The Tyrosine Kinase Negative Regulator c-Cbl as a RING-Type, E2-Dependent Ubiquitin-Protein Ligase

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Science  08 Oct 1999:
Vol. 286, Issue 5438, pp. 309-312
DOI: 10.1126/science.286.5438.309


Ubiquitination of receptor protein-tyrosine kinases (RPTKs) terminates signaling by marking active receptors for degradation. c-Cbl, an adapter protein for RPTKs, positively regulates RPTK ubiquitination in a manner dependent on its variant SRC homology 2 (SH2) and RING finger domains. Ubiquitin-protein ligases (or E3s) are the components of ubiquitination pathways that recognize target substrates and promote their ligation to ubiquitin. The c-Cbl protein acted as an E3 that can recognize tyrosine-phosphorylated substrates, such as the activated platelet-derived growth factor receptor, through its SH2 domain and that recruits and allosterically activates an E2 ubiquitin-conjugating enzyme through its RING domain. These results reveal an SH2-containing protein that functions as a ubiquitin-protein ligase and thus provide a distinct mechanism for substrate targeting in the ubiquitin system.

Specific and precise signaling by RPTKs requires that both the intensity and the duration of the elicited intracellular signals be tightly regulated. To terminate signaling, ubiquitination can mark receptors for degradation (1). Free ubiquitin (Ub) is recruited to ubiquitination pathways by the E1 Ub-activating enzyme through the formation of a thioester between a cysteine in E1 and the COOH-terminus of Ub (2). Ub is subsequently transferred, also as a thioester, to members of the E2 Ub-conjugating (Ubc) enzyme superfamily. Ub-protein ligases, or E3s, are defined as the pathway components responsible for substrate recognition and for promoting Ub ligation to substrate (2). HECT domain–containing E3s (homologous to E6-APCOOH-terminus) accept Ub from E2s in the form of a thioester and then catalyze the formation of stable Ub-substrate conjugates (2). E3s such as the Skp1/cullin/F box (SCF) and anaphase-promoting complexes (APC) (2) do not appear to form thioesters with Ub (3). Rather, these complexes act by bringing E2∼Ub and substrate together and by allosterically activating E2 (3, 4). The efficiency of poly-Ub chain synthesis may be increased by other factors, such as the E4 proteins (5).

c-Cbl, a 120-kD protein that contains a variant SH2 domain, a RING finger, a proline-rich region, and a Ub-associated domain, binds to, and stimulates the ubiquitination of, active platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony-stimulating factor–1 receptors (6–8). Moreover, the c-Cbl homolog in Caenorhabditis elegans, SLI-1, has been genetically defined as a negative regulator of RPTK signaling (9). The fact that c-Cbl can function as an adapter protein in tyrosine phosphorylation–dependent signaling raises the possibility that it recruits E3 or E4 proteins to ubiquitinate active receptors. A critical role for the RING finger in regulating this process is suggested by the observation that c-cbl alleles that are defective in promoting RPTK ubiquitination contain either an entire deletion (v-cbl), a small deletion (70Z/3 cbl), or a point mutation (Cys381→Ala) in the RING domain (8,9). Because RING finger subunits are essential components of SCF and APC ligases (3, 4, 10) and, together with cullins, these RING proteins recruit and activate E2s (3, 4), we tested whether c-Cbl might itself be an E3 ligase for RPTKs.

To determine whether the c-Cbl RING finger has intrinsic E3 activity, we generated glutathione-S-transferase (GST) fusion proteins encompassing residues 359 to 447 of wild-type (WT) human c-Cbl (11). This sequence includes the entire RING domain and 10 to 20 residues on either end. A similar construct was generated containing the 17–amino acid deletion (residues 366 to 382) found in the ubiquitination-deficient 70Z/3 Cbl, that includes the first cysteine in the RING consensus, Cys381. GST fusion proteins purified from Escherichia coli were assayed for the ability to promote substrate-independent Ub-protein ligation in the presence of human E1 and the E2 Ubc4 (both also purified from E. coli), adenosine 5′-triphosphate (ATP), and 125I-labeled Ub (12).

The WT c-Cbl RING activated the ligation of 125I -Ub to proteins in the reaction (Fig. 1A), whereas no ubiquitinated products were observed in the presence of GST or of GST-70Z/3 RING, even after prolonged incubation, at times when the reaction with the WT RING domain reached a plateau.

