A conserved DpYR motif in the juxtamembrane domain of the Met receptor family forms an atypical c-Cbl/Cbl-b tyrosine kinase binding domain binding site required for suppression of oncogenic activation

The activation and phosphorylation of Met, the receptor tyrosine kinase (RTK) for hepatocyte growth factor, initiates the recruitment of multiple signaling proteins, one of which is c-Cbl, a ubiquitin-protein ligase. c-Cbl promotes ubiquitination and enhances the down-modulation of the Met receptor and other RTKs, targeting them for lysosomal sorting and subsequent degradation. The ubiquitination of Met by c-Cbl requires the direct interaction of the c-Cbl tyrosine kinase binding (TKB) domain with tyrosine 1003 in the Met juxtamembrane domain. Although a consensus for c-Cbl TKB domain binding has been established ((D/N)XpYXX(D/E0phi), this motif is not present in Met, suggesting that other c-Cbl TKB domain binding motifs may exist. By alanine-scanning mutagenesis, we have identified a DpYR motif including Tyr(1003) as being important for the direct recruitment of the c-Cbl TKB domain and for ubiquitination of the Met receptor. The substitution of Tyr(1003) with phenylalanine or substitution of either aspartate or arginine residues with alanine impairs c-Cbl-recruitment and ubiquitination of Met and results in the oncogenic activation of the Met receptor. We demonstrate that the TKB domain of Cbl-b, but not Cbl-3, binds to the Met receptor and requires an intact DpYR motif. Modeling studies suggest the presence of a salt bridge between the aspartate and arginine residues that would position pTyr(1003) for binding to the c-Cbl TKB domain. The DpYR motif is conserved in other members of the Met RTK family but is not present in previously identified c-Cbl-binding proteins, identifying DpYR as a new binding motif for c-Cbl and Cbl-b.     

An adjacent arginine, and the phosphorylated tyrosine in the c-Met receptor target sequence, dictates the orientation of c-Cbl binding

Previously, we have demonstrated that the tyrosine phosphorylated hepatocyte growth factor receptor (Met) binds to the c-Cbl phosphotyrosine-recognition, tyrosine kinase binding (TKB) domain in a reverse orientation compared to other c-Cbl binding partners. A Met peptide with the DpYR motif changed to RpYD (MetRD) retains a similar TKB binding affinity as the native Met peptide. However, the TKB: MetRD complex crystal structure reveals a complete reversal of the binding orientation. Collated data indicates that both binding and orientation is dictated by the phosphorylated tyrosine and an adjacent arginine forming intra-peptide hydrogen bonds and aligning unidirectionally with complementary charges in the phosphotyrosine binding pocket of c-Cbl.

Structured summary

c-Cbl and MetRDbind: shown by x-ray crystallography (view interaction)c-Cbl and MetRDbind: shown by mass spectrometry studies of complexes (view interaction)c-Cblbind to Met: shown by surface plasmon resonance (view interactions 1,2)     

Negative regulation of EphA2 receptor by Cbl

The c-Cbl proto-oncogene product Cbl has emerged as a negative regulator of receptor and non-receptor tyrosine kinases, a function dependent on its recently identified ubiquitin ligase activity. Here, we report that EphA2, a member of Eph receptor tyrosine kinases is negatively regulated by Cbl. The negative regulation of EphA2 mediated by Cbl is dependent on the activity of EphA2, as the kinase inactive mutant of EphA2 cannot be regulated by Cbl. Moreover, a point mutation (G306E-Cbl) in TKB region of Cbl that has been reported to abolish Cbl binding to RTKs and non-receptor tyrosine kinases impaired the binding to active EphA2. The dominant negative mutant 70Z-Cbl, which has a 17-amino acids deletion in the N-boundary of the RING finger domain, defuncted negative regulatory function of Cbl to EphA2. These results demonstrate that the TKB domain and RING finger domain of Cbl are essential for this negative regulation.     

