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Proc Natl Acad Sci U S A. Dec 20, 2011; 108(51): 20579-20584.
Published online Dec 7, 2011. doi:  10.1073/pnas.1110712108
PMCID: PMC3251137
Biophysics and Computational Biology

Autoinhibition and phosphorylation-induced activation mechanisms of human cancer and autoimmune disease-related E3 protein Cbl-b

Abstract

Cbl-b is a RING-type E3 ubiquitin ligase that functions as a negative regulator of T-cell activation and growth factor receptor and nonreceptor-type tyrosine kinase signaling. Cbl-b dysfunction is related to autoimmune diseases and cancers in humans. However, the molecular mechanism regulating its E3 activity is largely unknown. NMR and small-angle X-ray scattering analyses revealed that the unphosphorylated N-terminal region of Cbl-b forms a compact structure by an intramolecular interaction, which masks the interaction surface of the RING domain with an E2 ubiquitin-conjugating enzyme. Phosphorylation of Y363, located in the helix-linker region between the tyrosine kinase binding and the RING domains, disrupts the interdomain interaction to expose the E2 binding surface of the RING domain. Structural analysis revealed that the phosphorylated helix-RING region forms a compact structure in solution. Moreover, the phosphate group of pY363 is located in the vicinity of the interaction surface with UbcH5B to increase affinity by reducing their electrostatic repulsion. Thus, the phosphorylation of Y363 regulates the E3 activity of Cbl-b by two mechanisms: one is to remove the masking of the RING domain from the tyrosine kinase binding domain and the other is to form a surface to enhance binding affinity to E2.

The Cbl proteins (c-Cbl, Cbl-b, and Cbl-3) belong to a family of RING-type ubiquitin ligases. Like other RING domain proteins, the Cbl proteins function as adaptor proteins, simultaneously binding to a cognate E2 ubiquitin-conjugating enzyme and a substrate protein, leading to transfer of ubiquitin to the substrate. This facilitates degradation of the target substrate by proteasomes or, in some cases, lysosomes. The Cbl proteins function as a negative regulator of T-cell activation, growth factor receptor [e.g., epidermal growth factor receptor (EGFR), c-KIT, and platelet-derived growth factor receptor (PDGFR)], and nonreceptor-type tyrosine kinase signaling (e.g., Src family kinases and Zap70) (1, 2), and dysfunctional mutations in Cbl proteins have been related to human cancer (37). Of the three Cbl proteins, Cbl-b plays a critical role in the down-regulation of immunological signaling to induce T-cell anergy (8, 9). Cbl-b knockout mice have been shown to exhibit severe autoimmune diseases (10), whereas, in humans, a dysfunctional mutation in Cbl-b was shown to be related to type I diabetes (11) and multiple sclerosis (12). Hence, the Cbl proteins are considered to be a potential therapeutic target.

Cbl-b and c-Cbl exhibit high sequence homology in the N-terminal region with 86% amino acid identity and share a conserved tyrosine kinase binding (TKB) domain comprised of a four-helix bundle, a Ca2+-binding EF hand domain and a variant SH2 domain (13, 14), as well as a short helix-linker region and a RING finger domain that directly associates with E2 proteins (Fig. 1A). The ubiquitin ligase activity of Cbl-b is known to be up-regulated by the phosphorylation of Y363 (Y371 in c-Cbl), which is located in the helix linker (15, 16). The mutation of this critical tyrosine residue to phenylalanine abolishes the E3 activity of Cbl (17). Moreover, the E3 activity of the Cbl proteins is reported to be negatively regulated by the TKB domain, showing that tyrosine phosphorylation removes Cbl protein autoinhibition due to the interaction between the RING and TKB domains. Protease susceptibility analysis has also revealed that the phosphorylation of the Y363 of Cbl-b induces a large conformational change that is sensitive to protease digestion (15). However, the manner in which the molecular mechanism underlying this conformational change leads to the upregulation of E3 activity remains elusive.

