c-Cbl and Cbl-b ubiquitin ligases: substrate diversity and the negative regulation of signalling responses

Abstract

The activation of signalling pathways by ligand engagement with transmembrane receptors is responsible for determining many aspects of cellular function and fate. While these outcomes are initially determined by the nature of the ligand and its receptor, it is also essential that intracellular enzymes, adaptor proteins and transcription factors are correctly assembled to convey the intended response. In recent years, it has become evident that proteins that regulate the amplitude and duration of these signalling responses are also critical in determining the function and fate of cells. Of these, the Cbl family of E3 ubiquitin ligases and adaptor proteins has emerged as key negative regulators of signals from many types of cell-surface receptors. The array of receptors and downstream signalling proteins that are regulated by Cbl proteins is diverse; however, in most cases, the receptors have a common link in that they either possess a tyrosine kinase domain or they form associations with cytoplasmic PTKs (protein tyrosine kinases). Thus Cbl proteins become involved in signalling responses at a time when PTKs are first activated and therefore provide an initial line of defence to ensure that signalling responses proceed at the desired intensity and duration.

Figure 1. Cbl protein family in mammals (c-Cbl, Cbl-b and Cbl-3), Drosophila (D-Cbl, long and short) and C. elegans (Sli-1)

All Cbl proteins share a high level of sequence conservation between their TKB, linker (L) and RING finger (RF) domains. c-Cbl and Cbl-b have extensive proline-rich regions (black) in their C-terminal halves that mediate interactions with numerous SH3-domain-containing proteins. The TKB domain is composed of three interacting regions comprising a four-helix bundle (4H), a calcium-binding EF hand and a variant SH2 domain that is connected to the RING finger by a short linker domain. The PR domain in the C-terminus of c-Cbl and Cbl-b refers to a PX(P/A)XXR motif that binds SH3 domains of the CIN85/RUK (regulator of ubiquitous kinase)/CD2AP (C2-associated protein) family of proteins. The UBA domain at the C-terminus of c-Cbl, Cbl-b and D-Cbl(long) refers to a region with homology to UBA domains. Conserved tyrosine residues are shown in purple.

Figure 2. Cbl-directed internalization, multi-ubiquitylation and degradation of activated RTKs, and modulation by Sprouty

(a) Growth factor (GF) binding induces RTK tyrosine phosphorylation and the recruitment of Cbl to the activated receptor by adaptor proteins such as Grb2, which is required to support receptor endocytosis [144]. This allows the TKB domain to engage a specific phosphotyrosine on the RTK [e.g. pTyr¹⁰⁴⁵ on the EGF receptor (EGFR)]. Activation of Src kinases after GF binding induces the tyrosine phosphorylation of Cbl and other proteins, including Sprouty. The association of Sprouty with the RING finger domain initially inhibits Cbl's recruitment of Ubc enzymes (E2s), but tyrosine phosphorylation of Sprouty removes this inhibition by displacing it from the RING finger to now interact with the TKB domain (b). This allows the RING finger to recruit an E2 conjugating enzyme which promotes the polyubiquitylation of Sprouty (c) and its subsequent proteasomal degradation (d). The TKB domain is freed to target the receptor (pTyr¹⁰⁴⁵ of the EGF receptor) and the E3 ligase function of Cbl can then catalyse the transfer of a ubiquitin molecule from the RING-finger-bound E2 to the RTK (e). This process has been found to be sufficient to mediate receptor internalization. Continued addition of ubiquitin moieties leads to multi-ubiquitylation which marks the RTK for intracellular trafficking to the lysosome, where the receptor is degraded. Tyrosine phosphorylation of Cbl also enhances the recruitment of a CIN85–endophilin complex through a novel proline-arginine motif (shown as PR). This protein complex helps to promote receptor internalization by causing the membrane to invaginate. Cbl tyrosine phosphorylation also recruits SH2-domain-containing proteins, such as Crk and p85 (not shown), and this recruitment can enhance signalling responses from the receptor. For simplicity, the Grb2–Cbl interaction is not shown in (e), although it still contributes to maintaining Cbl's association with the receptor. Ub, ubiquitin.

