Autoinhibition and phosphorylation-induced activationmechanisms of human cancer and autoimmunedisease-related E3 protein Cbl-bYoshihiro Kobashigawaa, Akira Tomitakab, Hiroyuki Kumetaa, Nobuo N. Nodaa,1,Masaya Yamaguchia, and Fuyuhiko Inagakia,2

aDepartment of Structural Biology, Faculty of Advanced Life Science, and bGraduate School of Life Science, Hokkaido University, Sapporo,Hokkaido 001-0021, Japan

Edited by* Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, and approved September 28, 2011 (received for review July 6, 2011)

Cbl-b is a RING-type E3 ubiquitin ligase that functions as a negativeregulator of T-cell activation and growth factor receptor and non-receptor-type tyrosine kinase signaling. Cbl-b dysfunction is relatedto autoimmune diseases and cancers in humans. However, themolecular mechanism regulating its E3 activity is largely unknown.NMR and small-angle X-ray scattering analyses revealed that theunphosphorylated N-terminal region of Cbl-b forms a compactstructure by an intramolecular interaction, which masks the inter-action surface of the RING domain with an E2 ubiquitin-conjugat-ing enzyme. Phosphorylation of Y363, located in the helix-linkerregion between the tyrosine kinase binding and the RING domains,disrupts the interdomain interaction to expose the E2 bindingsurface of the RING domain. Structural analysis revealed that thephosphorylated helix-RING region forms a compact structure insolution. Moreover, the phosphate group of pY363 is located in thevicinity of the interaction surface with UbcH5B to increase affinityby reducing their electrostatic repulsion. Thus, the phosphorylationof Y363 regulates the E3 activity of Cbl-b by two mechanisms:one is to remove the masking of the RING domain from the tyro-sine kinase binding domain and the other is to form a surface toenhance binding affinity to E2.

The Cbl proteins (c-Cbl, Cbl-b, and Cbl-3) belong to a familyof RING-type ubiquitin ligases. Like other RING domainproteins, the Cbl proteins function as adaptor proteins, simulta-neously binding to a cognate E2 ubiquitin-conjugating enzymeand a substrate protein, leading to transfer of ubiquitin to thesubstrate. This facilitates degradation of the target substrate byproteasomes or, in some cases, lysosomes. The Cbl proteins func-tion as a negative regulator of T-cell activation, growth factorreceptor [e.g., epidermal growth factor receptor (EGFR), c-KIT,and platelet-derived growth factor receptor (PDGFR)], and non-receptor-type tyrosine kinase signaling (e.g., Src family kinasesand Zap70) (1, 2), and dysfunctional mutations in Cbl proteinshave been related to human cancer (3–7). Of the three Cbl pro-teins, Cbl-b plays a critical role in the down-regulation of immu-nological signaling to induce T-cell anergy (8, 9). Cbl-b knockoutmice have been shown to exhibit severe autoimmune diseases(10), whereas, in humans, a dysfunctional mutation in Cbl-b wasshown to be related to type I diabetes (11) and multiple sclerosis(12). Hence, the Cbl proteins are considered to be a potentialtherapeutic target.

Cbl-b and c-Cbl exhibit high sequence homology in theN-terminal region with 86% amino acid identity and share a con-served tyrosine kinase binding (TKB) domain comprised of afour-helix bundle, a Ca2þ-binding EF hand domain and a variantSH2 domain (13, 14), as well as a short helix-linker region and aRING finger domain that directly associates with E2 proteins(Fig. 1A). The ubiquitin ligase activity of Cbl-b is known to beup-regulated by the phosphorylation of Y363 (Y371 in c-Cbl),which is located in the helix linker (15, 16). The mutation of thiscritical tyrosine residue to phenylalanine abolishes the E3 activityof Cbl (17). Moreover, the E3 activity of the Cbl proteins is

reported to be negatively regulated by the TKB domain, showingthat tyrosine phosphorylation removes Cbl protein autoinhibi-tion due to the interaction between the RING and TKB domains.Protease susceptibility analysis has also revealed that the phos-phorylation of the Y363 of Cbl-b induces a large conformationalchange that is sensitive to protease digestion (15). However, themanner in which the molecular mechanism underlying this con-formational change leads to the upregulation of E3 activityremains elusive.

The crystal structure of the c-Cbl N-terminal domain, consist-ing of the TKB domain, the helix linker, and RING in complexwith UbcH7 (E2) was reported (18). Although this study wasthe first to report on the complex between RING-type E3 and E2and provided significant insights into the interaction mechanism,the structural basis for the enhancement of c-Cbl ligation activitythrough Y371 phosphorylation remains largely unknown.

Here we report the structural analysis of the Cbl-b N-terminalhalf (Cbl-b39–426; hereafter CBLB-N) both in the unphosphory-lated and Y363-phosphorylated states (hereafter pY CBLB-N)using small-angle X-ray scattering (SAXS) and NMR spectro-scopy, and discuss the structural mechanism of autoinhibition andY363 phosphorylation induced activation of Cbl-b E3 ligase.