Fig. 1.
(A) Domain structures of c-Cbl and Cbl-b. (B) SAXS measurements of CBLB-N (red) and pY CBLB-N (blue). Inset represents the Guinier plots for CBLB-N (red) and pY CBLB-N (blue). (C) Overlay of the 1H-15N HSQC spectra between the segmental isotope-labeled ...

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

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

Results

Overall Structural Changes in CBLB-N Due to Y363 Phosphorylation.

First, we studied the overall structures of both unphosphorylated and phosphorylated CBLB-N in solution using SAXS, as Y363 was reported to be responsible for phosphorylation-induced ligation activity (15). The radii of gyration (Rg), as estimated by the Guinier approximation (19), were 24.0 and 26.7 Å for CBLB-N and pY CBLB-N, respectively (Fig. 1B), suggesting that CBLB-N is more elongated in the phosphorylated state than in the unphosphorylated state. Protease susceptibility analysis of Cbl-b revealed that phosphorylated Cbl-b is labile to protease cleavage (15). These results taken together support the idea that pY CBLB-N is more extended and more mobile than CBLB-N, indicating that the conformational changes in CBLB-N are induced by phosphorylation at Y363.

To obtain further insights into the conformational changes induced by phosphorylation at Y363, we prepared segmental isotope-labeled CBLB-N using the sortase-mediated protein ligation method (20, 21). Here, uniformly 15N-labeled helix-RING (H-RING) was enzymatically attached to the nonlabeled TKB domain. The detailed protocol for the preparation of segmental isotope-labeled CBLB-N is shown in Fig. S1 AC and SI Materials and Methods. We confirmed that the E3 activity of segmental isotope-labeled CBLB-N and pY CBLB-N was almost identical to the authentic proteins (Fig. S1 D and E) though there is an insertion sequence of LPETGG prior to H-RING. Fig. 1C shows a comparison of the 1H-15N heteronuclear single quantum coherence (HSQC) spectra of the segmental isotope-labeled CBLB-N in the unphosphorylated and phosphorylated states. In the unphosphorylated state, the H-RING moiety in CBLB-N exhibited broad NMR signals (red). Because CBLB-N was confirmed to be monomeric by SAXS at 300 uM (Fig. S1 F and G), signal broadening was caused by restricted mobility of the H-RING moiety through its possible involvement into the core structure of CBLB-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 independent from the TKB core. These NMR observations are consistent with the results from the SAXS and protease susceptibility analyses, indicating that pY CBLB-N contains a mobile H-RING moiety, whereas the H-RING moiety in CBLB-N is incorporated into the core structure. Thus, pY H-RING is considered to be a structural and functional unit possessing ligation activity.

Closed Structure of CBLB-N in the Unphosophorylated State.

The 1H-15N HSQC spectrum of the segmental isotope-labeled CBLB-N at the H-RING moiety was superimposed onto that of the isolated RING domain (Fig. 2A). Spectral overlay revealed that the RING moiety in CBLB-N exhibited broad signals (red). A comparison with the isolated RING domain (blue) showed that some peaks were absent, possibly due to line broadening by the intermediate exchange process. The residues absent in CBLB-N were mapped on the RING domain in the crystal structure of c-Cbl complexed with UbcH7 (18) (Fig. 2B). Some of the absent residues were located at the binding interface with E2 (enclosed by a dotted circle), supporting the notion that the RING moiety surface required for binding to E2 partially overlaps with that required for the interdomain interaction with the TKB domain and helix (hereafter TKB-H).

Fig. 2.
(A) Overlay of the 1H-15N HSQC spectra of the segmental isotope-labeled CBLB-N (red) at the H-RING and the isolated RING domain (blue). Signal assignment of the isolated RING is shown in the spectrum. (B) Lost residues (red) in the 1H-15N HSQC spectrum ...

