Structural basis for recruitment of the adaptor protein APS to the activated insulin receptor

The adaptor protein APS is a substrate of the insulin receptor and couples receptor activation with phosphorylation of Cbl to facilitate glucose uptake. The interaction with the activated insulin receptor is mediated by the Src homology 2 (SH2) domain of APS. Here, we present the crystal structure of the APS SH2 domain in complex with the phosphorylated tyrosine kinase domain of the insulin receptor. The structure reveals a novel dimeric configuration of the APS SH2 domain, wherein the C-terminal half of each protomer is structurally divergent from conventional, monomeric SH2 domains. The APS SH2 dimer engages two kinase molecules, with pTyr-1158 of the kinase activation loop bound in the canonical phosphotyrosine binding pocket of the SH2 domain and a second phosphotyrosine, pTyr-1162, coordinated by two lysine residues in beta strand D. This structure provides a molecular visualization of one of the initial downstream recruitment events following insulin activation of its dimeric receptor.     

Structural basis for phosphotyrosine recognition by the Src homology-2 domains of the adapter proteins SH2-B and APS

SH2-B, APS, and Lnk constitute a family of adapter proteins that modulate signaling by protein tyrosine kinases. These adapters contain an N-terminal dimerization region, a pleckstrin homology domain, and a C-terminal Src homology-2 (SH2) domain. SH2-B is recruited via its SH2 domain to various protein tyrosine kinases, including Janus kinase-2 (Jak2) and the insulin receptor. Here, we present the crystal structure at 2.35 A resolution of the SH2 domain of SH2-B in complex with a phosphopeptide representing the SH2-B recruitment site in Jak2 (pTyr813). The structure reveals a canonical SH2 domain-phosphopeptide binding mode, but with specific recognition of a glutamate at the +1 position relative to phosphotyrosine, in addition to recognition of a hydrophobic residue at the +3 position. Biochemical studies of SH2-B and APS demonstrate that, although the SH2 domains of these two adapter proteins share 79% sequence identity, the SH2-B SH2 domain binds preferentially to Jak2, whereas the APS SH2 domain has higher affinity for the insulin receptor. This differential specificity is attributable to the difference in the oligomeric states of the two SH2 domains: monomeric for SH2-B and dimeric for APS.     

Structural Requirements for Function of the Crkl Adapter Protein in Fibroblasts and Hematopoietic Cells

Crkl is an adapter protein and phosphotyrosine-containing substrate implicated in transformation by the bcr-abl oncogene and in signaling by cytokines. When phosphorylated, Crkl binds through its Src homology 2 (SH2) domain to other tyrosine phosphoproteins such as paxillin and Cbl. Overexpression of Crkl in fibroblasts induces transformation. Here we examine the role of Crkl in hematopoietic cells and find that overexpression of Crkl confers a signal leading to increased adhesion to fibronectin. In both fibroblasts and hematopoietic cells, individual mutations or deletions of each SH2 and SH3 domain abrogated transformation and adhesion, respectively, indicating that interactions with other proteins such as Cbl and paxillin (SH2 domain) and Abl, Sos, and C3G (N-terminal SH3 domain) are essential for biological activity. In vivo and in vitro tryptic phosphopeptide mapping studies show that Crkl is phosphorylated on multiple tyrosine residues when overexpressed or when activated by Bcr-Abl. Mutation at tyrosine 207, a residue conserved in c-Crk, abrogates all in vivo tyrosine phosphorylation of Crkl. Despite this loss of phosphotyrosine, mutation at this site enhanced Crkl function as measured by complex formation with SH2 binding proteins, signal transduction to Jun Kinase, and fibroblast transformation. These observations implicate Crkl in cellular adhesion and demonstrate that Y207 functions as a negative regulatory site.     