ResultsOverall Structural Changes in CBLB-N Due to Y363 Phosphorylation.First, we studied the overall structures of both unphosphorylatedand phosphorylated CBLB-N in solution using SAXS, as Y363was reported to be responsible for phosphorylation-induced liga-tion activity (15). The radii of gyration (Rg), as estimated by theGuinier approximation (19), were 24.0 and 26.7 Å for CBLB-Nand pYCBLB-N, respectively (Fig. 1B), suggesting that CBLB-Nis more elongated in the phosphorylated state than in the un-phosphorylated state. Protease susceptibility analysis of Cbl-brevealed that phosphorylated Cbl-b is labile to protease cleavage(15). These results taken together support the idea that pYCBLB-N is more extended and more mobile than CBLB-N,indicating that the conformational changes in CBLB-N areinduced by phosphorylation at Y363.

Author contributions: Y.K. and F.I. designed research; Y.K., A.T., H.K., N.N.N., and M.Y.performed research; Y.K. and A.T. contributed new reagents/analytic tools; Y.K., A.T.,H.K., and N.N.N. analyzed data; and Y.K. and F.I. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Freely available online through the PNAS open access option.

Data deposition: The NMR, atomic coordinates, chemical shifts, and restraints reported inthis paper have been deposited in the BioMagResBank, www.bmrb.wisc.edu (accessionno. Q28: BMR17680), and the atomic coordinates have been deposited in the Protein DataBank, www.pdb.org (PDB ID codes 2LDR and 3VGO).1Present address: Institute of Microbial Chemistry, Tokyo 141-0021, Japan.2To whom correspondence should be addressed. E-mail: finagaki@pharm.hokudai.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1110712108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1110712108 PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 ∣ 20579–20584

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To obtain further insights into the conformational changesinduced by phosphorylation at Y363, we prepared segmentalisotope-labeled CBLB-N using the sortase-mediated protein liga-tion method (20, 21). Here, uniformly 15N-labeled helix-RING(H-RING) was enzymatically attached to the nonlabeled TKBdomain. The detailed protocol for the preparation of segmentalisotope-labeled CBLB-N is shown in Fig. S1 A–C and SI Materialsand Methods. We confirmed that the E3 activity of segmentalisotope-labeled CBLB-N and pY CBLB-N was almost identicalto the authentic proteins (Fig. S1 D and E) though there is aninsertion sequence of LPETGG prior to H-RING. Fig. 1C showsa comparison of the 1H-15N heteronuclear single quantum coher-ence (HSQC) spectra of the segmental isotope-labeled CBLB-Nin the unphosphorylated and phosphorylated states. In the un-phosphorylated state, the H-RING moiety in CBLB-N exhibitedbroad NMR signals (red). Because CBLB-N was confirmed tobe monomeric by SAXS at 300 uM (Fig. S1 F and G), signalbroadening was caused by restricted mobility of the H-RINGmoiety through its possible involvement into the core structure ofCBLB-N rather than aggregation. In contrast, in pY CBLB-N,the H-RING moiety exhibited sharp, well-dispersed signals(blue), indicating that the H-RING moiety is mobile and inde-pendent from the TKB core. These NMR observations are con-sistent with the results from the SAXS and protease susceptibilityanalyses, indicating that pYCBLB-N contains a mobile H-RINGmoiety, whereas the H-RING moiety in CBLB-N is incorporatedinto the core structure. Thus, pY H-RING is considered to be astructural and functional unit possessing ligation activity.

Closed Structure of CBLB-N in the Unphosophorylated State. The1H-15N HSQC spectrum of the segmental isotope-labeledCBLB-N at the H-RING moiety was superimposed onto that ofthe isolated RING domain (Fig. 2A). Spectral overlay revealedthat the RING moiety in CBLB-N exhibited broad signals (red).A comparison with the isolated RING domain (blue) showed thatsome peaks were absent, possibly due to line broadening by theintermediate exchange process. The residues absent in CBLB-Nwere mapped on the RING domain in the crystal structure ofc-Cbl complexed with UbcH7 (18) (Fig. 2B). Some of the absentresidues were located at the binding interface with E2 (enclosedby a dotted circle), supporting the notion that the RING moietysurface required for binding to E2 partially overlaps with thatrequired for the interdomain interaction with the TKB domainand helix (hereafter TKB-H).

The interface of the RING moiety and TKB-H was alsostudied by comparing the 1H-13C heteronuclear multiple quan-tum coherence (HMQC) spectra of Ile δ1-methyl-labeledCBLB-N (red) and the isolated RING domain (black) (Fig. 2C).Spectral overlay revealed that there was an appreciable shift inthe δ1-methyl signals from I375 and I421 between these con-structs, which is consistent with the finding that the I375 mainchain amide (HN) signal also disappeared in the segmental iso-tope-labeled CBLB-N, indicating that I375 and I421 are involvedin the interface with TKB-H. The shifted (red) and nonshifted(yellow) Ile residues were mapped on the RING domain in thecrystal structure of c-Cbl complexed with UbcH7 (18) (Fig. 2D).It should be noted that Ile375 is located at the binding interfacewith E2 (enclosed by a dotted circle).