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

In order to determine the binding interface of TKB-H and the RING moiety, methionine 13C methyl-labeled samples under a 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 by acquiring the spectra of a series of mutants in which the methionine was replaced by Lys for surface exposed residues and by Ile or Leu for buried residues (Fig. S2). Differences in the positions of the methyl peaks of M214 and M365 were observed between CBLB-N and TKB-H, suggesting that M214 and M365 are involved in the binding with the RING moiety. However, as M365 is located close to the C terminus of TKB-H, the M365 peak shift could be due to a truncation effect. M365 exhibited a sharp peak, whereas that of M214 was appreciably broadened by an intermediate chemical exchange process, presumably between the open and closed states. Therefore, M214 is considered to be located at the interface with the RING moiety. The Met residues (M214 is colored red and the others yellow) were mapped on the TKB-H region in the crystal structure of c-Cbl complexed with UbcH7 (18) (Fig. 2F). Although M261 is located in the vicinity of M365, it did not exhibit any peak shift, supporting the idea that the 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 the CBLB-N crystal, as well as the dynamic feature of the RING moiety, we could not construct a model of the RING moiety, although a high electron density was observed around M214. This supports the NMR findings that there exists an interdomain interaction between TKB-H and RING in CBLB-N.

The interfaces between RING and TKB-H and UbcH7 partially overlap with each other (Fig. 2 B and D) so that the associations of RING with TKB-H and E2 are mutually exclusive. The conformational change induced by the phosphorylation of Y363 may be required to disrupt the interaction between RING and TKB-H, thereby facilitating the exposure of the RING domain and subsequent association with E2.

Solution Structure of the pY H-RING.

The 1H-15N HSQC signals of pY H-RING (Fig. 3A in red) almost entirely overlapped with those of the H-RING moiety in pY CBLB-N (Fig. 3A in blue). This supports the notion that the H-RING moiety in pY CBLB-N is exposed and independent from the TKB core and that its structure is similar to that of pY H-RING. We subsequently determined the solution structure of pY H-RING by NMR (Fig. 3B and Fig. S3A). Fig. 3B shows the structure of pY H-RING. The RING moiety is composed of two large Zn2+-binding loops, a short three-stranded antiparallel β-sheet, and a central α-helix, as shown in pink in Fig. 3B. The overall structure of the RING moiety is very similar to those of other RING domains (18, 2230). However, the phosphorylation of Y363 induces an additional structure that includes the formation of a parallel β-sheet between the N terminus of the helix linker and the C terminus of the RING domain so that the RING domain has extensive contact with the helix linker. Steady-state NOE analysis supported that RING as well as helix linker and the linker region of N and C terminus of pY H-RING formed rigid structures in solution (Fig. 3B, Fig. S3A, and Table S1). Moreover, we observed long range NOEs between K374 Hϵ and the aromatic ring proton of pY363, which was further supported by NOEs between surrounding residues. The interaction between the RING domain and the helix linker is stabilized by the electrostatic interaction between the phosphate group of pY363 on the helix linker and the positively charged cluster formed by K374 and K381 in the RING domain (Fig. 3B). These interactions make pY H-RING a single structural unit presenting the RING domain to E2. This notion was supported by the fact that Lys Hζ proton signals of both K374 and K381 were observed in the 1H-15N HSQC spectra (Fig. S3C). The Hζ proton signal from Lys can only be observed in cases where chemical exchange with solvent proton is highly restricted due to hydrogen bond formation. We also confirmed the interaction of K374 and K381 with the phosphate group of pY363 by the mutational analysis of H-RING in which K374 and K381 were replaced by Glu. These mutants did not show significant spectral changes upon Y363 phosphorylation (Fig. S3 E and F) and gave similar spectra to that of H-RING in contrast to the case of the wild type (Fig. S3D). In summary, the arrangement of the helix linker relative to the RING domain was markedly changed by the phosphorylation of Y363, thereby releasing the helix linker and RING from the TKB domain and exposing the H-RING moiety.