RING finger mutations that abolish c-Cbl-directed polyubiquitination and downregulation of the EGF receptor are insufficient for cell transformation

The c-Cbl protooncogene can function as a negative regulator of receptor protein tyrosine kinases (RPTKs) by targeting activated receptors for polyubiquitination and downregulation. This function requires its tyrosine kinase binding (TKB) domain for targeting RPTKs and RING finger domain to recruit E2 ubiquitin-conjugating enzymes. It has therefore been proposed that oncogenic Cbl proteins act in a dominant-negative manner to block this c-Cbl activity. In testing this hypothesis, we found that although mutations spanning the RING finger abolish c-Cbl-directed polyubiquitination and downregulation of RPTKs, they do not induce transformation. In contrast, it is mutations within a highly conserved alpha-helical structure linking the SH2 and RING finger domains that render Cbl proteins oncogenic. Thus, Cbl transformation involves effects additional to polyubiquitination of RPTKs that are independent of the RING finger and its ability to recruit E2-conjugating enzymes.     

Additional Serine/Threonine Phosphorylation Reduces Binding Affinity but Preserves Interface Topography of Substrate Proteins to the c-Cbl TKB Domain

The E3-ubiquitin ligase, c-Cbl, is a multi-functional scaffolding protein that plays a pivotal role in controlling cell phenotype. As part of the ubiquitination and downregulation process, c-Cbl recognizes targets, such as tyrosine kinases and the Sprouty proteins, by binding to a conserved (NX/R)pY(S/T)XXP motif via its uniquely embedded SH2 domain (TKB domain). We previously outlined the mode of binding between the TKB domain and various substrate peptide motifs, including epidermal growth factor receptor (EGFR) and Sprouty2 (Spry2), and demonstrated that an intrapetidyl hydrogen bond forms between the (pY-1) arginine or (pY-2) asparagine and the phosphorylated tyrosine, which is crucial for binding. Recent reports demonstrated that, under certain types of stimulation, the serine/threonine residues at the pY+1 and/or pY+2 positions within this recognition motif of EGFR and Sprouty2 may be endogenously phosphorylated. Using structural and binding studies, we sought to determine whether this additional phosphorylation could affect the binding of the TKB domain to these peptides and consequently, whether the type of stimulation can dictate the degree to which substrates bind to c-Cbl. Here, we show that additional phosphorylation significantly reduces the binding affinity between the TKB domain and its target proteins, EGFR and Sprouty2, as compared to peptides bearing a single tyrosine phosphorylation. The crystal structure indicates that this is accomplished with minimal changes to the essential intrapeptidyl bond and that the reduced strength of the interaction is due to the charge repulsion between c-Cbl and the additional phosphate group. This obvious reduction in binding affinity, however, indicates that Cbl''s interactions with its TKB-centered binding partners may be more favorable in the absence of Ser/Thr phosphorylation, which is stimulation and context specific in vivo. These results demonstrate the importance of understanding the environment in which certain residues are phosphorylated, and the necessity of including this in structural investigations.     

Structural basis for UBA-mediated dimerization of c-Cbl ubiquitin ligase

Ligand-induced down-regulation by the ubiquitin-protein ligases, c-Cbl and Cbl-b, controls signaling downstream from many receptor-tyrosine kinases (RTK). Cbl proteins bind to phosphotyrosine residues on activated RTKs to affect ligand-dependent ubiquitylation of these receptors targeting them for degradation in the lysosome. Both c-Cbl and Cbl-b contain a ubiquitin-associated (UBA) domain, which is important for Cbl dimerization and tyrosine phosphorylation; however, the mechanism of UBA-mediated dimerization and its requirement for Cbl biological activity is unclear. Here, we report the crystal structure of the UBA domain of c-Cbl refined to 2.1-A resolution. The structure reveals the protein is a symmetric dimer tightly packed along a large hydrophobic surface formed by helices 2 and 3. NMR chemical shift mapping reveals heterodimerization can occur with the related Cbl-b UBA domain via the same surface employed for homodimerization. Disruption of c-Cbl dimerization by site-directed mutagenesis impairs c-Cbl phosphorylation following activation of the Met/hepatocyte growth factor RTK and c-Cbl-dependent ubiquitination of Met. This provides direct evidence for a role of Cbl dimerization in terminating signaling following activation of RTKs.     