In order to determine the binding interface of TKB-H andthe RING moiety, methionine 13C methyl-labeled samples undera deuterium background were prepared for CBLB-N and TKB-H, and their 1H-13C HMQC spectra were overlaid (Fig 2E).Resonance assignments of Met methyl signals were obtained byacquiring the spectra of a series of mutants in which the methio-nine was replaced by Lys for surface exposed residues and by Ileor Leu for buried residues (Fig. S2). Differences in the positions

Cbl-b

c-Cbl 4H EF SH2 RING

Y371

PRR UBA 906

4H EF SH2 RING

Y363

PRR UBA 982

TKB domain Helix

Sequence Identity

83% 90%86%

13511.0 10.0 9.0 8.0 7.0

HN (ppm)

130

125

120

115

110

105

N (ppm

)

A

B C

0.01

0.1

1

10

100

0.05 0.1 0.15 0.2 0.25

q ( -1)

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nsity

0

1

2

0.002 0.003 0.004 0.0

Å

Å

05 0.006

ln (

Inte

nsity

)

q2 ( -2)

4H EF SH2 RING

Y368

39 42635115N14N

351 37539 426

CBLB-N

H-RINGTKB-H375

39 426

351 42639

343

Fig. 1. (A) Domain structures of c-Cbl and Cbl-b. (B) SAXS measurements ofCBLB-N (red) and pY CBLB-N (blue). Inset represents the Guinier plots forCBLB-N (red) and pY CBLB-N (blue). (C) Overlay of the 1H-15N HSQC spectrabetween the segmental isotope-labeled CBLB-N (red) and pY CBLB-N (blue) atthe H-RING moiety.

A

B

C

D

I375

I415

I421

I422I385

1.2 1.0 0.8 0.6 0.4 0.2

H (ppm)

14.0

12.0

10.0

8.0

C (ppm

)

180°I375

I422

I421

I415

I422

180°

I375

F370

K374

L372

E378

C411R412C413

L391K381 F370

C376

E378N379

D380

T394V383

R412

E

FM53

M132

M214

M115

M365

M261

180°

90°

M53

M115

M261

M153

2.2 2.0 1.8 1.6 1.4 1.2 1.0H (ppm)

17.0

16.0

15.0

14.0

13.0

C (ppm

)

M392

M132

*

M365

M214

M53 M214*

M115

M365

10.0 9.0 8.0 7.0HN (ppm)

130

125

120

115

110

N (ppm

)

M392

C393T394

K416

K374

C373

R412

I375

L372

F426

N379

C376E378

D380

F410

S368

Q371L391

C396

T398

K384I385

I415

C408

S403

G405G417

T418(s)

E419(s)

G389G407

T418

D404C411

E402

V383E414

A399I422(s)

W400

C388L397

E386

K381

Q401 C413

E419V423

V423(s)I422D424

A377

S395

H390

D382

F370

I421*

G367

Fig. 2. (A) Overlay of the 1H-15N HSQC spectra of the segmental isotope-la-beled CBLB-N (red) at the H-RING and the isolated RING domain (blue). Signalassignment of the isolated RING is shown in the spectrum. (B) Lost residues(red) in the 1H-15N HSQC spectrum of the segmental isotope-labeled CBLB-Nwere mapped on the structure of the c-Cbl-N RING domain (1FBV). Red: re-presents lost or shifted residues, Black: removed from the analysis due tobeing missed from the peaks in the 1H-15N HSQC spectrum of the isolatedRING domain or the N-terminal residue in the isolated RING domain, and Yel-low: represents nonshifted but observed peaks. The dotted circle representsthe E2 binding region. (C) Overlay of the 1H-13C HMQC spectra of the Ile δ1methyl selectively labeled CBLB-N (red) and the isolated RING domain (black).Shifted peaks are shown by arrows. Signal assignment of the isolated RING isshown in the spectrum. (D) Shifted (red) and nonshifted (yellow) peaks in Cwere mapped on the structure of the c-Cbl-N RING domain (1FBV). Thedotted circle represents the E2 binding region. (E) Overlay of the 1H-13CHMQC spectra of the Met methyl signal selectively labeled CBLB-N (red)and TKB-H (blue). (F) Methionine residues were mapped on the structureof the TKB-H region of c-CBL-N (1FBV). The helix region is colored pink.

20580 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1110712108 Kobashigawa et al.