Figure 1

Intrinsic Ub ligase activity of the c-Cbl RING finger. (A) Ubiquitination reactions were performed, as indicated, with GST or GST fusion proteins with c-Cbl and 70Z/3 Cbl RING fingers, E1, Ubc4, 125I-Ub, and ATP for the indicated time. Ub-protein conjugates were resolved by reducing SDS-PAGE and detected by autoradiography. The indicated 60-kD125I-labeled protein is present in unreacted samples. Free Ub was run off the gel. (B) Ubiquitination reactions were performed as in (A), except that unlabeled Ub was used and the reaction was developed by immunoblot with antibody to GST. SDS gel sample buffer (2×) was added to the reactions corresponding to lanes 1 and 3 before proteins. The positions of GST-RING and poly-ubiquitinated (Ubn) GST-RING are indicated. The asterisk indicates cross-reacting proteins that are present in certain unreacted GST preparations (27). Numbers at the right are molecular sizes (in kilodaltons). (C) Reactions performed as in (B) developed by immunoblot with antibody to His tag to detect His-Ubc4. S.B. refers to the addition of SDS gel sample buffer to the reaction before proteins. The positions of Ubc4 and ubiquitinated Ubc4 (Ubn-Ubc4) are indicated. The asterisk indicates the position of a weakly staining band present in both unreacted (lane 4) and reacted (lane 5) 70Z/3 samples that comigrates with a band present only in the reacted WT sample (lane 3).

The products of substrate-independent ubiquitination reactions with the Cbl RING finger were also analyzed by immunobloting with an antibody to GST (anti-GST) (Fig. 1B) (12). Incubation of the WT, but not the 70Z/3 RING-GST fusion protein, with E1, Ubc4, ATP, and Ub led to the formation of slower migrating proteins that were recognized by anti-GST (13). Therefore, in the absence of a substrate, the GST-RING fusion protein itself becomes poly-ubiquitinated on the GST or RING moiety, or both. Because the RING component of E3s does not appear to form a thioester with Ub (3), the products of the reactions shown in Fig. 1, A and B, presumably arise from a direct transfer of Ub from an E2∼Ub thioester to GST-RING lysyl residues. We conclude that the c-Cbl RING has E3 activity.

Two components of SCF ligases, the Hrt1/Roc1/Rbx1 RING finger protein and cullin/Cdc53, form a complex that allosterically stimulates the basal rate of auto-ubiquitination of the E2 Cdc34 (3,4). No auto-ubiquitination of the E2 Ubc4 was detected in the presence of E1, Ub, and ATP (Fig. 1C), but addition of GST-RING WT, but not of GST-RING 70Z/3, caused ubiquitination of Ubc4 (14). This result suggests that the c-Cbl RING can allosterically activate the E2 Ubc4.

We examined the effect of point mutations in three residues that lie in the region deleted in 70Z/3 Cbl (Fig. 2A) on c-Cbl RING Ub ligase activity: Cys381 fits the RING consensus and its mutation impairs c-Cbl's ability to stimulate ubiquitination of EGF receptor (EGF-R) in vivo (8); Cys372 might participate in metal binding, although it does not fit the RING consensus and is not conserved in Cbl-3; and Tyr368, whose deletion renders Cbl oncogenic (15). The Cys381→Ala mutant was defective in both ubiquitination and Ubc4 activation assays, whereas Cys372→Ala and ΔTyr368 were as active as the WT RING (Fig. 2, B and C). Therefore, loss of a RING finger consensus cysteine (Cys381) can account for the ubiquitination defect of 70Z/3 Cbl. In contrast, the nearly WT Ub ligase activity of ΔTyr368 RING does not correlate with the oncogenic potential of ΔTyr368Cbl. Thus, the ΔTyr368 mutation may indirectly affect RING function in the context of full-length c-Cbl in vivo.

Figure 2

Analysis of c-Cbl RING domain residues required for Ub ligase activity and Ubc4 activation. (A) Alignment of the RING finger [amino acids 357 to 425; boundaries as defined in (16)] and additional NH2-terminal sequences in human c-Cbl, Cbl-b, and Cbl-3,Drosophila melanogaster D-Cbl, and C. elegans SLI-1. Point mutations examined are indicated by the arrows under the alignment. Residue numbers are for human c-Cbl. The site of the v-cbl truncation and the amino acids removed by the 70Z/3 deletion are indicated at the top. The Cys and His residues that fit the RING consensus for zinc binding are indicated by dots. Nonconserved residues are boxed. (B) As in Fig. 1B. SDS sample buffer (S.B.) was added before proteins in the reaction analyzed in lane 1. (C) As in Fig. 1C. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Only the few residues thought to maintain the RING structural scaffold are conserved among RING proteins (16). A subset of these proteins, that either possess Ub ligase activity or are implicated in protein degradation, have a conserved tryptophan at a position where other hydrophobic residues are otherwise found (3, 4, 10, 16–18) (Fig. 3A). The sequence COOH-terminal to Trp408, Trp-Gln-Glu-Ser-Glu-Gly, also bears resemblance to a small portion of a domain (Trp-Gln-Ser-Asp-Gly) present in the Apc10 subunit of APC (19). c-Cbl RING Trp408 is required for E3 ligase activity, because its replacement by alanine reduced the ability of the RING to promote Ub ligation and Ubc4 activation (Fig. 3, B and C).