The evolutionarily conserved N-terminal region of Cbl is sufficient to enhance down-regulation of the epidermal growth factor receptor

The mammalian proto-oncoprotein Cbl and its homologues in Caenorhabditis elegans and Drosophila are evolutionarily conserved negative regulators of the epidermal growth factor receptor (EGF-R). Overexpression of wild-type Cbl enhances down-regulation of activated EGF-R from the cell surface. We report that the Cbl tyrosine kinase-binding (TKB) domain is essential for this activity. Whereas wild-type Cbl enhanced ligand-dependent EGF-R ubiquitination, down-regulation from the cell surface, accumulation in intracellular vesicles, and degradation, a Cbl TKB domain-inactivated mutant (G306E) did not. Furthermore, the transforming truncation mutant Cbl-N (residues 1-357), comprising only the Cbl TKB domain, functioned as a dominant negative protein. It colocalized with EGF-R in intracellular vesicular structures, yet it suppressed down-regulation of EGF-R from the surface of cells expressing endogenous wild-type Cbl. Therefore, Cbl-mediated down-regulation of EGF-R requires the integrity of both the N-terminal TKB domain and additional C-terminal sequences. A Cbl truncation mutant comprising amino acids 1-440 functioned like wild-type Cbl in down-regulation assays. This mutant includes the evolutionarily conserved TKB and RING finger domains but lacks the less conserved C-terminal sequences. We conclude that the evolutionarily conserved N terminus of Cbl is sufficient to effect enhancement of EGF-R ubiquitination and down-regulation from the cell surface.     

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.     

Direct binding of Cbl to Tyr568 and Tyr936 of the stem cell factor receptor/c-Kit is required for ligand-induced ubiquitination, internalization and degradation

The ubiquitin E3 ligase Cbl has been shown to negatively regulate tyrosine kinase receptors, including the stem cell factor receptor/c-Kit. Impaired recruitment of Cbl to c-Kit results in a deregulated positive signalling that eventually can contribute to carcinogenesis. Here, we present results showing that Cbl is activated by the SFKs (Src family kinases) and recruited to c-Kit in order to trigger receptor ubiquitination. We demonstrate that phosphorylated Tyr568 and Tyr936 in c-Kit are involved in direct binding and activation of Cbl and that binding of the TKB domain (tyrosine kinase binding domain) of Cbl to c-Kit is specified by the presence of an isoleucine or leucine residue in position +3 to the phosphorylated tyrosine residue on c-Kit. Apart from the direct association between Cbl and c-Kit, we show that phosphorylation of Cbl by SFK members is required for activation of Cbl to occur. Moreover, we demonstrate that Cbl mediates monoubiquitination of c-Kit and that the receptor is subsequently targeted for lysosomal degradation. Taken together, our findings reveal novel insights into the mechanisms by which Cbl negatively regulates c-Kit-mediated signalling.     

A phenylalanine zipper mediates APS dimerization

The APS, SH2-B and LNK proteins are adapters that activate and modulate receptor tyrosine kinase and JAK/STAT signaling. We now show that a conserved N-terminal domain mediates APS homodimerization. We determined the crystal structure of the dimerization domain at a resolution of 1.7 A using bromide ion MAD phasing. Each molecule contributes two helices to a compact four-helix bundle having a bisecting-U topology. Its most conspicuous feature is a stack of interdigitated phenylalanine side chains at the domain core. These residues create a new motif we refer to as a 'phenylalanine zipper,' which is critical to dimerization. A newly developed bridging yeast tri-hybrid assay showed that APS dimerizes JAK2, insulin receptor and IGF1 receptor kinases using its SH2 and dimerization domains. Dimerization via the phenylalanine zipper domain provides a mechanism for activating and modulating tyrosine kinase activity even in the absence of extracellular ligands.     