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of the methyl peaks of M214 and M365 were observed betweenCBLB-N and TKB-H, suggesting that M214 and M365 are in-volved in the binding with the RING moiety. However, as M365is located close to the C terminus of TKB-H, the M365 peak shiftcould be due to a truncation effect. M365 exhibited a sharp peak,whereas that of M214 was appreciably broadened by an inter-mediate chemical exchange process, presumably between theopen and closed states. Therefore, M214 is considered to belocated at the interface with the RING moiety. The Met residues(M214 is colored red and the others yellow) were mapped on theTKB-H region in the crystal structure of c-Cbl complexed withUbcH7 (18) (Fig. 2F). Although M261 is located in the vicinityof M365, it did not exhibit any peak shift, supporting the idea thatthe peak shift of M365 is due to a truncation effect.

We have also performed a crystallographic analysis of CBLB-N. Unfortunately, due to the perfectly twined nature of theCBLB-N crystal, as well as the dynamic feature of the RINGmoiety, we could not construct a model of the RING moiety,although a high electron density was observed around M214. Thissupports the NMR findings that there exists an interdomaininteraction between TKB-H and RING in CBLB-N.

The interfaces between RING and TKB-H and UbcH7 par-tially overlap with each other (Fig. 2 B and D) so that the associa-tions of RING with TKB-H and E2 are mutually exclusive. Theconformational change induced by the phosphorylation of Y363may be required to disrupt the interaction between RING andTKB-H, thereby facilitating the exposure of the RING domainand subsequent association with E2.

Solution Structure of the pY H-RING. The 1H-15N HSQC signals ofpY H-RING (Fig. 3A in red) almost entirely overlapped withthose of the H-RING moiety in pY CBLB-N (Fig. 3A in blue).This supports the notion that the H-RINGmoiety in pYCBLB-Nis exposed and independent from the TKB core and that its struc-ture is similar to that of pY H-RING. We subsequently deter-mined the solution structure of pY H-RING by NMR (Fig. 3Band Fig. S3A). Fig. 3B shows the structure of pY H-RING. TheRING moiety is composed of two large Zn2þ-binding loops, ashort three-stranded antiparallel β-sheet, and a central α-helix, asshown in pink in Fig. 3B. The overall structure of the RING moi-ety is very similar to those of other RING domains (18, 22–30).However, the phosphorylation of Y363 induces an additionalstructure that includes the formation of a parallel β-sheet be-tween the N terminus of the helix linker and the C terminus ofthe RING domain so that the RING domain has extensive con-tact with the helix linker. Steady-state NOE analysis supportedthat RING as well as helix linker and the linker region of N andC terminus of pY H-RING formed rigid structures in solution(Fig. 3B, Fig. S3A, and Table S1). Moreover, we observed longrange NOEs between K374 Hϵ and the aromatic ring protonof pY363, which was further supported by NOEs between sur-rounding residues. The interaction between the RING domainand the helix linker is stabilized by the electrostatic interactionbetween the phosphate group of pY363 on the helix linker andthe positively charged cluster formed by K374 and K381 in theRING domain (Fig. 3B). These interactions make pY H-RINGa single structural unit presenting the RING domain to E2. Thisnotion was supported by the fact that Lys Hζ proton signals ofboth K374 and K381 were observed in the 1H-15N HSQC spectra(Fig. S3C). The Hζ proton signal from Lys can only be observed incases where chemical exchange with solvent proton is highly re-stricted due to hydrogen bond formation. We also confirmed theinteraction of K374 and K381 with the phosphate group of pY363by the mutational analysis of H-RING in which K374 and K381were replaced by Glu. These mutants did not show significantspectral changes upon Y363 phosphorylation (Fig. S3 E and F)and gave similar spectra to that of H-RING in contrast to the caseof the wild type (Fig. S3D). In summary, the arrangement of the

helix linker relative to the RING domain was markedly changedby the phosphorylation of Y363, thereby releasing the helix linkerand RING from the TKB domain and exposing the H-RINGmoiety.

NMR Examination of the Interaction Between the E2 Protein UbcH5and pY H-RING. To elucidate the biological implications of thestructural changes induced by the phosphorylation of Y363 inH-RING, the interaction of pY H-RING with UbcH5B wasstudied by chemical-shift perturbation methods using solutionNMR (Fig. S3 G and H). First, unlabeled UbcH5B was titratedto 15N-labeled pY H-RING. Upon the addition of aliquots ofUbcH5B, some of the peaks in 15N-labeled pY H-RING weregradually shifted by a fast exchange process. From the chemical-shift change of S403, we estimated that the Kd value for the bind-ing of pY H-RING to UbcH5B is 1.2 μM (SDof � 0.4 μM).Next, we mapped the residues with large chemical-shift perturba-tions on the structure of pY H-RING. Most of the residueswith large chemical-shift perturbations in the NMR spectra uponcomplex formation are located in the two regions: residues 374–379 and 408–411 in the loop regions, and residues 397–408 in thehelix of the RING domain (Fig. 3C and Fig. S3G). On the otherhand, the helix linker was not perturbed, indicating that thisregion is not directly involved in the interaction with UbcH5B.The interaction region was similar to the previous report forc-Cbl H-RING in the unphosphorylated state (24).

