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.     

Differential regulation of the B cell receptor-mediated signaling by the E3 ubiquitin ligase Cbl

The E3 ubiquitin ligase Cbl has been implicated in intracellular signaling pathways induced by the engagement of the B cell antigen receptor (BCR) as a negative regulator. Here we showed that Cbl deficiency results in a reduction of B cell proliferation. Cbl-/- B cells show impaired tyrosine phosphorylation, reduced Erk activation, and attenuated calcium mobilization in response to BCR engagement. The phosphorylation of Syk and Btk is also down-modulated. Interestingly, Cbl-/- B cells display enhanced BCR-induced phosphorylation of CD19 and its association with phosphatidylinositol 3-kinase. Importantly, Lyn kinase activity is up-regulated in Cbl-/- B cells, which correlates inversely with the Cbl-mediated ubiquitination of Lyn. Because Lyn has both negative and positive roles in B cells, our results suggested that Cbl differentially modulates the BCR-mediated signaling pathways through targeting Lyn ubiquitination, which affects B cell development and activation.     

Signal transduction by human-restricted Fc gamma RIIa involves three distinct cytoplasmic kinase families leading to phagocytosis.

Recent experiments indicate an important role for Src family and Syk protein tyrosine kinases and phosphatidylinositol 3-kinase in the signal transduction process initiated by mouse receptors for IgG and leading to phagocytosis. Considerably less is known regarding signal transduction by the human-restricted IgG receptor, FcgammaRIIa. Furthermore, the relationship among the Src family, Syk, and phosphatidylinositol 3-kinase in phagocytosis is not understood. Here, we show that FcgammaRIIa is phosphorylated by an Src family member, which results in recruitment and concomitant activation of the distal enzymes Syk and phosphatidylinositol 3-kinase. Using a FcgammaRI-p85 receptor chimera cotransfected with kinase-inactive mutants of Syk or application of a pharmacological inhibitor of Syk, we show that Syk acts in parallel with phosphatidylinositol 3-kinase. Our results indicate that FcgammaRIIa-initiated monocyte or neutrophil phagocytosis proceeds from the clustered IgG receptor to Src to phosphatidylinositol 3-kinase and Syk.     

Negative regulation of phagocytosis in macrophages by the CD47-SHPS-1 system

Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that is expressed predominantly in macrophages. Its extracellular region interacts with the transmembrane ligand CD47 expressed on the surface of adjacent cells, and its cytoplasmic region binds the protein tyrosine phosphatases SHP-1 and SHP-2. Phagocytosis of IgG- or complement-opsonized RBCs by peritoneal macrophages derived from mice that express a mutant SHPS-1 protein that lacks most of the cytoplasmic region was markedly enhanced compared with that apparent with wild-type macrophages. This effect was not observed either with CD47-deficient RBCs as the phagocytic target or in the presence of blocking Abs to SHPS-1. Depletion of SHPS-1 from wild-type macrophages by RNA interference also promoted FcgammaR-mediated phagocytosis of wild-type RBCs. Ligation of SHPS-1 on macrophages by CD47 on RBCs promoted tyrosine phosphorylation of SHPS-1 and its association with SHP-1, whereas tyrosine phosphorylation of SHPS-1 was markedly reduced in response to cross-linking of FcgammaRs. Treatment with inhibitors of PI3K or of Syk, but not with those of MEK or Src family kinases, abolished the enhancement of FcgammaR-mediated phagocytosis apparent in macrophages from SHPS-1 mutant mice. In contrast, FcgammaR-mediated tyrosine phosphorylation of Syk, Cbl, or the gamma subunit of FcR was similar in macrophages from wild-type and SHPS-1 mutant mice. These results suggest that ligation of SHPS-1 on macrophages by CD47 promotes the tyrosine phosphorylation of SHPS-1 and thereby prevents the FcgammaR-mediated disruption of the SHPS-1-SHP-1 complex, resulting in inhibition of phagocytosis. The inhibition of phagocytosis by the SHPS-1-SHP-1 complex may be mediated at the level of Syk or PI3K signaling.     

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.     