Phosphorylation control of the ubiquitin ligase Cbl is conserved in choanoflagellates

Cbl proteins are E3 ubiquitin ligases specialized for the regulation of tyrosine kinases by ubiquitylation. Human Cbl proteins are activated by tyrosine phosphorylation, thus setting up a feedback loop whereby the activation of tyrosine kinases triggers their own degradation. Cbl proteins are targeted to their substrates by a phosphotyrosine‐binding SH2 domain. Choanoflagellates, unicellular eukaryotes that are closely related to metazoans, also contain Cbl. The tyrosine kinase complement of choanoflagellates is distinct from that of metazoans, and it is unclear if choanoflagellate Cbl is regulated similarly to metazoan Cbl. Here, we performed structure‐function studies on Cbl from the choanoflagellate species Salpingoeca rosetta and found that it undergoes phosphorylation‐dependent activation. We show that S. rosetta Cbl can be phosphorylated by S. rosetta Src kinase, and that it can ubiquitylate S. rosetta Src. We also compared the substrate selectivity of human and S. rosetta Cbl by measuring ubiquitylation of Src constructs in which Cbl‐recruitment sites are placed in different contexts with respect to the kinase domain. Our results indicate that for both human and S. rosetta Cbl, ubiquitylation depends on proximity and accessibility, rather than being targeted toward specific lysine residues. Our results point to an ancient interplay between phosphotyrosine and ubiquitin signaling in the metazoan lineage.     

Structural basis for dimerization of the Grb10 Src homology 2 domain. Implications for ligand specificity

Grb7, Grb10, and Grb14 are members of a distinct family of adapter proteins that interact with various receptor tyrosine kinases upon receptor activation. Proteins in this family contain several modular signaling domains including a pleckstrin homology (PH) domain, a BPS (between PH and SH2) domain, and a C-terminal Src homology 2 (SH2) domain. Although SH2 domains are typically monomeric, we show that the Grb10 SH2 domain and also full-length Grb10 gamma are dimeric in solution under physiologic conditions. The crystal structure of the Grb10 SH2 domain at 1.65-A resolution reveals a non-covalent dimer whose interface comprises residues within and flanking the C-terminal alpha helix, which are conserved in the Grb7/Grb10/Grb14 family but not in other SH2 domains. Val-522 in the BG loop (BG3) and Asp-500 in the EF loop (EF1) are positioned to interfere with the binding of the P+3 residue of a phosphopeptide ligand. These structural features of the Grb10 SH2 domain will favor binding of dimeric, turn-containing phosphotyrosine sequences, such as the phosphorylated activation loops in the two beta subunits of the insulin and insulin-like growth factor-1 receptors. Moreover, the structure suggests the mechanism by which the Grb7 SH2 domain binds selectively to pTyr-1139 (pYVNQ) in Her2, which along with Grb7 is co-amplified in human breast cancers.     

Mutational investigation of the specificity determining region of the Src SH2 domain

SH2 domains are protein modules which bind tyrosine phosphorylated sequences in many signaling pathways. These domains contain two regions with specialized functions: residues in one region form a deep pocket into which the phosphotyrosine of the target inserts, while the other region contains the so-called "specificity determining residues" which interact with the three residues C-terminal to the phosphotyrosine in the target. Here, titration calorimetry and site-directed mutagenesis have been used to probe the importance of eight specificity determining residues of the SH2 domain of the Src kinase involved in contacts with its tyrosine phosphorylated consensus peptide target (sequence pYEEI where pY indicates a phosphotyrosine). Mutating six of these eight residues to Ala individually, resulted in a threefold or less loss in binding affinity; hence the majority of the residues in the specificity determining region are by themselves of minimal importance for binding. Two residues were found to have significant effects on binding: Tyr betaD5 and Lys betaD3. Tyr betaD5 was the most crucial residue as evidenced by the 30-fold loss in affinity when Tyr betaD5 is mutated to Ile. However, while this mutation eliminated the specificity of the Src SH2 domain for the pYEEI peptide sequence, it was not sufficient to switch the specificity of the Src SH2 domain to that of a related SH2 domain which has an Ile at the betaD5 position. Mutation of Lys betaD3 to an Ala residue resulted in a modest reduction in binding affinity (sevenfold). It is interesting that this mutation resulted in a change of specificity affecting the selection of the +1 position residue C-terminal to the phosphotyrosine. Except for the Lys betaD3-+1 Glu interaction which is significantly coupled, only weak energetic coupling was observed across the binding interface, as assessed using double mutant cycles. The results of this study suggest that interactions involving the specificity determining region of SH2 domains may be insufficient by themselves to target single SH2 domains to particular phosphorylated sites.     

