Down-regulation of transcobalamin receptor TCblR/CD320 by siRNA inhibits cobalamin uptake and proliferation of cells in culture

The clinical phenotype of cobalamin (Cbl) deficiency is dictated by the essential role of this vitamin in two key enzymatic reactions. Multiple proteins and receptors participate in the absorption, transport and delivery of this vitamin to tissue cells. Cellular uptake of Cbl is mediated by transcobalamin (TC), a plasma protein and a transmembrane receptor (TCblR) with high affinity for TC saturated with Cbl. Knockdown of TCblR with siRNA results in decreased TC–Cbl uptake. The ensuing Cbl deficiency leads to an increase in doubling time and decreased proliferation of these cells. The study confirms the seminal role of this receptor in the cellular uptake of Cbl and its down-regulation as a potential strategy to inhibit proliferation of cancer cells.     

Context-dependence of T-loop Mediated Long-range RNA Tertiary Interactions

The architecture and folding of complex RNAs is governed by a limited set of highly recurrent structural motifs that form long-range tertiary interactions. One of these motifs is the T-loop, which was first identified in tRNA but is broadly distributed across biological RNAs. While the T-loop has been examined in detail in different biological contexts, the various receptors that it interacts with are not as well defined. In this study, we use a cell-based genetic screen in concert with bioinformatic analysis to examine three different, but related, T-loop receptor motifs found in the flavin mononucleotide (FMN) and cobalamin (Cbl) riboswitches. As a host for different T-loop receptors, we employed the env8 class-II Cbl riboswitch, an RNA that uses two T-loop motifs for both folding and supporting the ligand binding pocket. A set of libraries was created in which select nucleotides that participate in the T-loop/T-loop receptor (TL/TLR) interaction were fully randomized. Library members were screened for their ability to support Cbl-dependent expression of a reporter gene. While T-loops appear to be variable in sequence, we find that the functional sequence space is more restricted in the Cbl riboswitch, suggesting that TL/TLR interactions are context dependent. Our data reveal clear sequence signatures for the different types of receptor motifs that align with phylogenic analysis of these motifs in the FMN and Cbl riboswitches. Finally, our data suggest the functional contribution of various nucleobase-mediated long-range interactions within the riboswitch subclass of TL/TLR interactions that are distinct from those found in other RNAs.     

Structural characterization of a novel Cbl phosphotyrosine recognition motif in the APS family of adapter proteins

The Cbl adapter proteins typically function to down-regulate activated protein tyrosine kinases and other signaling proteins by coupling them to the ubiquitination machinery for degradation by the proteasome. Cbl proteins bind to specific tyrosine-phosphorylated sequences in target proteins via the tyrosine kinase-binding (TKB) domain, which comprises a four-helix bundle, an EF-hand calcium-binding domain, and a non-conventional Src homology-2 domain. The previously derived consensus sequence for phosphotyrosine recognition by the Cbl TKB domain is NXpY(S/T)XXP (X denotes lesser residue preference), wherein specificity is conferred primarily by residues C-terminal to the phosphotyrosine. Cbl is recruited to and phosphorylated by the insulin receptor in adipose cells through the adapter protein APS. APS is phosphorylated by the insulin receptor on a C-terminal tyrosine residue, which then serves as a binding site for the Cbl TKB domain. Using x-ray crystallography, site-directed mutagenesis, and calorimetric studies, we have characterized the interaction between the Cbl TKB domain and the Cbl recruitment site in APS, which contains a sequence motif, RA(V/I)XNQpY(S/T), that is conserved in the related adapter proteins SH2-B and Lnk. These studies reveal a novel mode of phosphopeptide interaction with the Cbl TKB domain, in which N-terminal residues distal to the phosphotyrosine directly contact residues of the four-helix bundle of the TKB domain.     