Fig. 3. (A) Overlay of the 1H-15N HSQC spectra of the H-RING moiety in pYCBLB-N (blue) and pY H-RING (red). (B) Ribbon (Left) and surface potential(Right) representations of the solution structure of pY H-RING. pY363 andits interacting positively charged residues, K374 and K381, are shown as astick model. (C) The pY H-RING residues shifted upon the addition of 1.5equivalent molar ratio of UbcH5B were mapped on the structure of pYH-RING. Red represents chemical-shift changes larger than 0.35 ppm, orangeof 0.25, and yellow of 0.15 ppm. Black represents Pro or missing residues.(D) The UbcH5B residues shifted upon addition of 1.5 equivalent molar of pYH-RING were mapped on the structure of UbcH5B. Green represents chemi-cal-shift changes larger than 0.05 ppm and magenta represents missing re-sidues. Black represents Pro residues. (E) Docking model between pY H-RINGand UbcH5B created by the structural overlay of the crystal structure c-CBL-Nin complex with the E2 protein UbcH7 (1FBV). (F) UbcH5B is shown as a sur-face charge model, and H-RING (model based on c-Cbl from 1FBV) and pYH-RING as a ribbon model. H-RING is colored blue and pY H-RING pink. Sidechains of pY363, K374, and K381 are shown as a stick model.

Kobashigawa et al. PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 ∣ 20581

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Next, unlabeled pY H-RING was titrated to 15N-labeledUbcH5B (Fig. 3D and Fig. S3H). Upon addition of aliquots ofpY H-RING, some of the UbcH5B peaks disappeared in inter-mediate exchange process or gradually shifted in fast exchangeprocess. Slight precipitation of UbcH5B was observed upon ad-dition of pY H-RING so that the Kd value could not be estimatedfrom this experiment. The largely affected residues in UbcH5Bwere located in the first helix, the loop between strands β3 andβ4, and the loop region connecting the second and third helices(Fig. 3D and Fig. S3H). Interaction regions were similar to theprevious report for c-Cbl H-RING in the unphosphorylated state(24). To our knowledge, there are five RING domains for whichthe complex structures with E2 have been reported (18, 22, 23, 25,26); CBL RING/UbcH7 [Protein Data Bank (PDB) ID: 1FBV],cIAP2 RING/UbcH5B (PDB ID: 3EB6), TRAF6 RING/Ubc13(PDB ID: 3HCT), Ring1b/UbcH5C (PDB ID: 3RPG), and IDOLRING/UbcH5A (PDB ID: 2YHO). The interaction regions be-tween the RING domain and E2 in these protein complexes werefound to be essentially the same (Fig. S3I), which was also sup-ported by NMR and/or mutational analysis for other RING do-mains of MDM2 (27), CNOT4 (28, 29), and BRCA1 (30). Thus,we constructed the complex model between pY H-RING andUbcH5B by overlaying the RING domain of pY H-RING andUbcH5B to the RING region and UbcH7 of the c-Cbl/UbcH7complex structure (1FBV) (18), respectively (Fig. 3E). The com-plex model is consistent with and supported by the results ofthe NMR titration analysis, despite the fact NMR data were notconsidered at all for the model construction.

Affinity of CBLB-N and CBLB-N Variants Toward UbcH5B.Fluorescencepolarization spectroscopy (hereafter FP) was next used to eval-uate the affinity of CBLB-N and CBLB-N variants for UbcH5B(Fig. S4). Titration measurements of the affinity of CBLB-Ntoward Alexa 488-labeled UbcH5B revealed the estimated Kdvalue of 97.5 μM (SDof � 3.3 μM). CBLB-N has a much loweraffinity to UbcH5B than isolated H-RING (22.7 μM: SDof�1.0 μM), indicating the masking of the RING moiety inCBLB-N. The affinity of pY CBLB-N toward UbcH5B was mea-sured, and the Kd value was estimated to be 1.1 μM (SDof�0.1 μM), which is similar to that of isolated pY H-RING(1.2 μM: SDof � 0.4 μM) obtained by NMR, indicating that themasking was released in pY CBLB-N. This is consistent with theresults of the SAXS and NMR measurements. It should be notedthat a comparison of the Kd values between H-RING and pYH-RING revealed that the phosphorylation of Y363 increasesthe affinity toward UbcH5B by about 20-fold. It can be concludedthat the phosphorylation of Y363 in CBLB-N facilitates theassociation of the E2 protein, not only through unmasking butalso through formation of a proper E2 binding surface. Indeed,the phosphate group of pY363 in pY H-RING interacts withK374 and K381 to fix the structure of the H-RING moiety andto reduce its basic surface potential (Fig. 3 B and F). This surfaceis partially involved in the binding to the conserved, positivelycharged surface of the α1 helix in UbcH5B so that neutralizationof the positively charged surface by the phosphate group of Y363increases the binding affinity between RING and UbcH5B(Fig. 3F).

