRGS3 function, we studied the 14-3-3f protein-induced conforma-tional changes of RGS3 using the time-resolved tryptophan fluores-cence spectroscopy.

In this work, we have used a construct of RGS3 (isoform 1) con-sisting of residues 229–513 (Fig. 1). This construct covers the re-gion containing phosphorylation site/14-3-3 binding motifSer264 and the C-terminal RGS domain (sequence 384–513). Theexpressed protein was fairly soluble and stable to be prepared insufficient quantity for spectroscopic studies. The phosphorylationsite Ser264 was quantitatively phosphorylated by PKA (Fig. 2)and the FRET-based binding assay showed that this phosphoryla-tion is necessary for the high affinity binding to the 14-3-3f protein(Fig. 3).

The sequence of RGS3 protein (fragment 229–513) containsthree tryptophan residues. The first one (Trp295) is located closeto the phosphorylation site/14-3-3 binding motif Ser264, thereforeits fluorescence was used to study the conformational changes inthis region upon the phosphorylation and the 14-3-3f proteinbinding (Table 1). Our results clearly show that both the phosphor-ylation of Ser264 itself and the binding of 14-3-3fnoW to the phos-phorylated pRGS3229–513W295 protein affect the structure of RGS3in the vicinity of Trp295. This is in agreement with previous re-ports that the motif around Ser264 is the binding site for the 14-3-3 protein (Niu et al., 2002; Ward and Milligan, 2005). In addition,our data also show that the 14-3-3fnoW protein interacts, to someextent, with unphosphorylated RGS3229–513W295.

To monitor conformational changes within the RGS domain atthe C-terminus of RGS3 molecule, we used the tryptophanTrp424. This residue is located in the middle of the domain closeto the Ga interaction surface (Fig. 8B). Results of both the time-re-solved tryptophan fluorescence intensity and anisotropy decays re-vealed that the 14-3-3fnoW protein binding changes the structureof the RGS domain of RGS3 in the region around Trp424 (Table 2).According to our best knowledge, this is the first demonstrationthat the 14-3-3 protein binding affects the structure of RGS do-main. Thus it is reasonable to speculate that such structural changein the close vicinity of the Ga interaction surface could be respon-sible for the inhibition of RGS3 interaction with Ga protein. Ourdata, similarly as in the case of Trp295 mutant, also suggest thatthe unphosphorylated RGS3229–513W424 weakly interacts withthe 14-3-3fnoW protein.

The observed structural change in the RGS domain of RGS3could be the result of either direct interaction between the 14-3-3f protein and the RGS domain or the interaction between theRGS domain and some other region of RGS3 molecule affected bythe 14-3-3 protein binding. Since both the FRET-based binding as-say (Fig. 3) and the tryptophan fluorescence measurements sug-gested interaction between the 14-3-3fnoW protein andunphosphorylated RGS3, it is entirely possible that the 14-3-3fprotein might directly interact with the RGS domain itself in aphosphorylation-independent manner. To test this possibility, westudied the interaction between the isolated RGS domain of RGS3(sequence 384–513) and the 14-3-3fnoW protein (Table 3,Fig. 7). These experiments strongly indicate that the isolated RGSdomain of RGS3 can interact, although rather weakly, with the14-3-3f protein. It is unclear whether this interaction has anyphysiological importance, but many of the 14-3-3 protein bindingpartners contain two or more 14-3-3 binding motifs. It has beensuggested that for these multi-site ligands, one binding site islikely to function as a ‘gatekeeper’ whose phosphorylation is neces-

sary for the 14-3-3 protein binding but may not always be suffi-cient for full biological activity (Yaffe, 2002). Thus thephosphorylation site Ser264 might be such ‘gatekeeper’ responsi-ble for stable association of 14-3-3 with RGS3 and the RGS domaincontains second site (unphosphorylated) whose interaction withthe 14-3-3 protein induces structural modulation of RGS domainand inhibits its GAP activity.

Acknowledgments

This work was supported by Grant Agency of the Academy ofSciences of the Czech Republic Grant IAA501110801; Ministry ofEducation, Youth, and Sports of the Czech Republic Research Pro-jects MSM0021620857 and MSM0021620835 and Center of Neuro-sciences LC554; and Academy of Sciences of the Czech RepublicResearch Project AV0Z50110509. We thank Dr. Ondrej Julinek forhelping with the measurements of circular dichroism spectra. Wealso thank Dr. Jiri Brynda for helping with the collection of X-raydiffraction data.

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14-3-3 protein interacts with and affects the structure of rgs domain of regulator of g protein...

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