Protein tyrosine kinase regulation by ubiquitination: critical roles of Cbl-family ubiquitin ligases

Abstract

Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell-cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant "activated PTK-selective" ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease.

Fig. 1

Alternate routes of endocytic traffic of activated tyrosine kinase coupled cell surface receptors and the role of Cbl-family protein dependent ubiquitination. The schematic shows the EGF-induced activation and endocytosis of EGF receptor (EGFR) as a prototype; for simplicity, only clathrin-dependent endocytic pathway is shown. EGF activation leads to receptor dimerization, auto-/trans-phosphorylation and recruitment of signaling proteins including Cbl proteins. The activated receptors accumulate in clathrin-coated pits followed by internalization into early endosomes (EE) and traffic to sorting endosomes (SE), late endosome/multi-vesicular body (MVB) and finally lysosomes (degradative pathway). Alternatively, internalized receptors can be sorted into recycling pathway and returned for further ligand binding and activation (Recycling Pathway). Cbl-dependent ubiquitination is thought to serve as an internalization signal and as a sorting signal at the level of MVBs. The latter role involves the recognition of the ubiquitin modification on the receptor by components of the ESCRT complexes to facilitate the sorting of ubiquitinated receptors into internal vesicles of the MVB, an event required for lysosomal degradation of activated receptors. Non-ubiquitinated receptors or those from which ubiquitin species have been removed by deubiquitinases (DUBs) are thought to enter the recycling pathway. Signaling proteins that interact with Cbl proteins and are ubiquitinated may also be targeted for an alternate mode of degradation in the proteasome.

Fig. 2

Evolutionary conservation of the primary structure and domain organization of Cbl proteins. The comparison includes: the three human (Homo sapiens) Cbl proteins (Cbl or c-Cbl; Cbl-b; and Cbl-c, Cbl-3 or Cbl- SL) as representative mammalian Cbl proteins; Chicken (Gallus gallus) Cbl; Zebra fish (Danio rerio) Cbl; Frog (Xenopus tropicalis) Cbl; Fly (Drosophila melanogaster) long and short Cbl; Worm (Caenorhabditis elegans) Cbl (SLI-1); and Dicty (Dictyostelium discoideum) Cbl (Cbl-A). * Xenopus tropicalis was used for comparison rather than Xenopus laevis, as Cbl sequences in the databases for the latter species were partial. Domain designations: TKB, Tyrosine Kinase-Binding; 4H, four-helical bundle; SH2, Src-Homology 2; RF, RING Finger; L, Linker helical region; P, Proline-rich region; U, Ubiquitin-associated (UBA) domain; The amino acid sequences were compared to human Cbl and Cbl-b and are shown as two values separated by “/” under % identity and similarity for whole protein (or available partial sequence; shown with // across C-terminal end) and for the N-terminal domains (TKB, Linker and RF). The latter emphasizes the higher evolutionary conservation of the N-terminal domains that constitute the core PTK-directed E3 activity of Cbl proteins. V-Cbl corresponds to amino acids 1–357 of mouse Cbl that are present in viral Cbl oncogene. Dicty Cbl 4H region (inferred in UniProt) was confirmed using the YASARA structure program (www.yasara.org); however, a linker helical region has not been identified in Dicty Cbl, making it an exception in the entire Cbl protein family. N and C refer to amino and carboxyl termini.

Fig. 3

Phosphotyrosine-containing sequence motifs that mediate Cbl TKB domain binding to its targets. The consensus motifs and individual target protein sequence motifs with critical amino acids that contribute to binding affinity are indicated [–210]. Φ indicates hydrophobic residue. The motifs and sequences are adapted from [106].

Fig. 4

Domain architecture of Cbl proteins and major protein-protein interactions involving various domains/motifs. The N-terminal Tyrosine Kinase-Binding (TKB) domain binds to phosphotyrosine (pY)-containing sequence motifs in target proteins, that typically include activated receptor and non-receptor tyrosine kinases. The Linker region and the RING finger (RF) domain bind to ubiquitin conjugating enzymes (E2). The proline-rich motifs (Pro-rich) bind to SH3 domain containing signaling and endocytic proteins. Induced tyrosine phosphorylation sites (major sites at Y700, Y731 and Y774 are shown) recruit SH2 domain-containing signaling proteins. The Ubiquitin-associated (UBA) domain/leucine zipper near the C-terminus is involved in ubiquitin binding and dimerization. N and C refer to amino and carboxyl termini.

Fig. 5

Schematic representation of the basic role of Cbl-family proteins as ubiquitin ligases (E3s) towards components of tyrosine kinase signaling pathways. Human Cbl is shown as a prototype of the family. The TKB domain, the proline-rich motifs and the induced tyrosine phosphorylation sites recruit targets for ubiquitin modification. The linker/RF-associated ubiquitin conjugating enzyme (E2) serves as an acceptor of activated ubiquitin from a ubiquitin-activating enzyme (E1) and transfers it to targets bound to various domain/motifs of Cbl to promote mono-ubiquitination (shown as a single UB subunit) or poly-ubiquitination (shown as four UB subunits).