The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells

The protooncogene product Cbl has emerged as a negative regulator of tyrosine kinases. We have shown previously that Cbl binds to ZAP-70 through its N-terminal tyrosine kinase binding (TKB) domain. In this study, we demonstrate that overexpression of Cbl in Jurkat T cells decreases the TCR-induced phosphorylation of ZAP-70 and other cellular phosphoproteins. Coexpression of Cbl with ZAP-70 in COS cells reproduced the Cbl-induced reduction in the level of phosphorylated ZAP-70. The effect of Cbl was eliminated by the TKB-inactivating G306E mutation in Cbl as well as by a phenylalanine mutation of Tyr292 within the TKB domain binding site on ZAP-70. Notably, the oncogenic Cbl-70Z/3 mutant associated with ZAP-70, but did not reduce the levels of phosphorylated ZAP-70. Overexpression of Cbl, but not Cbl-G306E, in Jurkat T cells led to a decrease in the TCR-induced NF-AT luciferase reporter activity. Overexpression of the TKB domain itself, but not its G306E mutant, functioned in a dominant-negative manner and led to an increase in NF-AT reporter activity. Cbl-70Z/3-overexpressing cells exhibited an increase in both basal and TCR-induced NF-AT luciferase reporter activity, and this trend was reversed by the G306E mutation. Finally, by reconstituting a ZAP-70-deficient Jurkat T cell line, p116, we demonstrate that wild-type ZAP-70 is susceptible to the negative regulatory effect of Cbl, whereas the ZAP-70-Y292F mutant is resistant. Together, our results establish that the linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells.     

FcgammaRII tyrosine phosphorylation differs between FcgammaRII cross-linking and platelet-activating anti-platelet monoclonal antibodies.

Using glutathione S-transferase Syk fusion proteins, we evaluated the mode of platelet FcgammaRII tyrosine phosphorylation induced by FcgammaRII cross-linking or anti-CD9 monoclonal antibodies (mAb). The N-terminal SH2 domain of Syk (Syk-N-SH2), the C-terminal SH2 domain of Syk (Syk-C-SH2), and the domain having both the N- and C-terminal SH2 of Syk (Syk-NC-SH2) all bound to tyrosine-phosphorylated FcgammaRII with FcgammaRII cross-linking. In the case of anti-CD9 mAb-induced platelet activation, only Syk-C-SH2 and Syk-NC-SH2 bound to tyrosine-phosphorylated FcgammaRII. Since the SH2 domain is specific for a particular structure containing phosphotyrosine, these findings suggest that only one tyrosine residue in the immunoreceptor tyrosine-based activation motif (ITAM) is phosphorylated with anti-CD9 mAb, and that both are phosphorylated with FcgammaRII cross-linking. Synthetic peptides corresponding to the ITAM of human platelet FcgammaRII with the N-terminal tyrosine residue phosphorylated (N-P) or the C-terminal tyrosine residue phosphorylated (C-P), were used. N-P more potently dissociated Syk-C-SH2 from tyrosine-phosphorylated FcgammaRII than C-P, suggesting that the N-terminal tyrosine residue is phosphorylated upon anti-CD9 mAb-induced activation. Furthermore, these findings imply that Syk-N-SH2 binds to the phosphorylated C-terminal tyrosine residue of ITAM, and Syk-C-SH2 to the N-terminal tyrosine. Taken together, our findings suggest that FcgammaRII-dependent platelet activation without FcgammaRII dimerization, such as with anti-CD9 mAb, is distinct from that induced by FcgammaRII cross-linking.     