Growth factor receptor binding protein 2-mediated recruitment of the RING domain of Cbl to the epidermal growth factor receptor is essential and sufficient to support receptor endocytosis

Knockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR). To gain insights into the function of Grb2 in EGFR endocytosis, we have generated cell lines in which endogenous Grb2 was replaced by yellow fluorescent protein (YFP)-tagged Grb2 expressed at the physiological level. In these cells, Grb2-YFP fully reversed the inhibitory effect of Grb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis. Overexpression of Grb2-binding protein c-Cbl did not restore endocytosis in Grb2-depleted cells. However, EGFR endocytosis was rescued in Grb2-depleted cells by chimeric proteins consisting of the Src homology (SH) 2 domain of Grb2 fused to c-Cbl. The "knockdown and rescue" analysis revealed that the expression of Cbl-Grb2/SH2 fusions containing RING finger domain of Cbl restores normal ubiquitylation and internalization of the EGFR in the absence of Grb2, consistent with the important role of the RING domain in EGFR endocytosis. In contrast, the carboxy-terminal domain of Cbl, when attached to Grb2 SH2 domain, had 4 times smaller endocytosis-rescue effect compared with the RING-containing chimeras. Together, the data suggest that the interaction of Cbl carboxy terminus with CIN85 has a minor and a redundant role in EGFR internalization. We concluded that Grb2-mediated recruitment of the functional RING domain of Cbl to the EGFR is essential and sufficient to support receptor endocytosis.     

A requirement for calcium in the transport of cobalamin across the outer membrane of Escherichia coli

Cobalamin (Cbl) transport across the outer membrane of cells of Escherichia coli consists of high affinity Cbl binding to the btuB protein of the Cbl receptor, followed by the proton motive force- and tonB-dependent release of the Cbl into the periplasmic space. During a search for experimental conditions that would mimic this release in vitro with isolated cell envelope particles, we found that calcium was required for the high affinity Cbl binding, and subsaturating calcium concentrations resulted in the decreased affinity of the Cbl receptor for Cbl. The apparent affinity of the Cbl receptor for calcium (KD, approximately 30 nM at pH 6.6) decreased with decreasing pH, resulting in decreased affinity for Cbl at lower pH values. With suboptimal levels of calcium, Cbl binding was decreased by millimolar levels of magnesium.     

Leucine zipper-mediated homodimerization of the adaptor protein c-Cbl. A role in c-Cbl's tyrosine phosphorylation and its association with epidermal growth factor receptor.

The 120-kDa proto-oncogenic protein c-Cbl is a multidomain adaptor protein that is phosphorylated in response to the stimulation of a broad range of cell surface receptors and participates in the assembly of signaling complexes that are formed as a result of the activation of various signal transduction pathways. Several structural features of c-Cbl, including the phosphotyrosine-binding domain, proline-rich domain, and motifs containing phosphotyrosine and phosphoserine residues, mediate the association of c-Cbl with other components of these complexes. In addition to those domains that have been demonstrated to play a role in the binding of c-Cbl to other signaling molecules, c-Cbl also contains a RING finger motif and a putative leucine zipper. In this study, we demonstrate that the previously identified putative leucine zipper mediates the formation of Cbl homodimers. Using the yeast two-hybrid system, we show that deletion of the leucine zipper domain is sufficient to abolish Cbl homodimerization, while Cbl mutants carrying extensive N-terminal truncations retain the ability to dimerize with the full-length Cbl. The requirement of the leucine zipper for the homodimerization of Cbl was confirmed by in vitro binding assays, using deletion variants of the C-terminal half of Cbl with and without the leucine zipper domain, and in cells using Myc and green fluorescent protein (GFP) N-terminal-tagged Cbl variants. In cells, the deletion of the leucine zipper caused a decrease in both the tyrosine phosphorylation of Cbl and its association with the epidermal growth factor receptor following stimulation with epidermal growth factor, thus demonstrating a role for the leucine zipper in c-Cbl's signaling functions. Thus, the leucine zipper domain enables c-Cbl to homodimerize, and homodimerization influences Cbl's signaling function, modulating the activity of Cbl itself and/or affecting Cbl's associations with other signaling proteins in the cell.     

The cDNA sequence and the deduced amino acid sequence of human transcobalamin II show homology with rat intrinsic factor and human transcobalamin I.

