Cbl protein (for Casitas B-lineage lymphoma) was discovered as the cellular form of v-Cbl, a retroviral oncoprotein. v-Cbl is a 357 amino acid-long, gag-fusion transforming protein of Cas NS-1 retrovirus, which causes pre- and pro-B lymphomas in mice (Langdon et al. 1989). The cellular Cbl has a length of 906 or 913 amino acids in humans and mice, respectively, so v-Cbl corresponds to a large C-terminal truncation of Cbl (Blake et al. 1991). Following this finding, other Cbl-family members were identified and the role of Cbl-family proteins as key players in cellular regulation was established. The history of research in this area has been outlined in multiple review articles, for example, Tsygankov et al. (2001) and Swaminathan and Tsygankov (2006), etc. During the first years following the discovery of c-Cbl, the prevalent view was that it functions as an adaptor protein. The discovery of the E3 ubiquitin-protein ligase activity of c-Cbl, which was also demonstrated in other Cbl-family members – Cbl-b and Cbl-c, revealed that Cbl ubiquitylates specific substrates, thus promoting their degradation or modifying their functions (reviewed in Nau and Lipkowitz 2008; Reddi et al. 2008; Rubin and Yarden 2008). As a result of this discovery and subsequent dramatic expansion of the original findings to multiple cellular targets, the pendulum moved in the direction of accepting E3 activity as the major if not the only mode of action for Cbl-family proteins. It appears, however, that Cbl functions not only as an E3 ligase but as a multivalent adaptor as well (reviewed in Tsygankov et al. 2001; Tsygankov 2008). Currently, it is thought that Cbl plays a key regulatory role in multiple biological systems.
Structure and Diversity of Cbl
The TKB domain consists of a four-helix bundle (4H), a calcium-binding EF-hand, and a modified SH2 domain and was so named for its ability to bind to phosphotyrosine (pTyr) residues of multiple protein tyrosine kinases (PTKs). The consensus sequence for recognition of most PTKs to Cbl TKB appears to be (N/D)XpY(S/T)XXP, although many exceptions to this rule are known (Mohapatra et al. 2013). To summarize, TKB mediates interactions of Cbl with multiple proteins, among which several non-PTK proteins are clearly present, via several different mechanisms, among which the pTyr-dependent ones play a major while not the only role.
The RING finger mediates the E3 ubiquitin ligase activity of Cbl by virtue of being able to bind to E2, a ubiquitin-conjugating enzyme. The RING finger is separated from the TKB domain by a short linker sequence, which plays a critical role in the regulation of Cbl E3 ligase activity. Multiple mutations in the linker sequence render Cbl transforming (reviewed in Kales et al. 2010; Mohapatra et al. 2013).
The C-terminal region of mammalian Cbl contains multiple proline-rich motifs, tyrosine phosphorylation sites, and the ubiquitin-associated (UBA) domain. The tyrosine phosphorylation sites are generated in Cbl as a result of PTK-dependent receptor signaling or coexpression of Cbl with activated PTKs (reviewed in Tsygankov et al. 2001; Swaminathan and Tsygankov 2006; Nau and Lipkowitz 2008).
Cbl-like proteins also exist in single-cellular organisms. Thus, the protein termed CblA, which was found in several species of the order Dictyosteliida, contains a TKB-type domain and a RING finger and regulates tyrosine phosphorylation-dependent signaling through the mechanisms exhibiting similarities with those mediating the effects of metazoan Cbl (reviewed in Nau and Lipkowitz 2008; Mohapatra et al. 2013).
Functions of Cbl as an E3 Ligase
Downregulation of multiple receptor PTKs other than EGFR by Cbl has also been shown. Cbl downregulates kinase receptors for the platelet-derived growth factor PDGF (PDGFR), colony-stimulating factor CSF-1 (Fms), hepatocyte growth factor HGF (Met), neurotrophin (p75NTR), stem cell factor (Kit), vascular endothelial growth factor VEGF (Flk-1 and Flt-1), macrophage-stimulating protein MSP (Ron), glial cell-derived neurotrophic factor GDNF (Ret), fibroblast growth factor FGF (FGFR), insulin (IR), and ephrin (reviewed in Swaminathan and Tsygankov 2006; Rubin and Yarden 2008).