c-Cbl is involved in Met signaling in B cells and mediates hepatocyte growth factor-induced receptor ubiquitination

Hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase Met are key regulators of epithelial motility and morphogenesis. Recent studies indicate that the HGF/Met pathway also plays a role in B cell differentiation, whereas uncontrolled Met signaling may lead to B cell neoplasia. These observations prompted us to explore HGF/Met signaling in B cells. In this study, we demonstrate that HGF induces strong tyrosine phosphorylation of the proto-oncogene product c-Cbl in B cells and increases Cbl association with the Src family tyrosine kinases Fyn and Lyn, as well as with phosphatidylinositol-3 kinase and CrkL. In addition, we demonstrate that c-Cbl mediates HGF-induced ubiquitination of Met. This requires the juxtamembrane tyrosine Y1001 (Y2) of Met, but not the multifunctional docking site (Y14/15) or any additional C-terminal tyrosine residues (Y13-16). In contrast to wild-type c-Cbl, the transforming mutants v-Cbl and 70Z/3 Cbl, which lack the ubiquitin ligase RING finger domain, suppress Met ubiquitination. Our findings identify c-Cbl as a negative regulator of HGF/Met signaling in B cells, mediating ubiquitination and, consequently, proteosomal degradation of Met, and suggest a role for Cbl in Met-mediated tumorigenesis.     

Intramolecular regulatory switch in ZAP-70: analogy with receptor tyrosine kinases

ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.     

A role of the protein Cbl in FGF-2-induced angiogenesis in murine brain endothelial cells

The Cbl protein functions both as a multivalent adaptor and a negative regulator of receptor tyrosine kinases (RTKs), the latter by directing polyubiquitination of RTKs. To study the function of Cbl in endothelial cell signalling and angiogenesis, wild-type Cbl and tyrosine kinase binding (TKB) domain mutated Cbl (G306E) were overexpressed in murine immortalised brain endothelial (IBE) cells. Wild-type Cbl cells exhibited enhanced proliferation in low serum compared with the control and G306E Cbl cells. Furthermore, up-regulated phosphorylation of fibroblast growth factor receptor 1 (FGFR-1) and Akt were observed in wild-type Cbl cells upon FGF-2 stimulation. A Cbl TKB domain mutant, G306E, disrupted the phosphorylation of the FGFR-1 but not that of FRS2. In the tubular morphogenesis assay, cells expressing wild-type Cbl initially formed tubular structures. These showed decreased stability and converted into cell aggregates, possibly due to a failure to cease proliferating. Our data support the idea that the wild-type Cbl cells exhibit enhanced proliferation, and thus lose their ability to differentiate appropriately. The present study reveals a role of the Cbl protein in FGF-2 dependent signalling in endothelial cells by its destabilisation of tubular structures.     

Activation of receptor protein-tyrosine kinases from the cytoplasmic compartment

It is widely accepted that receptor protein-tyrosine kinases (RTKs) are activated upon dimerization by binding to their extracellular ligands. However, EGF receptor (EGFR) dimerization per se does not require ligand binding. Instead, its cytoplasmic kinase domains have to form characteristic head-to-tail asymmetric dimers to become active, where one 'activator' domain activates the other 'receiver' domain. The non-catalytic, cytoplasmic regions of RTKs, namely the juxtamembrane and carboxy terminal portions, also regulate kinase activity. For instance, the juxtamembrane region of the RTK MuSK inhibits the kinase domain probably together with a cellular factor(s). These findings suggest that RTKs could be activated by cytoplasmic proteins. Indeed, Dok-7 and cytohesin have recently been identified as such activators of MuSK and EGFR, respectively. Given that failure of Dok-7 signaling causes myasthenia, and inhibition of cytohesin signaling reduces the proliferation of EGFR-dependent cancer cells, cytoplasmic activators of RTKs may provide new therapeutic targets.     