The cellular uptake of cobalamin (Cbl, vitamin B12) is mediated by transcobalamin II (TCII), a plasma protein that binds Cbl and is secreted by human umbilical vein endothelial (HUVE) cells. These cells synthesize and secrete TCII and, therefore, served as the source of the complementary DNA (cDNA) library from which the TCII cDNA was isolated. This full-length cDNA consists of 1866 nucleotides that code for a leader peptide of 18 amino acids, a secreted protein of 409 amino acids, a 5'-untranslated segment of 37 nucleotides, and a 3'-untranslated region of 548 nucleotides. A single 1.9-kilobase species of mRNA corresponding to the size of the cDNA was identified by Northern blot analysis of the RNA isolated from HUVE cells. TCII has 20% amino acid homology and greater than 50% nucleotide homology with human transcobalamin I (TCI) and with rat intrinsic factor (R-IF). TCII has no homology with the amino-terminal region of R-IF that has been reported to have significant primary as well as secondary structural homology with the nucleotide-binding domain of NAD-dependent oxidoreductases. The regions of homology that are common to all three proteins are located in seven domains of the amino acid sequence. One or more of these conserved domains is likely to be involved in Cbl binding, a function that is common to all three proteins. However, the difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl analogues indicates, a priori, that structural differences in the ligand-binding site of these proteins exist and these probably resulted from divergence of a common ancestral gene.     

The adaptor molecule CIN85 regulates Syk tyrosine kinase level by activating the ubiquitin-proteasome degradation pathway

Triggering of mast cells and basophils by IgE and Ag initiates a cascade of biochemical events that lead to cell degranulation and the release of allergic mediators. Receptor aggregation also induces a series of biochemical events capable of limiting FcepsilonRI-triggered signals and functional responses. Relevant to this, we have recently demonstrated that Cbl-interacting 85-kDa protein (CIN85), a multiadaptor protein mainly involved in the process of endocytosis and vesicle trafficking, regulates the Ag-dependent endocytosis of the IgE receptor, with consequent impairment of FcepsilonRI-mediated cell degranulation. The purpose of this study was to further investigate whether CIN85 could alter the FcepsilonRI-mediated signaling by affecting the activity and/or expression of molecules directly implicated in signal propagation. We found that CIN85 overexpression inhibits the FcepsilonRI-induced tyrosine phosphorylation of phospholipase Cgamma, thus altering calcium mobilization. This functional defect is associated with a substantial decrease of Syk protein levels, which are restored by the use of selective proteasome inhibitors, and it is mainly due to the action of the ubiquitin ligase c-Cbl. Furthermore, coimmunoprecipitation experiments demonstrate that CIN85 overexpression limits the ability of Cbl to bind suppressor of TCR signaling 1 (Sts1), a negative regulator of Cbl functions, while CIN85 knockdown favors the formation of Cbl/Sts1 complexes. Altogether, our findings support a new role for CIN85 in regulating Syk protein levels in RBL-2H3 cells through the activation of the ubiquitin-proteasome pathway and provide a mechanism for this regulation involving c-Cbl ligase activity.     

APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes

APS is a Cbl-binding protein that is tyrosine phosphorylated by the insulin receptor kinase. Insulin-stimulated phosphorylation of tyrosine 618 in APS is necessary for its association with c-Cbl and the subsequent tyrosine phosphorylation of Cbl by the insulin receptor in both 3T3-L1 adipocytes and CHO-IR cells. When overexpressed in these cells, wild-type APS but not an APS/Y(618)F mutant facilitated the tyrosine phosphorylation of coexpressed Cbl and its association with Crk upon insulin stimulation. APS-facilitated phosphorylation occurred on tyrosines 371, 700, and 774 in the Cbl protein. APS also interacted directly with the c-Cbl-associated protein (CAP) and colocalized with the protein in cells. The association was dependent on the SH3 domains of CAP and was independent of insulin treatment. Overexpression of the APS/Y(618)F mutant in 3T3-L1 adipocytes blocked the insulin-stimulated tyrosine phosphorylation of endogenous Cbl and binding to Crk. Moreover, the translocation of GLUT4 from intracellular vesicles to the plasma membrane was also inhibited by overexpression of the APS/Y(618)F mutant. These data suggest that APS serves as an adapter protein linking the CAP/Cbl pathway to the insulin receptor and, further, that APS-facilitated Cbl tyrosine phosphorylation catalyzed by the insulin receptor is a crucial event in the stimulation of glucose transport by insulin.     