Cbl also downregulates a number of nonreceptor PTKs, including Syk Zap-70, Fyn, Lck, Hck, Fgr, Lyn, and c-Abl. In most cases downregulation of the protein level and kinase activity of activated nonreceptor PTKs correlates with their ubiquitylation, consistent with a detailed study of the effect of Cbl on the specific activity of Syk through ubiquitylation and subsequent proteasome-mediated degradation of the phosphorylation fraction of Syk (reviewed in Swaminathan and Tsygankov 2006; Reddi et al. 2008).
Furthermore, Cbl can downmodulate not only PTKs but non-PTK proteins that belong to several distinct groups. Thus, Cbl promotes ubiquitylation and degradation of tyrosine-phosphorylated Vav, a guanine nucleotide-exchange factor (GEF) specific for Rho-family GTPases (reviewed in Swaminathan and Tsygankov 2006; Mohapatra et al. 2013), and the adaptor protein LAT (Matalon et al. 2016). Cbl downregulates STAT transcription factor-mediated signaling that is initiated by growth hormone and G-CSF as well as signaling mediated by the IRF-8/ICSBP transcription factor (reviewed in Swaminathan and Tsygankov 2006). Cbl also induces degradation of β-catenin, a key component of the Wnt signaling pathway (Chitalia et al. 2013).
Cbl downregulates non-PTK cell surface receptors, such as FcεRIγ chain in mast cells and TCRς chain in T cells. Cbl mediates constitutive downmodulation of pre-TCR from the surface of thymocytes, a key event in T-cell development, and ligand-induced TCR downregulation. Cbl is also involved in the termination of signaling through GPCRs, since it mediates ubiquitylation and degradation of protease-activated receptor 2 (PAR-2). Cbl appears to downregulate integrin receptors and the transmembrane receptor/transcription factor Notch. Cbl regulates the level of proapoptotic protein Bim through its ubiquitylation and degradation (reviewed in Swaminathan and Tsygankov 2006).
Regulation of PTKs by Cbl-mediated ubiquitylation may also be independent of ubiquitylation-driven degradation. Thus, Cbl induces ubiquitylation of Syk in platelets in response to signaling through the GPVI receptor for collagen and the lack of Cbl greatly augments platelet response to this simulation. However, this ubiquitylation of Syk is not followed by its degradation, suggesting that a degradation-independent mechanism is critically involved (Dangelmaier et al. 2005). It is possible that Syk downregulation in this case is mediated by a protein tyrosine phosphatase binding to Cbl and/or ubiquitin. TULA-2, a novel protein tyrosine phosphatase, may play this role (Thomas et al. 2010).
Finally, it is also possible that Cbl acts as a Nedd8 E3 ligase mediating neddylation of EGFR (Oved et al. 2006) and the TGF-β receptor (Zuo et al. 2013). The functional consequence of neddylation differs in these systems: neddylation enhances subsequent ubiquitylation and degradation of EGFR, while preventing ubiquitylation and degradation of the TGF-β receptor, hence supporting sustained TGF-β signaling. It is possible that the effects of neddylation on degradation are specific for individual proteins.
Regulation of Cbl E3 Ligase Activity
The RING domain is absolutely essential for the E3 activity of Cbl. This requirement is independent of the mode of interaction of Cbl proteins with their ubiquitylation targets. The RING finger of c-Cbl serves as a binding site for an E2 ubiquitin-conjugating enzyme, many of which have been identified to support the Cbl-mediated ubiquitylation. The structures of several Cbl-E2 and Cbl-E2-substrate complexes have been determined (reviewed in Reddi et al. 2008; Rubin and Yarden 2008; Mohapatra et al. 2013; Li et al. 2016).
Another essential step of the Cbl-driven ubiquitylation is binding of Cbl to a protein target. Initial studies established a critical role of the TKB domain in Cbl-dependent ubiquitylation. The G306E mutation in TKB, which recapitulates a naturally occurring loss-of-function mutation in Sli-1, a C. elegans homolog of c-Cbl, disrupts TKB-dependent binding and Cbl-driven ubiquitylation in many experimental systems (reviewed in Swaminathan and Tsygankov 2006; Reddi et al. 2008; Rubin and Yarden 2008; Mohapatra et al. 2013). Later, it was shown that Cbl interacts with many of its targets through domains other than TKB. Thus, downregulation of Src-family PTKs and other protein targets depends on their binding to the C-terminal region of Cbl, including its proline-rich motifs and pTyr residues (Tsygankov et al. 2001; Swaminathan and Tsygankov 2006; Reddi et al. 2008; Rubin and Yarden 2008; Chitalia et al. 2013; Mohapatra et al. 2013).