Fig. 3.
(A) Overlay of the 1H-15N HSQC spectra of the H-RING moiety in pY CBLB-N (blue) and pY H-RING (red). (B) Ribbon (Left) and surface potential (Right) representations of the solution structure of pY H-RING. pY363 and its interacting positively charged residues, ...

NMR Examination of the Interaction Between the E2 Protein UbcH5 and pY H-RING.

To elucidate the biological implications of the structural changes induced by the phosphorylation of Y363 in H-RING, the interaction of pY H-RING with UbcH5B was studied by chemical-shift perturbation methods using solution NMR (Fig. S3 G and H). First, unlabeled UbcH5B was titrated to 15N-labeled pY H-RING. Upon the addition of aliquots of UbcH5B, some of the peaks in 15N-labeled pY H-RING were gradually shifted by a fast exchange process. From the chemical-shift change of S403, we estimated that the Kd value for the binding of pY H-RING to UbcH5B is 1.2 μM (SD of  ± 0.4 μM). Next, we mapped the residues with large chemical-shift perturbations on the structure of pY H-RING. Most of the residues with large chemical-shift perturbations in the NMR spectra upon complex formation are located in the two regions: residues 374–379 and 408–411 in the loop regions, and residues 397–408 in the helix of the RING domain (Fig. 3C and Fig. S3G). On the other hand, the helix linker was not perturbed, indicating that this region is not directly involved in the interaction with UbcH5B. The interaction region was similar to the previous report for c-Cbl H-RING in the unphosphorylated state (24).

Next, unlabeled pY H-RING was titrated to 15N-labeled UbcH5B (Fig. 3D and Fig. S3H). Upon addition of aliquots of pY H-RING, some of the UbcH5B peaks disappeared in intermediate exchange process or gradually shifted in fast exchange process. Slight precipitation of UbcH5B was observed upon addition of pY H-RING so that the Kd value could not be estimated from this experiment. The largely affected residues in UbcH5B were located in the first helix, the loop between strands β3 and β4, and the loop region connecting the second and third helices (Fig. 3D and Fig. S3H). Interaction regions were similar to the previous report for c-Cbl H-RING in the unphosphorylated state (24). To our knowledge, there are five RING domains for which the 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 IDOL RING/UbcH5A (PDB ID: 2YHO). The interaction regions between the RING domain and E2 in these protein complexes were found to be essentially the same (Fig. S3I), which was also supported by NMR and/or mutational analysis for other RING domains of MDM2 (27), CNOT4 (28, 29), and BRCA1 (30). Thus, we constructed the complex model between pY H-RING and UbcH5B by overlaying the RING domain of pY H-RING and UbcH5B to the RING region and UbcH7 of the c-Cbl/UbcH7 complex structure (1FBV) (18), respectively (Fig. 3E). The complex model is consistent with and supported by the results of the NMR titration analysis, despite the fact NMR data were not considered at all for the model construction.

Affinity of CBLB-N and CBLB-N Variants Toward UbcH5B.

Fluorescence polarization spectroscopy (hereafter FP) was next used to evaluate the affinity of CBLB-N and CBLB-N variants for UbcH5B (Fig. S4). Titration measurements of the affinity of CBLB-N toward Alexa 488-labeled UbcH5B revealed the estimated Kd value of 97.5 μM (SD of  ± 3.3 μM). CBLB-N has a much lower affinity to UbcH5B than isolated H-RING (22.7 μM: SD of  ± 1.0 μM), indicating the masking of the RING moiety in CBLB-N. The affinity of pY CBLB-N toward UbcH5B was measured, and the Kd value was estimated to be 1.1 μM (SD of  ± 0.1 μM), which is similar to that of isolated pY H-RING (1.2 μM: SD of  ± 0.4 μM) obtained by NMR, indicating that the masking was released in pY CBLB-N. This is consistent with the results of the SAXS and NMR measurements. It should be noted that a comparison of the Kd values between H-RING and pY H-RING revealed that the phosphorylation of Y363 increases the affinity toward UbcH5B by about 20-fold. It can be concluded that the phosphorylation of Y363 in CBLB-N facilitates the association of the E2 protein, not only through unmasking but also through formation of a proper E2 binding surface. Indeed, the phosphate group of pY363 in pY H-RING interacts with K374 and K381 to fix the structure of the H-RING moiety and to reduce its basic surface potential (Fig. 3 B and F). This surface is partially involved in the binding to the conserved, positively charged surface of the α1 helix in UbcH5B so that neutralization of the positively charged surface by the phosphate group of Y363 increases the binding affinity between RING and UbcH5B (Fig. 3F).