The proto-oncogene p120(Cbl) is a downstream substrate of the Hck protein-tyrosine kinase

Hematopoietic cell kinase (Hck) is a member of the Src-family of protein tyrosine kinases. We have found that upon enzymatic activation of Hck by the heavy metal mercuric chloride, there was a rapid increase in the levels of tyrosine phosphorylation of several proteins including the proto-oncogene p120(Cbl). Fibroblasts that are transformed with an activated allele of Hck exhibit constitutive Cbl phosphorylation. Upon Fcgamma receptor activation, a more physiologically relevant extracellular signal, Cbl is tyrosine phosphorylated and the Src-family selective inhibitor, PP1, can prevent this phosphorylation on Cbl. Hck phosphorylates Cbl in vitro and the interaction between Cbl and Hck is direct, requiring Hck's unique, SH3 and SH2 domains for optimal binding. Using a novel estrogen-regulated chimera of Hck we have shown a hormone-dependent association between Hck and Cbl in murine fibroblasts. This work suggests that Cbl serves as a key mediator of Hck induced signalling in hematopoietic cells.     

Observations on the calcium dependence and reversibility of cobalamin transport across the outer membrane of Escherichia coli

The calcium dependence of cobalamin (Cbl) binding to the BtuB protein of Escherichia coli and the reversibility of its function in the transport of Cbl across the outer membrane have been examined. The results show that the two calcium-binding sites in BtuB that were identified previously by others are responsible for the calcium dependence of high affinity Cbl binding. The affinity of the pure BtuB protein for Cbl was approximately 1000-fold higher in the presence of saturating levels of calcium than in its absence. The affinities of BtuB for both Cbl and calcium were decreased by insertion of alanine residues at position 51 of the mature protein and were increased by several mutations and deletions in the TonB box. Experiments on the uptake of Cbl into the periplasmic space showed that this process is reversible and that the exit of Cbl back into the medium does not require the protonmotive force. Our interpretation of these results is that the role of the TonB-ExbB-ExbD complex, potentiated by the protonmotive force, is to reduce the affinity of the Cbl-binding site, thus increasing the rate of Cbl release into the periplasmic space. The evidence also indicates that access of the Cbl-binding site of BtuB to the periplasmic space does not require removal of the hatch domain from the barrel.     

Met kinase-dependent loss of the E3 ligase Cbl in gastric cancer

Strict regulation of signaling by receptor tyrosine kinases (RTKs) is essential for normal biological processes, and disruption of this regulation can lead to tumor initiation and progression. Signal duration by the Met RTK is mediated in part by the E3 ligase Cbl. Cbl is recruited to Met upon kinase activation and promotes ubiquitination, trafficking, and degradation of the receptor. The Met RTK has been demonstrated to play a role in various types of cancer. Here, we show that Met-dependent loss of Cbl protein in MET-amplified gastric cancer cell lines represents another mechanism contributing to signal dysregulation. Loss of Cbl protein is dependent on Met kinase activity and is partially rescued with a proteasome inhibitor, lactacystin. Moreover, Cbl loss not only uncouples Met from Cbl-mediated negative regulation but also releases other Cbl targets, such as the EGF receptor, from Cbl-mediated signal attenuation. Thus, Met-dependent Cbl loss may also promote cross-talk through indirect enhancement of EGF receptor signaling.     

Cobalamin transport proteins and their cell-surface receptors

The primary function of cobalamin (Cbl; vitamin B12) is the formation of red blood cells and the maintenance of a healthy nervous system. Before cells can utilise dietary Cbl, the vitamin must undergo cellular transport using two distinct receptor-mediated events. First, dietary Cbl bound to gastric intrinsic factor (IF) is taken up from the apical pole of ileal epithelial cells via a 460 kDa receptor, cubilin, and is transported across the cell bound to another Cbl-binding protein, transcobalamin II (TC II). Second, plasma TC II-Cbl is taken up by cells that need Cbl via the TC II receptor (TC II-R), a 62 kDa protein that is expressed as a functional dimer in cellular plasma membranes. Human Cbl deficiency can develop as a result of acquired or inherited dysfunction in either of these two transmembrane transport events. This review focuses on the biochemical, cellular and molecular aspects of IF and TC II and their cell-surface receptors.     

