Regulation of Ubiquitin Protein Ligase Activity in c-Cbl by Phosphorylation-induced Conformational Change and Constitutive Activation by Tyrosine to Glutamate Point Mutations

Abstract

c-Cbl down-regulates receptor tyrosine kinases by conjugating ubiquitin to them, leading to receptor internalization and degradation. The ubiquitin protein ligase activity of c-Cbl (abbreviated as E3 activity) is mediated by its RING finger domain. We show here that the E3 activity of c-Cbl is negatively regulated by other domains present in the amino-terminal half of the protein (the TKB and linker helix domains) and that this negative regulation is removed when the protein is phosphorylated on tyrosine residues. Protease digestion studies indicate that tyrosine phosphorylation alters the conformation of c-Cbl. We also show that mutation of certain conserved tyrosine residues to glutamate can constitutively activate the E3 activity of c-Cbl. In particular, a Y371E mutant shows constitutive E3 activity while retaining the ability to bind epidermal growth factor receptor (EGFR). The Y371E mutant also has altered protease sensitivity from wild type, instead resembling the proteolytic pattern seen with tyrosine-phosphorylated c-Cbl. Mutation of the homologous tyrosine residue in Cbl-b to glutamate also leads to E3 activation while retaining EGFR-binding ability. These studies argue that Tyr-371 plays a key role in activating the E3 activity of c-Cbl and that the Y371E mutant may partially mimic phosphorylation at that site. However, Tyr-371 point mutants of c-Cbl are still able to undergo phosphorylation-induced E3 activation, and we show that Tyr-368 can also be phosphorylated in addition to Tyr-371, and contributes to activation.The c-Cbl proto-oncogene was first discovered as the cellular homologue of v-Cbl, a viral transforming gene from the Cas NS-1 murine retrovirus, which causes pre-B cell lymphomas and myelogenous leukemias in mice (1). The transforming gene v-Cbl is a truncation mutant of c-Cbl, which itself does not transform cells (2). Human c-Cbl encodes a widely expressed cytosolic protein of 906 amino acids, which is a prominent substrate of a variety of tyrosine kinases and undergoes binding interactions with a large number of intracellular signaling molecules (3, reviewed in Refs. 4 -8). Two other related genes exist in mammals, Cbl-b (9) and Cbl-3 (10), and Cbl homologues have been identified in Caenorhabditis elegans (11) and Drosophila (12).The amino terminus of c-Cbl contains a conserved functional domain that binds phosphotyrosine, composed of a four-helix bundle, a Ca 2ϩ -binding EF hand domain, and a variant SH2 domain (13). These structural components together comprise a functional unit that has been called the TKB domain (for tyrosine-kinase binding). A short helical linker region connects the TKB domain to a RING finger domain that contains two bound zinc ions. The TKB, linker helix, and RING domains are well conserved among all Cbl family members.Important clues regarding Cbl function have come from genetic studies in C. elegans and Drosophila, in which it was found that Cbl family members act to negatively regulate receptor tyrosine kinases (11,12). Subsequent studies in mammalian cells ...

ROMK1 channel activity is regulated by monoubiquitination

“…It is possible that monoubiquitination of ROMK channels may be required for initiating endocytosis induced by stimulation of PTK activity. Interestingly, it has been demonstrated that activity of c-Cbl, an E3 ubiquitin ligase with RING finger domain, is stimulated by tyrosine phosphorylation (41). Fig.…”

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

“…The domain is required for recruitment of E2 enzymes, and functions together with the linker sequence that connects the TKB domain and the RING finger domain in this recruitment (Joazeiro et al, 1999;Levkowitz et al, 1999;Yokouchi et al, 1999;Zheng et al, 2000). The E3 activity is regulated by phosphorylation of residues Y368 and Y371 in c-Cbl, and phosphorylation probably results in conformational changes in c-Cbl favoring E3 activity Kassenbrock and Anderson, 2004). Additionally, c-Cbl activity is proposed to be (Yokouchi et al, 2001).…”

Reduced Cbl phosphorylation and degradation of the ζ‐chain of the T‐cell receptor/CD3 complex in T cells with low Lck levels

“…Notable targets for c-Cbl-mediated ubiquitination are Lck [9], Vav [10], Fyn [11][12][13], , as well as the already mentioned TCR/ CD3-complex. c-Cbl itself is activated by tyrosine phosphorylation on several residues, most importantly Y700, Y731 and Y774 [13,[15][16][17]. These residues are not, however, substrates of Lck or but of Fyn and Syk [13,16,18,19], which are activated directly or indirectly by Lck [20].…”