Next, the E3 activity of the Cbl-b variants was studied by measuring their autoubiquitination activity. Consistent with previous data (15, 16), the ubiquitination activity of CBLB-N was markedly enhanced by Y363 phosphorylation (Fig. 4 B and C), which also supports our structural data for phosphorylation-induced CBLB-N unmasking and activation.

Fig. 4.
Autoubiquitination assay of CBLB-N variants. (A) The mutation sites in the TKB-H region were mapped and colored yellow. The helix region is colored pink (Left). Mutation sites in the RING moiety were mapped and colored yellow (Right). (B) Time course ...

The Closed Structure of CBLB-N Based on a Docking Study, the Design of Mutants, and Ubiquitination Assay.

According to the NMR and FP 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 which the interaction between TKB-H and RING was disrupted. Prior to mutant design, the CBLB-N structure was modeled by rigid body docking between TKB-H and RING using the HADDOCK program (31), incorporating the NMR chemical-shift perturbation data (Fig. 2). A detailed description of the docking calculations between TKB-H and RING is given in Table S2 and the docking structure is shown in Fig. S5 A and B and statistics are shown in Table S2. In the crystallographic analysis, we could observe a low but extensive electron density around the RING region in the HADDOCK model (Fig. S5C and Table S3), which supports the NMR- and HADDOCK-derived model. The RING moiety fitted into a shallow groove on TKB-H, and the interaction between TKB-H and the RING moiety seemed to be mainly electrostatic in nature (Fig. S5B). We, therefore, focused on the exposed charged residues on the TKB-H surface. Four mutants in the TKB region, D112R, K129E/R131E, K195E/R198E, and D226R (Fig. 4A), were prepared and applied to the autoubiquitination assay. These mutants exhibited two- to sevenfold higher E3 activity than the wild type. This supported the idea that the E3 activity of CBLB-N is autoinhibited by the interdomain interaction with the TKB region.

We next designed RING domain mutants that would disrupt the masked structure. From the docking study and the data for the TKB mutants, the masking was expected to be maintained mainly by electrostatic interaction. Hence, we focused on the charged residues, the signals of which disappeared in the segmental-labeled CBLB-N (Fig. 2B). We then performed autoubiquitination assay of three RING mutants, E378R, K381E, and R412E. Among these mutants, K381E exhibited markedly higher activity, whereas E378R and R412E exhibited lower activity compared to CBLB-N. As E378 and R412 are located on the interface between RING and E2 (18), the lower activity of these mutants is thought to be disruption of association with UbcH5B. Intriguingly, R420 in c-CBL (R412 in Cbl-b) is a mutational hot spot in human cancer malignancies (37). On the other hand, CBLB-N (K381E) exhibited the highest activity among the mutants we studied. From these observations, K381 may be located in the interface region between RING and TKB-H domains. Thus, the results of E3 activity, as well as the FP and SAXS analyses, support our view that the dynamic structural change induced by Y363 phosphorylation enhances the E3 activity of Cbl-b.