Epidermal growth factor receptor internalization through clathrin-coated pits requires Cbl RING finger and proline-rich domains but not receptor polyubiquitylation

Cbl proteins have been implicated in the regulation of endocytic trafficking of epidermal growth factor receptor. However, the precise role of Cbl in epidermal growth factor receptor endocytosis is not defined. To directly visualize Cbl in cells and perform structure-function analysis of Cbl's role in epidermal growth factor receptor internalization, a yellow fluorescent protein-fusion of c-Cbl was constructed. Upon epidermal growth factor receptor activation, Cbl-yellow fluorescent protein moved with epidermal growth factor receptor to clathrin-coated pits and endosomes. Localization of Cbl-yellow fluorescent protein to these endocytic organelles was dependent on a proline-rich domain of c-Cbl that interacts with Grb2 as shown by fluorescence resonance energy transfer microscopy. In contrast, direct binding of Cbl to phosphotyrosine 1045 of the epidermal growth factor receptor was required for epidermal growth factor receptor polyubiquitination, but was not essential for Cbl-yellow fluorescent protein localization in epidermal growth factor receptor-containing compartments. These data suggest that the binding of Cbl to epidermal growth factor receptor through Grb2 is necessary and sufficient for Cbl function during clathrin-mediated endocytosis. Overexpression of c-Cbl mutants that are capable of Grb2 binding but defective in linker/RING finger domain function severely inhibited epidermal growth factor receptor internalization. The same dominant-negative mutants of Cbl did not block epidermal growth factor receptor recruitment into coated pits but retained receptors in coated pits, thus preventing receptor endocytosis and transport to endosomes. These data suggest that the linker and RING finger domain of Cbl may function during late steps of coated vesicle formation. We propose that the RING domain of Cbl facilitates endocytosis either by epidermal growth factor receptor monoubiquitylation or by ubiquitylation of proteins associated with the receptor.     

Role of protein kinase C in signal attenuation following T cell receptor engagement.

T lymphocyte activation through stimulation of the T cell receptor complex and co-stimulatory receptors is associated with acute tyrosine phosphorylation of intracellular proteins, which in turn mediate downstream signaling events that regulate interleukin-2 expression and cell proliferation. The extent of protein tyrosine phosphorylation is rapidly attenuated after only 1-2 min of stimulation as a means of tightly controlling the initial signaling response. Here we show that this attenuation of tyrosine phosphorylation of Shc, CrkL, and the proto-oncogene Cbl is mimicked by treatment of mouse T lymphocytes or cultured Jurkat cells with phorbol 12-myristate 13-acetate. This effect is blocked by the specific protein kinase C inhibitor GF109203X, but not by PD98059, an inhibitor of MEK1/2 kinase. Activation of protein kinase C by phorbol ester also causes rapid (t(1)/(2) = 2 min) dissociation of both CrkL and p85/phosphoinositide 3-kinase from Cbl concomitant with Cbl tyrosine dephosphorylation. More important, GF109203X treatment of Jurkat cells prior to T cell receptor stimulation by anti-CD3/CD4 antibodies results in an enhanced (2-fold) peak of Cbl phosphorylation compared with that observed in control cells. Furthermore, the rate of attenuation of both Cbl tyrosine phosphorylation and its association with CrkL following stimulation with anti-CD3/CD4 antibodies is much slower in Jurkat cells treated with GF109203X. Taken together, these data provide strong evidence that one or more isoforms of phorbol ester-responsive protein kinase C play a key role in a feedback mechanism that attenuates tyrosine phosphorylation of proteins and reverses formation of signaling complexes in response to T cell receptor activation.     

Selective modulation of the CAP/Cbl pathway in the adipose tissue of high fat diet treated rats

A high-fat diet (HFD) is associated with reduced glucose uptake in muscle, but not in adipose tissue. In the present study, we investigated whether a HFD can modulate glucose uptake in adipose tissue by increasing signal transduction through the CAP/Cbl pathway, independently of the PI3-K/Akt pathway. Our results suggest that, in HFD, the differential regulation of insulin-induced glucose uptake between skeletal muscle and adipose tissue may, in part, be a consequence of the CAP/Cbl/C3G pathway, since the expression of CAP and Cbl, and also the activation of this pathway were increased in adipose tissue but not in muscle.     