Structural insights into FRS2α PTB domain recognition by neurotrophin receptor TrkB

The fibroblast growth factor receptor (FGFR) substrate 2 (FRS2) family proteins function as scaffolding adapters for receptor tyrosine kinases (RTKs). The FRS2α proteins interact with RTKs through the phosphotyrosine‐binding (PTB) domain and transfer signals from the activated receptors to downstream effector proteins. Here, we report the nuclear magnetic resonance structure of the FRS2α PTB domain bound to phosphorylated TrkB. The structure reveals that the FRS2α‐PTB domain is comprised of two distinct but adjacent pockets for its mutually exclusive interaction with either nonphosphorylated juxtamembrane region of the FGFR, or tyrosine phosphorylated peptides TrkA and TrkB. The new structural insights suggest rational design of selective small molecules through targeting of the two conjunct pockets in the FRS2α PTB domain. Proteins 2014; 82:1534–1541. © 2014 Wiley Periodicals, Inc.     

Rotational coupling of the transmembrane and kinase domains of the Neu receptor tyrosine kinase

Ligand binding to receptor tyrosine kinases (RTKs) regulates receptor dimerization and activation of the kinase domain. To examine the role of the transmembrane domain in regulation of RTK activation, we have exploited a simplified transmembrane motif, [VVVEVVV](n), previously shown to activate the Neu receptor. Here we demonstrate rotational linkage of the transmembrane domain with the kinase domain, as evidenced by a periodic activation of Neu as the dimerization motif is shifted across the transmembrane domain. These results indicate that activation requires a specific orientation of the kinase domains with respect to each other. Results obtained with platelet-derived growth factor receptor-beta suggest that this rotational linkage of the transmembrane domain to the kinase domain may be a general feature of RTKs. These observations suggest that activating mutations in RTK transmembrane and juxtamembrane domains will be limited to those residues that position the kinase domains in an allowed rotational conformation.     

Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7.

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.     

Autoregulation by the juxtamembrane region of the human ephrin receptor tyrosine kinase A3 (EphA3)

Ephrin receptors (Eph) affect cell shape and movement, unlike other receptor tyrosine kinases that directly affect proliferative pathways. The kinase domain of EphA3 is activated by ephrin binding and receptor oligomerization. This activation is associated with two tyrosines in the juxtamembrane region; these tyrosines are sites of autophosphorylation and interact with the active site of the kinase to modulate activity. This allosteric event has important implications both in terms of understanding signal transduction pathways mediated by Eph kinases as well as discovering specific therapeutic ligands for receptor kinases. In order to provide further details of the molecular mechanism through which the unphosphorylated juxtamembrane region blocks catalysis, we studied wild-type and site-specific mutants in detail. High-resolution structures of multiple states of EphA3 kinase with and without the juxtamembrane segment allowed us to map the coupled pathway of residues that connect the juxtamembrane segment, the activation loop, and the catalytic residues of the kinase domain. This highly conserved set of residues likely delineates a molecular recognition pathway for most of the Eph RTKs, helping to characterize the dynamic nature of these physiologically important enzymes.     

The 2.7 A crystal structure of the autoinhibited human c-Fms kinase domain

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 A resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.     

Ligand-induced downregulation of TrkA is partly regulated through ubiquitination by Cbl

Nerve growth factor (NGF) binding to its receptor TrkA, which belongs to the family of receptor tyrosine kinases (RTKs), is known to induce its internalization, endosomal trafficking and subsequent lysosomal degradation. The Cbl family of ubiquitin ligases plays a major role in mediating ubiquitination and degradation of RTKs. However, it is not known whether Cbl participates in mediating ubiquitination of TrkA. Here we report that c-Cbl mediates ligand-induced ubiquitination and degradation of TrkA. TrkA ubiquitination and degradation required direct interactions between c-Cbl and phosphorylated TrkA. c-Cbl and ubiquitinated TrkA are found in a complex after NGF stimulation and are degraded in lysosomes. Taken together, our data demonstrate that c-Cbl can induce downregulation of NGF-TrkA complexes through ubiquitination and degradation of TrkA.     