Next, the E3 activity of the Cbl-b variants was studied by mea-suring their autoubiquitination activity. Consistent with previousdata (15, 16), the ubiquitination activity of CBLB-N was mark-edly enhanced by Y363 phosphorylation (Fig. 4 B and C), whichalso supports our structural data for phosphorylation-inducedCBLB-N unmasking and activation.

The Closed Structure of CBLB-N Based on a Docking Study, the Designof Mutants, and Ubiquitination Assay. According to the NMR andFP data, the RING domain appears to be masked by TKB-H,thereby reducing the binding affinity of the RING moiety to E2.

We, therefore, designed several TKB domain mutants in whichthe interaction between TKB-H and RING was disrupted. Priorto mutant design, the CBLB-N structure was modeled by rigidbody docking between TKB-H and RING using the HADDOCKprogram (31), incorporating the NMR chemical-shift perturba-tion data (Fig. 2). A detailed description of the docking calcula-tions between TKB-H and RING is given in Table S2 and thedocking structure is shown in Fig. S5 A and B and statisticsare shown in Table S2. In the crystallographic analysis, we couldobserve a low but extensive electron density around the RINGregion in the HADDOCK model (Fig. S5C and Table S3), whichsupports the NMR- and HADDOCK-derived model. The RINGmoiety fitted into a shallow groove on TKB-H, and the interac-tion between TKB-H and the RING moiety seemed to be mainlyelectrostatic in nature (Fig. S5B). We, therefore, focused on theexposed charged residues on the TKB-H surface. Four mutantsin the TKB region, D112R, K129E/R131E, K195E/R198E, andD226R (Fig. 4A), were prepared and applied to the autoubiqui-tination assay. These mutants exhibited two- to sevenfold higherE3 activity than the wild type. This supported the idea that theE3 activity of CBLB-N is autoinhibited by the interdomain inter-action with the TKB region.

We next designed RING domain mutants that would disruptthe masked structure. From the docking study and the data forthe TKB mutants, the masking was expected to be maintainedmainly by electrostatic interaction. Hence, we focused on thecharged residues, the signals of which disappeared in the segmen-tal-labeled CBLB-N (Fig. 2B). We then performed autoubiquiti-nation assay of three RINGmutants, E378R, K381E, and R412E.Among these mutants, K381E exhibited markedly higher activity,whereas E378R and R412E exhibited lower activity comparedto CBLB-N. As E378 and R412 are located on the interfacebetween RING and E2 (18), the lower activity of these mutantsis thought to be disruption of association with UbcH5B. Intrigu-ingly, R420 in c-CBL (R412 in Cbl-b) is a mutational hot spot in

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Fig. 4. Autoubiquitination assay of CBLB-N variants. (A) The mutation sitesin the TKB-H region were mapped and colored yellow. The helix region iscolored pink (Left). Mutation sites in the RING moiety were mapped andcolored yellow (Right). (B) Time course of the autoubiquitination of CBLB-N, pY CBLB-N, and CBLB-N mutants. Ubiquitination was monitored by theappearance of a ladder pattern in the Western blotting. For all autoubiqui-tination experiments, N terminus hexahistidine-tag-attached ubiquitin wasused and detected using anti-histidine-tag antibody. (C) Autoubiquitinationactivity of pY CBLB-N and CBLB-N mutants relative to that of the wild type.Samples from the autoubiquitination assay at a time course of 60 min wereapplied for Western blotting in single SDS-PAGE gel, and the band densitywas quantified using ImageJ software. Reaction reached a plateau within15 min for pY CBLB-B, thus underestimate the relative activity obtainedat 60 min. Error bar indicates standard deviation.

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human cancer malignancies (3–7). On the other hand, CBLB-N(K381E) exhibited the highest activity among the mutants westudied. From these observations, K381 may be located in theinterface region between RING and TKB-H domains. Thus, theresults of E3 activity, as well as the FP and SAXS analyses,support our view that the dynamic structural change induced byY363 phosphorylation enhances the E3 activity of Cbl-b.

DiscussionThe present structural analyses of CBLB-N revealed that theinterdomain interaction between RING and TKB-H leads to theformation of a compact structure in the unphosphorylated statein which the binding site between the RING domain and E2 ismasked by TKB-H. In contrast, pY CBLB-N has an extendedstructure in which the phosphorylated H-RING moiety is freelyaccessible to the E2 proteins. Moreover, the H-RING moietymarkedly changes its structure upon phosphorylation, therebyincreasing its affinity to the E2 protein. This was confirmed bybiophysical and biochemical analyses together with mutationalanalysis. However, this process is not merely a closed-to-openconformation change induced by Y363 phosphorylation.