Characterization of Drosophila insulin receptor substrate

Insulin receptor substrate (IRS) proteins are phosphorylated by multiple tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream signaling pathways. The Drosophila insulin receptor (dIR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that dIR might not require an IRS protein to accomplish its signaling functions. However, we obtained a cDNA encoding Drosophila IRS (dIRS), and we demonstrated expression of dIRS in a Drosophila cell line. Like mammalian IRS proteins, the N-terminal portion of dIRS contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with dIRS in COS-7 cells, a chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic domain of dIR) mediated insulin-stimulated tyrosine phosphorylation of dIRS. Mutating the juxtamembrane NPXY motif markedly reduced the ability of the receptor to phosphorylate dIRS. In contrast, the NPXY motifs in the C-terminal extension of dIR were required for stable association with dIRS. Coimmunoprecipitation experiments demonstrated insulin-dependent binding of dIRS to phosphatidylinositol 3-kinase and SHP2. However, we did not detect interactions with Grb2, SHC, or phospholipase C-gamma. Taken together with published genetic studies, these biochemical data support the hypothesis that dIRS functions directly downstream from the insulin receptor in Drosophila.     

The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase

The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.     

Mapping of multiple phosphorylation sites within the structural and catalytic domains of the Fujinami avian sarcoma virus transforming protein.

The phosphorylation sites of the P140gag-fps gene product of Fujinami avian sarcoma virus have been identified and localized to different regions of this transforming protein. FSV P140gag-fps isolated from transformed cells is phosphorylated on at least three distinct tyrosine residues and one serine residue, in addition to minor phosphorylation sites shared with Pr76gag. Partial proteolysis with virion protease p15 or with Staphylococcus aureus V8 protease has been used to generate defined peptide fragments of P140gag-fps and thus to map its phosphorylation sites. The amino-terminal gag-encoded region of P140gag-fps contains a phosphotyrosine residue in addition to normal gag phosphorylation sites. The two major phosphotyrosine residues and the major phosphorserine residue are located in the carboxy-terminal portion of the fps-encoded region of P140gag-fps. P140gag-fps radiolabeled in vitro in an immune complex kinase reaction is phosphorylated at only one of the two C-terminal tyrosine residues phosphorylated in vivo and weakly phosphorylated at the gag-encoded tyrosine and at a tyrosine site not detectably phosphorylated in vivo. Thus, the in vitro tyrosine phosphorylation of P140gag-fps is distinct from that seen in the transformed cell. A comparative tryptic phosphopeptide analysis of the gag-fps proteins of three Fujinami avian sarcoma virus variants showed that the phosphotyrosine-containing peptides are invariant, and this high degree of sequence conservation suggests that these sites are functionally important or lie within important regions. The P105gag-fps transforming protein of PRCII avian sarcoma virus lacks one of the C-terminal phosphotyrosine sites found in Fujinami avian sarcoma virus P140gag-fps. Partial trypsin cleavage of FSV P140gag-fps immunoprecipitated with anti-gag serum releases C-terminal fragments of 45K and 29K from the immune complex that retain an associated tyrosine-specific protein kinase activity. This observation, and the localization of the major P140gag-fps phosphorylation sites to the C-terminal fps region, indicate that the kinase domain of P140gag-fps is located at its C terminus. The phosphorylation of P140gag-fps itself is complex, suggesting that it may itself interact with several protein kinases in the transformed cell.     