HIV-1 down-modulates gamma signaling chain of Fc gamma R in human macrophages: a possible mechanism for inhibition of phagocytosis

HIV-1 infection impairs a number of macrophage effector functions, thereby contributing to development of opportunistic infections and the pathogenesis of AIDS. FcgammaR-mediated phagocytosis by human monocyte-derived macrophages (MDM) is inhibited by HIV-1 infection in vitro, and the underlying mechanism was investigated in this study. Inhibition of phagocytosis directly correlated with the multiplicity of HIV-1 infection. Expression of surface FcgammaRs was unaffected by HIV-1 infection, suggesting that inhibition of phagocytosis occurred during or after receptor binding. HIV-1 infection of MDM markedly inhibited tyrosine phosphorylation of the cellular proteins, which occurs following engagement of FcgammaRs, suggesting a defect downstream of initial receptor activation. FcgammaR-mediated phagocytosis in HIV-infected MDM was associated with inhibition of phosphorylation of tyrosine kinases from two different families, Hck and Syk, defective formation of Syk complexes with other tyrosine-phosphorylated proteins, and inhibition of paxillin activation. Down-modulation of protein expression but not mRNA of the gamma signaling subunit of FcgammaR (a docking site for Syk) was observed in HIV-infected MDM. Infection of MDM with a construct of HIV-1 in which nef was replaced with the gene for the gamma signaling subunit augmented FcgammaR-mediated phagocytosis, suggesting that down-modulation of gamma-chain protein expression in HIV-infected MDM caused the defective FcgammaR-mediated signaling and impairment of phagocytosis. This study is the first to demonstrate a specific alteration in phagocytosis signal transduction pathway, which provides a mechanism for the observed impaired FcgammaR-mediated phagocytosis in HIV-infected macrophages and contributes to the understanding of how HIV-1 impairs cell-mediated immunity leading to HIV-1 disease progression.     

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.     

Fc gamma R-mediated phagocytosis stimulates localized pinocytosis in human neutrophils

Engulfment of IgG-coated particles by neutrophils and macrophages is an essential component of the innate immune response. This process, known as phagocytosis, is triggered by clustering of FcgammaR at sites where leukocytes make contact with the opsonized particles. We found that phagocytosis is accompanied by a burst of fluid phase pinocytosis, which is largely restricted to the immediate vicinity of the phagosomal cup. FcgammaR-induced pinocytosis preceded and appeared to be independent of phagosomal sealing. Accordingly, fluid phase uptake was accentuated by actin depolymerization, which precludes phagocytosis. Stimulation of pinocytosis required phosphatidylinositol 3-kinase activity and was eliminated when changes in the cytosolic free Ca(2+) concentration were prevented. Because stimulation of FcgammaR also induces secretion, which is similarly calcium and phosphatidylinositol 3-kinase dependent, we studied the possible relationship between these events. Neutrophil fragments devoid of secretory granules (cytoplasts) were prepared by sedimentation through Ficoll gradients. Cytoplasts could perform FcgammaR-mediated phagocytosis, which was not accompanied by activation of pinocytosis. This observation suggests that granule exocytosis is required for stimulation of pinocytosis. Analysis of the cytosolic Ca(2+) dependence of secretion and pinocytosis suggests that primary (lysosomal) granule exocytosis is the main determinant of pinocytosis during FcgammaR stimulation. Importantly, primary granules are secreted in a polarized fashion near forming phagosomes. Focal pinocytosis during particle engulfment may contribute to Ag processing and presentation and/or to retrieval of components of the secretory machinery. Alternatively, it may represent an early event in the remodeling of the phagosomal membrane, leading to phagosomal maturation.     

The adapter protein LAT enhances fcgamma receptor-mediated signal transduction in myeloid cells

FcgammaR clustering in monocytes initiates a cascade of signaling events that culminate in biological responses such as phagocytosis, production of inflammatory cytokines, and generation of reactive oxygen species. We have identified and determined the function of the adapter protein linker of activation of T cell (LAT) in FcgammaR-mediated signaling and function. Clustering of FcgammaRs on the human monocytic cell line, THP-1, induces phosphorylation of a major 36-kDa protein which immunoreacts with anti-LAT antisera. Our data indicate that although both the 36-kDa and 38-kDa isoforms of LAT are expressed in THP-1 and U937 human monocytic cells, FcgammaR clustering induces phosphorylation of the 36-kDa isoform only. Co-immunoprecipitation experiments revealed a constitutive association of p36 LAT with both FcgammaRI and FcgammaRIIa immunoprecipitates, and an activation-induced association of LAT with PLCgamma1, Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Transient transfection experiments in COS-7 cells indicated that overexpression of a wild type but not a dominant-negative LAT, that is incapable of binding to p85, enhances phagocytosis by FcgammaRI. Furthermore, bone marrow-derived macrophages from LAT-deficient mice displayed reduced phagocytic efficiency in comparison to the macrophages from wild-type mice. Thus, we conclude that p36 LAT serves to enhance FcgammaR-induced signal transduction in myeloid cells.     