Discussion

The present structural analyses of CBLB-N revealed that the interdomain interaction between RING and TKB-H leads to the formation of a compact structure in the unphosphorylated state in which the binding site between the RING domain and E2 is masked by TKB-H. In contrast, pY CBLB-N has an extended structure in which the phosphorylated H-RING moiety is freely accessible to the E2 proteins. Moreover, the H-RING moiety markedly changes its structure upon phosphorylation, thereby increasing its affinity to the E2 protein. This was confirmed by biophysical and biochemical analyses together with mutational analysis. However, this process is not merely a closed-to-open conformation change induced by Y363 phosphorylation.

First, we considered the structure of CBLB-N in the closed state. We modeled a closed CBLB-N structure by docking the RING domain with the crystal structure of TKB-H (modeled from c-Cbl: 1FBV) on the basis of chemical-shift perturbation studies (Fig. 2) using the HADDOCK program. Most of the RING residues on the TKB-H binding interface disappeared, indicating that these residues are in an intermediate exchange process and showing that CBLB-N in the unphosphorylated state is in an equilibrium between a closed and a partially open state (Fig. 5A). This view is consistent with the basal E3 activity of Cbl-b in the unphosphorylated state as well as the crystal structure of CBLB-N, where the electron density of the RING domain is diffusive. This suggests that the RING domain is not fixed but is in a dynamic equilibrium between several forms. The partially open state may be considered to be an ensemble of multiple states, one of which may be similar to the crystal structure of c-Cbl in which the RING moiety is released from the TKB region while the helix region is fixed on the TKB domain (18). In some of the partially open states, both the helix and the RING regions may be released from the TKB domain so that the Y363 of Cbl-b or the Y371 of c-Cbl becomes susceptible to phosphorylation. Once phosphorylated, the helix region is detached from the TKB domain and interacts with the RING domain through ionic interactions between the phosphate group of pY363 and the positively charged cluster in the RING domain. A comparison of the 1H-15N HSQC spectra of the segmental isotope-labeled pY CBLB-N at the H-RING moiety with the isolated pY H-RING domain clearly supports the idea that the H-RING moiety in pY CBLB-N is exposed and mobile, in a manner similar to the isolated pY H-RING. Thus, the helix region and the RING domain work together as a structural and functional unit to recruit E2 proteins. The structure of pY CBLB-N was subsequently modeled based on the structure of TKB and pY H-RING. Because of the β-sheet formation between the N and C termini of the H-RING moiety, the location of the H-RING moiety relative to the TKB domain may be significantly different from that observed in the crystal structure of c-Cbl complexed with UbcH7, and close to the Zap70 phosphotyrosine-containing peptide binding site, particularly as the linker between TKB and H-RING is comprised of four residues (Fig. 5A and Fig. S6).

Fig. 5.
(A) Schematic representation of the regulation of E3 activity of Cbl-b by phosphorylation. CBLB-N is in equilibrium between the closed inactive and open partially active states. In the closed state, the E2 binding region is masked by the TKB region, but ...

From the present structural and functional analyses, the following model for Cbl-b signaling can be proposed (Fig. 5B). Upon activation of the cell surface receptors, including the T-cell receptor, EGFR, PDGFR, c-KIT, and so on, the tyrosine phosphorylation of the receptors by protein tyrosine kinases creates a binding site for the Cbl-b TKB domain, thus recruiting Cbl-b. Because of the intramolecular interaction of the RING domain with TKB-H, Cbl-b is in a closed state that masks the binding site for the E2 protein. However, when Cbl-b is phosphorylated, the H-RING becomes exposed, thereby enhancing the affinity for the E2 protein. Thus, activated Cbl-b catalyzes ubiquitination of the receptor- and nonreceptor-type tyrosine kinases to down regulate signaling. In conclusion, our structural and biochemical studies have revealed a regulatory mechanism of the E3 activity of Cbl-b, which is closely related to cancer as well as autoimmune diseases in humans.

Materials and Methods

See detail also for SI Materials and Methods.

Protein Expression and Purification.

All the proteins were expressed as hexahistidine tag fusion proteins using Escherichia coli strain Rossetta (DE3) at 25 °C, purified using Ni2+-affinity column chromatography, followed by HRV3C protease digestion to remove the tag, and further purified by gel filtration chromatography using Superdex 75 (GE Healthcare).