Composite organization of the cobalamin binding and cubilin recognition sites of intrinsic factor

Intrinsic factor (IF(50)) is a cobalamin (Cbl)-transporting protein of 50 kDa, which can be cleaved into two fragments: the 30 kDa N-terminal peptide IF(30) and the 20 kDa C-terminal glycopeptide IF(20). Experiments on binding of Cbl to IF(30), IF(20), and IF(50) revealed comparable association rate constants (k(+)(Cbl) = 4 x 10(6), 14 x 10(6), and 26 x 10(6) M(-1) s(-1), respectively), but the equilibrium dissociation constants were essentially different (K(Cbl) = 200 microM, 0.2 microM, and 相似文献   

Phosphotyrosine binding domain-dependent upregulation of the platelet-derived growth factor receptor alpha signaling cascade by transforming mutants of Cbl: implications for Cbl's function and oncogenicity.

Recent studies have demonstrated that Cbl, the 120-kDa protein product of the c-cbl proto-oncogene, serves as a substrate of a number of receptor-coupled tyrosine kinases and forms complexes with SH3 and SH2 domain-containing proteins, pointing to its role in signal transduction. Based on genetic evidence that the Caenorhabditis elegans Cbl homolog, SLI-1, functions as a negative regulator of the LET-23 receptor tyrosine kinase and our demonstration that Cbl's evolutionarily conserved N-terminal transforming region (Cbl-N; residues 1 to 357) harbors a phosphotyrosine binding (PTB) domain that binds to activated ZAP-70 tyrosine kinase, we examined the possibility that oncogenic Cbl mutants may activate mitogenic signaling by deregulating cellular tyrosine kinase machinery. Here, we show that expression of Cbl-N and two other transforming Cbl mutants (CblY368 delta and Cbl366-382 delta or Cb170Z), but not wild-type Cbl, in NIH 3T3 fibroblasts leads to enhancement of endogenous tyrosine kinase signaling. We identified platelet-derived growth factor receptor alpha (PDGFR alpha) as one target of mutant Cbl-induced deregulation. In mutant Cbl transfectants, PDGFR alpha was hyperphosphorylated and constitutively complexed with a number of SH2 domain-containing proteins. PDGFR alpha hyperphosphorylation and enhanced proliferation of mutant Cbl-transfected NIH 3T3 cells were drastically reduced upon serum starvation, and PDGF-AA substituted for the maintenance of these traits. PDGF-AA stimulation of serum-starved Cbl transfectants induced the in vivo association of transfected Cbl proteins with PDGFR alpha. In vitro, Cbl-N directly bound to PDGFR alpha derived from PDGF-AA-stimulated cells but not to that from unstimulated cells, and this binding was abrogated by a point mutation (G306E) corresponding to a loss-of-function mutation in SLI-1. The Cbl-N/G306E mutant protein, which failed to induce enhanced growth and transformation of NIH 3T3 cells, also failed to induce hyperphosphorylation of PDGFR alpha. Altogether, these findings identify a novel mechanism of Cbl's physiological function and oncogenesis, involving its PTB domain-dependent direct interaction with cellular tyrosine kinases.     

Abi-1 forms an epidermal growth factor-inducible complex with Cbl: role in receptor endocytosis

The Abl-interactor (Abi) proteins are involved in the regulation of actin polymerization and have recently been shown to modulate epidermal growth factor receptor (EGFR) endocytosis. Here we describe the identification of a novel complex between Abi-1 and the Cbl ubiquitin ligase that is induced by stimulation with EGF. Notably, an Abi-1 mutant lacking the SH3 domain (DeltaSH3) fails to interact with Cbl and inhibits EGFR internalization. We show that expression of the Abi-1DeltaSH3 mutant inhibits Cbl accumulation at the plasma membrane after EGF treatment. We have previously shown that the oncogenic Abl tyrosine kinase inhibits EGFR internalization. Here we report that the oncogenic Abl kinase disrupts the EGF-inducible Abi-1/Cbl complex, highlighting the importance of Abl kinases and downstream effectors in the regulation of EGFR internalization. Thus, our work reveals a new role for oncogenic Abl tyrosine kinases in the regulation of the Abi-1/Cbl protein complex and uncovers a role for the Abi-1/Cbl complex in the regulation of EGFR endocytosis.     

Intersectin 1 enhances Cbl ubiquitylation of epidermal growth factor receptor through regulation of Sprouty2-Cbl interaction

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.