Binding of Cbl to its targets may also be indirect and mediated by adaptor proteins. Thus, Cbl is recruited to the activated EGFR via Grb2, a key adaptor protein of the EGFR signaling axis. This route leads to ubiquitylation and degradation of EGFR lacking Tyr-1045, a site binding to Cbl TKB, as a result of binding of both Cbl and phosphorylated EGFR to Grb2 (Fig. 3). This route is also important in C. elegans, where Sli-1 acts in conjunction with SEM-5, a Grb2 homolog, to inhibit LET-23, an EGFR homolog (reviewed in Rubin and Yarden 2008).
A similar role may be played by other proteins linking Cbl to EGFR; for instance, LRIG1 can interact with all members of the ErbB family, while its juxtamembrane region binds to the TKB domain of Cbl. As a result, LRIG1 recruits Cbl to EGFR and promotes ubiquitylation and degradation of EGFR. FRS2 and Grb2 form a complex linking Cbl to FGFR, while the Grb2-Shc complex recruits Cbl to RET; ArgBP2 links Cbl to c-Abl, and CD2AP facilitates binding of Cbl to VEGFR-1/Flt-1 (reviewed in Swaminathan and Tsygankov 2006; Rubin and Yarden 2008).
The TKB-independent mode of Cbl binding is often sufficient for ubiquitylation of protein targets. The relative contribution of TKB-dependent and independent interactions of Cbl to its biological effects likely differ for individual targets. For example, the G304E Cbl knockin mouse (the mouse G304E corresponds to G306E in humans) does not recapitulate the phenotype of the Cbl-null mouse, whereas the RING mutant knockin (C379A, which corresponds to human C381A) exhibits an effect exceeding that of the Cbl-null mutation. Thus, the negative regulatory effect of Cbl in T cells is dependent on the RING finger while largely independent of the intact TKB. Likewise, Cbl-driven ubiquitylation essential for the effect of Cbl on migration of v-Abl-transformed fibroblasts is TKB-independent (reviewed in Thien and Langdon 2005; Swaminathan and Tsygankov 2006; Lee and Tsygankov 2013).
Another factor essential for the E3 activity of Cbl is tyrosine phosphorylation of its linker between TKB and RING. It was suggested in the early studies that tyrosine phosphorylation of Tyr-371 of Cbl is crucial for its E3 activity. These results were consistent with the transforming potential of Cbl mutants lacking Tyr-371 or Tyr-368. It was shown using mass spectrometry that Tyr-371 and Tyr-368 are phosphorylated in Cbl and that Tyr-371 (and possibly, Tyr-368) cause conformational changes in Cbl and is essential for Cbl E3 activity. Further studies indicated that phosphorylation of Tyr-371, which increases the efficiency of Cbl E3 activity, disrupts the auto-inhibited conformation in which the E2-binding surface of RING is occluded, thus allowing the RING finger to bind to an E2 and placing the RING/E2 complex in the proximity of the protein substrate (reviewed in Reddi et al. 2008; Rubin and Yarden 2008; Mohapatra et al. 2013; Li et al. 2016).
Cbl as a Multivalent Adaptor
Although the E3 activity is critically important for the biological role of Cbl, some cellular events mediated or regulated by Cbl are dependent on its adaptor functions, since Cbl forms complexes with numerous proteins via its various domains (Fig. 1). The TKB domain binds not only ubiquitylation targets of Cbl but also the adaptor protein SLAP and tubulin. The proline-rich region of Cbl interacts with SH3-containing proteins, including multiple adaptors, Src-family PTKs, TULA-family proteins, and PI-3′ kinase p85. The C-terminal phosphorylated tyrosine residues of Cbl, among which pTyr-700, pTyr-731, and pTyr-774 are prominent, provide docking sites for various SH2-containing proteins, including adaptors, p85, Vav-family GEFs, Src-family, and other PTKs. The C-terminal region also contains an atypical proline/arginine-rich sequence through which it binds to CIN85. The UBA/LZ domain mediates dimerization of Cbl (reviewed in Swaminathan and Tsygankov 2006; Tsygankov 2008; Lee and Tsygankov 2013).
Cbl appears to be involved in the activation of MAP kinases in response to stimulation of various receptors. These effects appear to be mediated by Cbl acting as an adaptor by virtue of its association with PI-3′ kinase or Crk (reviewed in Swaminathan and Tsygankov 2006; Tsygankov 2008; Lee and Tsygankov 2013).