Fluorescence Labeling of the Protein and Fluorescence Polarization Measurement.

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

Protein Ligation and Purification of the Product.

Protein ligation was carried out at room temperature as described previously (20). Ligation product was digested by HRV3C protease, followed by pass through the Ni-nitrilotriacetate, and further purified by gel filtration using Superdex 75 (GE Healthcare) (Fig. S1).

Phosphorylation of CBLB-N and H-RING.

The phosphorylation reaction was carried out using fusion protein between c-Src kinase domain and Zap70 (SDGYTPEP) fragment to enhance phosphorylation efficiency (33). The phosphorylation reaction was monitored by SDS-PAGE and immunoblotting using PY20 (Zymed), a mouse monoclonal antibody against phosphotyrosine. Specificity of the phosphorylation at Y363 was confirmed using the Y363F mutant protein (Fig. S7).

In Vitro Ubiquitination Assay.

Ubiquitination reaction was carried out at 30 °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 terminus hexahistidine tag attached ubiquitin, and 10 μg creatine kinase. Reaction was terminated at 0, 15, 30, 60, and 120 min for Western blotting analysis. Ubiquitination was monitored by immunoblotting using peroxidase conjugated antipolyhistidine antibody (Sigma). Samples from autoubiquitination time course at 60 min were applied for Western blotting in the single SDS-PAGE gel and the band density was quantified by using ImageJ software.

NMR Spectroscopy.

All NMR experiments were carried out at 25 °C on a Varian Inova 500, 600, or 800 MHz NMR spectrometer. The segmental 15N-labeled CBLB-N and pY CBLB-N; 15N or 13C/15N-labeled pY H-RING and H-RING; An external file that holds a picture, illustration, etc.
Object name is pnas.1110712108eq1.jpg-Met-labeled CBLB-N, TKB-H, and RING; and Ile δ1-methyl An external file that holds a picture, illustration, etc.
Object name is pnas.1110712108eq2.jpg-labeled TKB-H, RING, and CBLB-N were dissolved in 20 mM MES (pH 6.3), 1 mM CaCl2, 2 mM DTT, 150 mM NaCl in 90% H2O/10% An external file that holds a picture, illustration, etc.
Object name is pnas.1110712108eq3.jpg or 20 mM deuterated MES (pH meter direct read of 6.3), 1 mM CaCl2, 2 mM deuterated DTT, and 150 mM NaCl in 100% D2O. The An external file that holds a picture, illustration, etc.
Object name is pnas.1110712108eq4.jpg resonances of the Met residues were assigned using a series of mutants of CBLB-N and TKB-H (Fig. S2) and the isolated RING domain.

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

Small-Angle X-ray Scattering Measurements.

All samples were dissolved in 20 mM Tris·HCl buffer (pH 8.0) and 150 mM NaCl. A protein concentration of 6 mg/mL was used for all SAXS measurements. The SAXS data were collected at 25 °C using the Nano-viewer (RIGAKU) at the Open Facility, Hokkaido University Sousei Hall. Solvent scattering was corrected for the use of buffer solutions identical to that used for the sample. Scattering data were analyzed using the Guinier approximation (19). I(0), intensity at zero scattering angle, and Rg, the radius of gyration, were calculated using the AutoRg software (34).

Supplementary Material

Supporting Information:

ACKNOWLEDGMENTS.

This work was supported by the Targeted Proteins Research Program, the matching Program for Innovations in Future Drug Discovery and Medical Care, the Funding Program for World-Leading Innovative R&D on Science and Technology, and a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Science, and Culture, Japan.

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The NMR, atomic coordinates, chemical shifts, and restraints reported in this paper have been deposited in the BioMagResBank, www.bmrb.wisc.edu (accession no. Q28: BMR17680), and the atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2LDR and 3VGO).

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

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