Figure 3

Requirement of a tryptophan residue for the Ub ligase function of c-Cbl. (A) Alignment of the sequences between the third and fourth pairs of zinc-chelating residues (indicated by dots) of human c-Cbl, S. cerevisiae Apc11, human Roc1 and Roc2, equine herpes virus ENX, herpes simplex virus Vmw110, and S. cerevisiae Hrd1. Conserved residues and conservative substitutions are boxed. Cbl Trp408 is indicated by an asterisk. (B) As in Fig. 1B. (C) As in Fig. 1C. The asterisk indicates antibody cross-reacting proteins that were also present in the unreacted samples shown in lane 1. (D) GST pulldown assay. The association between Ubc4 and the indicated GST fusion proteins was measured as described (20). The asterisks indicate cross-reacting GST and GST-RING fusion proteins (28).

Binding of c-Cbl RING-GST fusion protein to Ubc4 was examined (Fig. 3D) (20). The GST-RING bound to Ubc4, whereas GST did not. We also determined the effect of c-Cbl RING mutations on Ubc4 binding (Fig. 3D). The ΔTyr368 mutant was almost as efficient as the WT RING domain in Ubc4 binding. The Cys381→Ala mutant bound Ubc4 much less efficiently than the WT protein. Trp408→Ala showed similarly weak binding to Ubc4. Therefore, the reduction in Cys381→Ala and Trp408→Ala binding to Ubc4 can account for their defects in ubiquitination and E2 activation. The Cys372→Ala mutant was also less efficient than the WT RING domain in Ubc4 binding. However, Cys372→Ala was indistinguishable from the WT protein in Ub ligase activity and Ubc4 activation (Figs. 2, B and C) even under lower Ubc4 concentrations and shorter reaction times (21). Possibly, Ala372 weakens the association with Ubc4, but makes activation of Ubc4 more efficient.

To determine whether the PDGF β-receptor (PDGF-Rβ) can serve as a direct substrate for ubiquitination by c-Cbl, GST-RING or a GST fusion protein with the c-Cbl 480 NH2-terminal residues, containing both the variant SH2 and the RING domains (GST-SH2-RING), were tested for the ability to ubiquitinate PDGF-Rβ. In vitro32P-phosphorylated receptor was incubated with E1, Ubc4, Ub, ATP, and GST fusion proteins (22). Cbl RING·Ubc4 complexes did not cause ubiquitination of the receptor (Fig. 4, A and B). In contrast, efficient PDGF-Rβ ubiquitination was observed in the presence of GST-SH2-RING·Ubc4 (Fig. 4, A and B), suggesting that the c-Cbl variant SH2 domain is required for substrate recognition (23).

Figure 4

Ubiquitination of the PDGF β-receptor (Rβ) by the c-Cbl RING in an SH2-dependent manner. (A) Immunoprecipitated Rβ was labeled with [γ-32P]ATP in an in vitro kinase reaction, extensively washed, and subsequently incubated with the indicated GST fusion proteins in ubiquitination reactions (22). The position of polyubiquitinated (Ubn) Rβ is indicated at the left, as well as the 190-kD mature (m) and 160-kD precursor (p) forms of Rβ (29). Lane 1 represents the unreacted control, in which SDS sample buffer was added to the reaction before proteins. (B) Results in (A) were quantified and normalized as described in (30). In a separate experiment with smaller amounts of proteins, receptor poly-ubiquitination was 9% with GST-SH2-RING and 0% with GST alone (21).

The small RING components of E3 ligases that consist mostly of the RING domain require an accessory cullin subunit to activate E2s and additional subunits for substrate recognition (3,4, 10). However, cullins are not required for the Ub ligase activity of Cbl (24) or Ubr1p (25), and these larger proteins can also recognize substrate.

The results of this work suggest how phosphorylated tyrosine can serve as a targeting signal for the Ub pathway in vivo: by mediating interactions either with SH2 domain–containing E3s such as c-Cbl or with SH2-containing proteins that in turn recruit E3s, as may occur with Grb2, a major Cbl-associated protein (23,26).

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