Cbl was shown to be involved in the activation of small GTPases, Rap1 and Rac1, via PI-3′ kinase and Crk/C3G pathway and in the resulting cytoskeletal rearrangements in several experimental systems (reviewed in Swaminathan and Tsygankov 2006; Tsygankov 2008; Lee and Tsygankov 2013).
PI-3′ kinase-mediated effects of Cbl depend on binding of the p85 subunit of this kinase to pTyr-731. In several studies, a link between PI-3′ kinase-mediated Rac1 activation and cytoskeletal rearrangements was shown. The exact targets of Cbl/PI-3′ kinase signaling are not always identified, but a key role of the Cbl-PI-3′ kinase interaction was demonstrated in cytoskeleton-dependent events in multiple biological systems. PI-3′ kinase-mediated effects of Cbl on signaling contribute not only to the cytoskeletal events but also to proliferation and survival, involving MAP kinase, JAK/STAT, and AKT pathways (reviewed in Swaminathan and Tsygankov 2006; Tsygankov 2008; Lee and Tsygankov 2013).
Cbl upregulates Rap1 not through PI-3′ kinase but through C3G, a Rap1 GEF. It appears that Cbl exerts an effect on C3G-mediated signaling by recruiting this GEF through Crk-family adaptors binding to both C3G and Cbl; pTyr-700 and -774 act as the Crk-binding sites of Cbl (reviewed in Tsygankov et al. 2001; Lee and Tsygankov 2013).
Another effect of Cbl, which may be classified as adaptor-like, is to increase stability of microtubules by binding to tubulin. This function is in part explained by displacing histone deacetylase 6 (HDAC6), which destabilizes microtubules, by Cbl. The binding of Cbl to tubulin is mediated by the four-helix bundle region of TKB and is tyrosine phosphorylation-independent (reviewed in Lee and Tsygankov 2013).
Cbl in Cancer
The discovery of Cbl as a cellular version of a viral oncoprotein causing myeloid leukemia in mice indicated its proto-oncogenic nature. Further studies of the molecular basis of Cbl functions suggested a link between the mutation-induced loss of the ability to downregulate PTKs and the transforming potential of Cbl. Although this link remained elusive for some time, multiple mutations of Cbl linked to transformation were eventually identified in human cancers. First, mutations of Cbl were found in cells from a wide variety of myeloid neoplasms including acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML) in blast crisis, chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML). The frequency of Cbl mutations in patient samples is ~5% overall, being highest in JMML and CMML (>10%). Most of the mutations described, among which both point substitutions (~85%) and deletions (~15%) are present, occur within the linker region or the RING finger (reviewed in Kales et al. 2010; Katzav and Schmitz 2015).
All Cbl missense mutations found in myeloid neoplasms that have been studied abrogate the ability of Cbl to ubiquitylate receptor PTKs. Furthermore, cells expressing these mutant Cbl proteins show enhanced activation of the PI3K/AKT and STAT5 pathways, which appears to be important for proliferation. These findings are consistent with the ability of Cbl to facilitate activation of the JAK-STAT and PI3K-AKT pathways to transmit mitogenic and/or survival signals (reviewed in Kales et al. 2010; Katzav and Schmitz 2015).
Overall, Cbl has both tumor suppressor and oncogene potential and the transforming effects of its mutations are due to the loss of the tumor suppressor E3 function and the maintenance of the adaptor function, which acts as oncogenic (Kales et al. 2010).
Cbl is ubiquitously expressed in various cell types in all multicellular animals. Genetic, biochemical, structural, and cell biological studies of Cbl have established the dual functional role of this signaling protein. Cbl is both an E3 ligase, mediating ubiquitylation and less frequently neddylation of protein substrates, and a multivalent adaptor capable of recruiting and juxtaposing various proteins involved in cellular signaling and other biological functions. Ubiquitylation typically downregulates protein targets of Cbl, although this downregulation is not always dependent on protein degradation. Most of the ubiquitylation targets of Cbl are PTKs but many non-PTK proteins can also be targeted. The high diversity of ubiquitylation targets of Cbl depends on the presence of a number of various interactive sites in Cbl. Many of the same sites are also involved in the adaptor functions of Cbl, which typically exert a positive effect on signaling that promotes proliferation, survival, and cytoskeletal rearrangement. Cbl is involved in the regulation of embryonic development, immune responses, platelet functions, and other physiological processes as well as in the development of cancer, especially of myeloid nature.
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