Figure 3. Different types of ubiquitylation determine alternative fates for substrates targeted by E3 ligases

The substrates targeted by Cbl proteins can either be multi-ubiquitylated, such as the EGF receptor following activation by ligand binding, or poly-ubiquitylated, as is the case for Sprouty proteins. The multi-ubiquitylated EGFR receptor is destined for lysosomal degradation, whereas Sprouty proteins are degraded in the 26 S proteasome. The mechanisms that enable Cbl proteins to direct both types of ubiquitylation remain to be determined. Ubiquitin linkages formed through Lys⁴⁸ of ubiquitin direct proteins for proteasomal degradation, whereas linkages through Lys⁶³ and Lys²⁹ result in differing substrate fates (reviewed in [29]).

Figure 4. c-Cbl preferentially regulates TCR and Lck levels in CD4/CD8 DP thymocytes and negatively regulates tyrosine kinase signalling following anti-CD3/4 cross-linking

(a) CD4⁺CD8⁺ DP thymocytes and T-cells from lymph nodes of wild-type (+/+), c-Cbl^−/− (c−/−) and Cbl-b^−/− (b−/−) mice were analysed by flow cytometry for surface expression of TCRβ and intracellular levels of Lck. The results show that, in thymocytes, the negative regulation of surface TCR levels and intracellular Lck levels is carried out by c-Cbl and not by Cbl-b, and that this regulation by c-Cbl is not evident in peripheral T-cells. (b) Thymocytes from the above mice were left unstimulated or stimulated for 5 min at 37 °C with anti-CD3 and anti-CD4 antibodies, and total cell lysates were immunoblotted with anti-phosphotyrosine antibodies (4G10 monoclonal). The results show that c-Cbl, but not Cbl-b, has a marked effect on negatively regulating the activity of ZAP-70 and the tyrosine phosphorylation of its substrates SLP-76 and LAT. Molecular-mass sizes are shown in kDa.

Figure 5. Distinct mechanisms of negative regulation of TCR signalling by c-Cbl in thymocytes (a) and Cbl-b in peripheral T-cells (b)

(a) In CD4/CD8 DP thymocytes, c-Cbl negatively regulates the surface expression levels of the TCRα/β and CD3ε/γ/δ receptors, as well as the intracellular levels of CD3ζ and the Src family kinases Lck and Fyn. c-Cbl also negatively regulates the activity of ZAP-70; however, the mechanism of this regulation has not been determined, as it does not involve the TKB or RING finger domains. c-Cbl also plays a positive role by enhancing PI3K activity through the recruitment of the p85 regulatory subunit to the cell membrane. (b) In peripheral T-cells, Cbl-b negatively regulates a distinct set of signalling proteins to those targeted by c-Cbl in thymocytes. Cbl-b ubiquitylates Vav, which suppresses its tyrosine phosphorylation and activity as a GDP/GTP-exchange factor for Cdc42. This inhibits the interaction of Cdc42 with WASP, thus reducing the extent of actin reorganization and TCR clustering. Cbl-b also ubiquitylates p85, which inhibits its recruitment to CD28 and TCRζ, therefore suppressing the activation of PI3K. PLCγ1 and PKCθ are ubiquitylated by Cbl-b, and their reduced activity suppresses calcium mobilization and the activation of transcription factors that lead to IL-2 production. It is important to note that none of the above substrates show evidence of reduced protein levels following Cbl-b directed ubiquitylation. (a) and (b) Broken arrows represent pathways downstream of ZAP-70 leading to Ras, Rac, PLCγ or Cdc42 activation, but where intermediate effectors have not been shown. Plus or minus signs indicate the resultant activating or inhibitory effect on various pathways as a consequence of Cbl protein activity towards ZAP-70, Vav or PI3K. RF, RING finger.