Regulation of ubiquitin protein ligase activity in c-Cbl by phosphorylation-induced conformational change and constitutive activation by tyrosine to glutamate point mutations

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.     

A juxtamembrane tyrosine in the colony stimulating factor-1 receptor regulates ligand-induced Src association, receptor kinase function, and down-regulation

Recent literature implicates a regulatory function of the juxtamembrane domain (JMD) in receptor tyrosine kinases. Mutations in the JMD of c-Kit and Flt3 are associated with gastrointestinal stromal tumors and acute myeloid leukemias, respectively. Additionally, autophosphorylated Tyr559 in the JMD of the colony stimulating factor-1 (CSF-1) receptor (CSF-1R) binds to Src family kinases (SFKs). To investigate SFK function in CSF-1 signaling we established stable 32D myeloid cell lines expressing CSF-1Rs with mutated SFK binding sites (Tyr559-TFI). Whereas binding to I562S was not significantly perturbed, Y559F and Y559D exhibited markedly decreased CSF-1-dependent SFK association. All JMD mutants retained intrinsic kinase activity, but Y559F, and less so Y559D, showed dramatically reduced CSF-1-induced autophosphorylation. CSF-1-mediated wild-type (WT)-CSF-1R phosphorylation was not markedly affected by SFK inhibition, indicating that lack of SFK binding is not responsible for diminished Y559F phosphorylation. Unexpectedly, cells expressing Y559F were hyperproliferative in response to CSF-1. Hyperproliferation correlated with prolonged activation of Akt, ERK, and Stat5 in the Y559F mutant. Consistent with a defect in receptor negative regulation, c-Cbl tyrosine phosphorylation and CSF-1R/c-Cbl co-association were almost undetectable in the Y559F mutant. Furthermore, Y559F underwent reduced multiubiquitination and delayed receptor internalization and degradation. In conclusion, we propose that Tyr559 is a switch residue that functions in kinase regulation, signal transduction and, indirectly, receptor down-regulation. These findings may have implications for the oncogenic conversion of c-Kit and Flt3 with JMD mutations.     

Evidence for direct interaction between Sprouty and Cbl

Sprouty (SPRY) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with their Drosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. Here, we demonstrate that hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogated an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. Our results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis.     

Autoinhibition of the kit receptor tyrosine kinase by the cytosolic juxtamembrane region

Genetic studies have implicated the cytosolic juxtamembrane region of the Kit receptor tyrosine kinase as an autoinhibitory regulatory domain. Mutations in the juxtamembrane domain are associated with cancers, such as gastrointestinal stromal tumors and mastocytosis, and result in constitutive activation of Kit. Here we elucidate the biochemical mechanism of this regulation. A synthetic peptide encompassing the juxtamembrane region demonstrates cooperative thermal denaturation, suggesting that it folds as an autonomous domain. The juxtamembrane peptide directly interacted with the N-terminal ATP-binding lobe of the kinase domain. A mutation in the juxtamembrane region corresponding to an oncogenic form of Kit or a tyrosine-phosphorylated form of the juxtamembrane peptide disrupted the stability of this domain and its interaction with the N-terminal kinase lobe. Kinetic analysis of the Kit kinase harboring oncogenic mutations in the juxtamembrane region displayed faster activation times than the wild-type kinase. Addition of exogenous wild-type juxtamembrane peptide to active forms of Kit inhibited its kinase activity in trans, whereas the mutant peptide and a phosphorylated form of the wild-type peptide were less effective inhibitors. Lastly, expression of the Kit juxtamembrane peptide in cells which harbor an oncogenic form of Kit inhibited cell growth in a Kit-specific manner. Together, these results show the Kit kinase is autoinhibited through an intramolecular interaction with the juxtamembrane domain, and tyrosine phosphorylation and oncogenic mutations relieved the regulatory function of the juxtamembrane domain.