First, we considered the structure of CBLB-N in the closedstate. We modeled a closed CBLB-N structure by docking theRING domain with the crystal structure of TKB-H (modeledfrom c-Cbl: 1FBV) on the basis of chemical-shift perturbationstudies (Fig. 2) using the HADDOCK program. Most of theRING residues on the TKB-H binding interface disappeared,indicating that these residues are in an intermediate exchangeprocess and showing that CBLB-N in the unphosphorylated stateis in an equilibrium between a closed and a partially open state(Fig. 5A). This view is consistent with the basal E3 activity ofCbl-b in the unphosphorylated state as well as the crystal struc-ture of CBLB-N, where the electron density of the RING domainis diffusive. This suggests that the RING domain is not fixed but isin a dynamic equilibrium between several forms. The partiallyopen state may be considered to be an ensemble of multiplestates, one of which may be similar to the crystal structure ofc-Cbl in which the RING moiety is released from the TKB regionwhile the helix region is fixed on the TKB domain (18). In some ofthe partially open states, both the helix and the RING regionsmay be released from the TKB domain so that the Y363 ofCbl-b or the Y371 of c-Cbl becomes susceptible to phosphoryla-tion. Once phosphorylated, the helix region is detached from theTKB domain and interacts with the RING domain through ionicinteractions between the phosphate group of pY363 and thepositively charged cluster in the RING domain. A comparison ofthe 1H-15N HSQC spectra of the segmental isotope-labeled pY

CBLB-N at the H-RING moiety with the isolated pY H-RINGdomain clearly supports the idea that the H-RING moiety inpY CBLB-N is exposed and mobile, in a manner similar to theisolated pY H-RING. Thus, the helix region and the RING do-main work together as a structural and functional unit to recruitE2 proteins. The structure of pYCBLB-N was subsequently mod-eled based on the structure of TKB and pY H-RING. Becauseof the β-sheet formation between the N and C termini of theH-RING moiety, the location of the H-RING moiety relative tothe TKB domain may be significantly different from that ob-served in the crystal structure of c-Cbl complexed with UbcH7,and close to the Zap70 phosphotyrosine-containing peptide bind-ing site, particularly as the linker between TKB and H-RINGis comprised of four residues (Fig. 5A and Fig. S6).

From the present structural and functional analyses, the fol-lowing model for Cbl-b signaling can be proposed (Fig. 5B).Upon activation of the cell surface receptors, including the T-cellreceptor, EGFR, PDGFR, c-KIT, and so on, the tyrosine phos-phorylation of the receptors by protein tyrosine kinases createsa binding site for the Cbl-b TKB domain, thus recruiting Cbl-b.Because of the intramolecular interaction of the RING domainwith TKB-H, Cbl-b is in a closed state that masks the binding sitefor the E2 protein. However, when Cbl-b is phosphorylated, theH-RING becomes exposed, thereby enhancing the affinity forthe E2 protein. Thus, activated Cbl-b catalyzes ubiquitination ofthe receptor- and nonreceptor-type tyrosine kinases to down reg-ulate signaling. In conclusion, our structural and biochemicalstudies have revealed a regulatory mechanism of the E3 activityof Cbl-b, which is closely related to cancer as well as autoimmunediseases in humans.

Materials and MethodsSee detail also for SI Materials and Methods.

Protein Expression and Purification. All the proteins were expressed as hexa-histidine tag fusion proteins using Escherichia coli strain Rossetta (DE3) at25 °C, purified using Ni2þ-affinity column chromatography, followed byHRV3C protease digestion to remove the tag, and further purified by gelfiltration chromatography using Superdex 75 (GE Healthcare).

Fluorescence Labeling of the Protein and Fluorescence Polarization Measure-ment. Alexa 488-conjugated UbcH5B was prepared as described previously(32). Fluorescence polarization was measured at 22 °C with a buffer contain-ing 20 mM MES (pH 7.0) and 150 mM NaCl with 1 μM of Alexa 488-labeledUbcH5B using an RF-5300PC (Shimadu) fluorescence spectrometer. Aliquotsof the solution containing wild-type or mutant CBLB-N, pY CBLB-N, pYH-RING, or H-RING were added to this sample and the fluorescence anisotro-py was measured.

A

B

Fig. 5. (A) Schematic representation of the regulation ofE3 activity of Cbl-b by phosphorylation. CBLB-N is in equi-librium between the closed inactive and open partially ac-tive states. In the closed state, the E2 binding region ismasked by the TKB region, but in the open partially activestate, the E2 binding region is open. Upon phosphoryla-tion of Y363, the H-RING region shows a marked changein its structure to completely expose the E2 binding sur-face and moves to an open fully active state. (B) Schematicrepresentation of the regulation of E3 activity of Cbl-b byphosphorylation. Cbl-b is in a closed state and the E2 bind-ing region of RING is masked by the TKB region. Uponstimulation, the receptor is phosphorylated and subse-quently recruits Cbl-b via association with the TKB do-main. The Y363 of Cbl-b is phosphorylated by kinasesthat are also recruited to the receptor site, and Cbl-b be-comes open. Phosphorylation-induced unmasking andconformation change enhances the affinity of H-RINGto the E2 protein, facilitating the ubiquitination of the re-ceptor to down regulate the signals.