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.     

c-Cbl associates directly with the C-terminal tail of the receptor for the macrophage colony-stimulating factor, c-Fms, and down-modulates this receptor but not the viral oncogene v-Fms

The receptor for the macrophage colony-stimulating factor (CSF-1, also termed M-CSF), the tyrosine kinase c-Fms, was originally determined to be the oncogene product of the McDonough strain of feline sarcoma virus, v-Fms. The structural difference between c-Fms and v-Fms amounts to only five point mutations in the extracellular domain, two mutations in the cytoplasmic domain, and the replacement of 50 amino acids by 14 unrelated amino acids at the C-terminal tail. Here, we have identified c-Cbl as the direct binding partner for c-Fms. c-Cbl binds to phosphotyrosine residue 977 at the C-terminal end of feline c-Fms, which is absent in v-Fms. The replacement of the C-terminal end of v-Fms by the corresponding part of c-Fms (vc-Fms) restored the binding potential. As a result, vc-Fms reduced the transforming potency of v-Fms. The overexpression of Cbl did not influence the v-Fms-transformed phenotype, although c-Cbl forms a complex with v-Fms indirectly. In contrast, the expression of Cbl drastically reduced the vc-Fms-transformed phenotype and the activation of Erk and enhanced Fms ubiquitination via phosphotyrosine residue 977. Furthermore, the replacement of tyrosine 977 into phenylalanine in feline c-Fms and vc-Fms reduced the Cbl-dependent ubiquitination. These data suggest that an indirect association of c-Cbl via multimeric complex induced a different signaling pathway from the pathway induced by c-Cbl direct interaction.     

The roles of Cbl-b and c-Cbl in insulin-stimulated glucose transport

Previous studies suggest that the stimulation of glucose transport by insulin involves the tyrosine phosphorylation of c-Cbl and the translocation of the c-Cbl/CAP complex to lipid raft subdomains of the plasma membrane. We now demonstrate that Cbl-b also undergoes tyrosine phosphorylation and membrane translocation in response to insulin in 3T3-L1 adipocytes. Ectopic expression of APS facilitated insulin-stimulated phosphorylation of tyrosines 665 and 709 in Cbl-b. The phosphorylation of APS produced by insulin drove the translocation of both c-Cbl and Cbl-b to the plasma membrane. Like c-Cbl, Cbl-b associates constitutively with CAP and interacts with Crk upon insulin stimulation. Cbl proteins formed homo- and heterodimers in vivo, which required the participation of a conserved leucine zipper domain. A Cbl mutant incapable of dimerization failed to interact with APS and to undergo tyrosine phosphorylation in response to insulin, indicating an essential role of Cbl dimerization in these processes. Thus, both c-Cbl and Cbl-b can initiate a phosphatidylinositol 3-kinase/protein kinase B-independent signaling pathway critical to insulin-stimulated GLUT4 translocation.     

Binding of SH2-B family members within a potential negative regulatory region maintains JAK2 in an active state

The tyrosine kinase Janus kinase 2 (JAK2) transduces signaling for the majority of known cytokine receptor family members and is constitutively activated in some cancers. Here we examine the mechanisms by which the adapter proteins SH2-Bbeta and APS regulate the activity of JAK2. We show that like SH2-Bbeta, APS binds JAK2 at multiple sites and that binding to phosphotyrosine 813 is essential for APS to increase active JAK2 and to be phosphorylated by JAK2. Binding of APS to a phosphotyrosine 813-independent site inhibits JAK2. Both APS and SH2-Bbeta increase JAK2 activity independent of their N-terminal dimerization domains. SH2-Bbeta-induced increases in JAK2 dimerization require only the SH2 domain and only one SH2-Bbeta to be bound to a JAK2 dimer. JAK2 mutations and truncations revealed that amino acids 809 to 811 in JAK2 are a critical component of a larger regulatory region within JAK2, most likely including amino acids within the JAK homology 1 (JH1) and JH2 domains and possibly the FERM domain. Together, our data suggest that SH2-Bbeta and APS do not activate JAK2 as a consequence of their own dimerization, recruitment of an activator of JAK2, or direct competition with a JAK2 inhibitor for binding to JAK2. Rather, they most likely induce or stabilize an active conformation of JAK2.