Kobashigawa et al. PNAS ∣ December 20, 2011 ∣ vol. 108 ∣ no. 51 ∣ 20583

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Protein Ligation and Purification of the Product. Protein ligation was carriedout at room temperature as described previously (20). Ligation product wasdigested by HRV3C protease, followed by pass through the Ni-nitrilotriace-tate, and further purified by gel filtration using Superdex 75 (GE Healthcare)(Fig. S1).

Phosphorylation of CBLB-N and H-RING. The phosphorylation reaction wascarried out using fusion protein between c-Src kinase domain and Zap70(SDGYTPEP) fragment to enhance phosphorylation efficiency (33). The phos-phorylation reaction was monitored by SDS-PAGE and immunoblotting usingPY20 (Zymed), a mouse monoclonal antibody against phosphotyrosine.Specificity of the phosphorylation at Y363 was confirmed using the Y363Fmutant protein (Fig. S7).

In Vitro Ubiquitination Assay. Ubiquitination reaction was carried out at30 °C in 25 μL of reaction solution containing 20 mM Hepes-KOH (pH 7.5),50 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM ATP, 10 mM creatine phosphate,0.25 μg E1 (Sigma), 0.5 μg UbcH5B, 5.0 μg CBLB-N variant, 0.5 μg N terminushexahistidine tag attached ubiquitin, and 10 μg creatine kinase. Reaction wasterminated at 0, 15, 30, 60, and 120 min for Western blotting analysis. Ubi-quitination was monitored by immunoblotting using peroxidase conjugatedantipolyhistidine antibody (Sigma). Samples from autoubiquitination timecourse at 60 min were applied for Western blotting in the single SDS-PAGEgel and the band density was quantified by using ImageJ software.

NMR Spectroscopy.All NMR experiments were carried out at 25 °C on a VarianInova 500, 600, or 800 MHz NMR spectrometer. The segmental 15N-labeledCBLB-N and pY CBLB-N; 15N or 13C∕15N-labeled pY H-RING and H-RING; 13CH3-

Met-labeled CBLB-N, TKB-H, and RING; and Ile δ1-methyl 13CH3-labeledTKB-H, RING, and CBLB-N were dissolved in 20 mM MES (pH 6.3), 1 mMCaCl2, 2 mM DTT, 150 mM NaCl in 90% H2O∕10% 2H2O or 20 mM deuteratedMES (pH meter direct read of 6.3), 1 mM CaCl2, 2 mM deuterated DTT, and150 mM NaCl in 100% D2O. The 13CH3 resonances of the Met residueswere assigned using a series of mutants of CBLB-N and TKB-H (Fig. S2) andthe isolated RING domain.

Titration measurements were carried out at 25 °C. Small aliquots ofnonlabeled protein (pY H-RING or UbcH5B) were added to the 15N-labeledprotein solution. The dissociation constant (Kd) of pY H-RING for UbcH5Bwas estimated from the amide proton chemical-shift changes.

Small-Angle X-ray Scattering Measurements. All samples were dissolved in20 mM Tris·HCl buffer (pH 8.0) and 150 mM NaCl. A protein concentrationof 6 mg∕mL was used for all SAXS measurements. The SAXS data werecollected at 25 °C using the Nano-viewer (RIGAKU) at the Open Facility,Hokkaido University Sousei Hall. Solvent scattering was corrected for the useof buffer solutions identical to that used for the sample. Scattering data wereanalyzed using the Guinier approximation (19). Ið0Þ, intensity at zero scatter-ing angle, and Rg, the radius of gyration, were calculated using the AutoRgsoftware (34).

ACKNOWLEDGMENTS. This work was supported by the Targeted ProteinsResearch Program, the matching Program for Innovations in Future DrugDiscovery and Medical Care, the Funding Program for World-Leading Inno-vative R&D on Science and Technology, and a Grant-in-Aid for ScientificResearch on Innovative Areas from the Ministry of Education, Science, andCulture, Japan.

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http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1110712108/-/DCSupplemental/pnas.1110712108_SI.pdf?targetid=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1110712108/-/DCSupplemental/pnas.1110712108_SI.pdf?targetid=SF1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1110712108/-/DCSupplemental/pnas.1110712108_SI.pdf?targetid=SF7http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1110712108/-/DCSupplemental/pnas.1110712108_SI.pdf?targetid=SF7http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1110712108/-/DCSupplemental/pnas.1110712108_SI.pdf?targetid=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1110712108/-/DCSupplemental/pnas.1110712108_SI.pdf?targetid=SF2