Molecular basis for a direct interaction between the Syk protein-tyrosine kinase and phosphoinositide 3-kinase

After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcgammaRIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase.     

Critical role for scaffolding adapter Gab2 in Fc gamma R-mediated phagocytosis

Grb2-associated binder 2 (Gab2), a member of the Dos/Gab subfamily scaffolding molecules, plays important roles in regulating the growth, differentiation, and function of many hematopoietic cell types. In this paper, we reveal a novel function of Gab2 in Fcgamma receptor (FcgammaR)-initiated phagocytosis in macrophages. Upon FcgammaR activation, Gab2 becomes tyrosyl phosphorylated and associated with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and the protein-tyrosine phosphatidylinositol Shp-2. FcgammaR-mediated phagocytosis is severely impaired in bone marrow-derived macrophages from Gab2-/- mice. The defect in phagocytosis correlates with decreased FcgammaR-evoked activation of Akt, a downstream target of PI3K. Using confocal fluorescence microscopy, we find that Gab2 is recruited to the nascent phagosome, where de novo PI3K lipid production occurs. Gab2 recruitment requires the pleckstrin homology domain of Gab2 and is sensitive to treatment with the PI3K inhibitor wortmannin. The Grb2 binding site on Gab2 also plays an auxiliary role in recruitment to the phagosome. Because PI3K activity is required for FcgammaR-mediated phagocytosis, our results indicate that Gab2 acts as a key component of FcgammaR-mediated phagocytosis, most likely by amplifying PI3K signaling in the nascent phagosome.     

Cbl-mediated negative regulation of platelet-derived growth factor receptor-dependent cell proliferation. A critical role for Cbl tyrosine kinase-binding domain.

The Cbl proto-oncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases. Our previous observations that Cbl overexpression in NIH3T3 cells enhanced the ubiquitination and degradation of the platelet-derived growth factor receptor-alpha (PDGFRalpha) and that the expression of oncogenic Cbl mutants up-regulated the PDGFRalpha signaling machinery strongly suggested that Cbl negatively regulates PDGFRalpha signaling. Here, we show that, similar to PDGFRalpha, selective stimulation of PDGFRbeta induces Cbl phosphorylation, and its physical association with the receptor. Overexpression of wild type Cbl in NIH3T3 cells led to an enhancement of the ligand-dependent ubiquitination and subsequent degradation of the PDGFRbeta, as observed with PDGFRalpha. We show that Cbl-dependent negative regulation of PDGFRalpha and beta results in a reduction of PDGF-induced cell proliferation and protection against apoptosis. A point mutation (G306E) that inactivates the tyrosine kinase binding domain in the N-terminal transforming region of Cbl compromised the PDGF-inducible tyrosine phosphorylation of Cbl although this mutant could still associate with the PDGFR. More importantly, the G306E mutation abrogated the ability of Cbl to enhance the ligand-induced ubiquitination and degradation of the PDGFR and to inhibit the PDGF-dependent cell proliferation and protection from apoptosis. These results demonstrate that Cbl can negatively regulate PDGFR-dependent biological responses and that this function requires the conserved tyrosine kinase binding domain of Cbl.     

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.     

Requirements for pYXXM motifs in Cbl for binding to the p85 subunit of phosphatidylinositol 3-kinase and Crk, and activation of atypical protein kinase C and glucose transport during insulin action in 3T3/L1 adipocytes

Cbl is phosphorylated by the insulin receptor and reportedly functions within the flotillin/CAP/Cbl/Crk/C3G/TC10 complex during insulin-stimulated glucose transport in 3T3/L1 adipocytes. Cbl, via pYXXM motifs at tyrosine-371 and tyrosine-731, also activates phosphatidylinositol (PI) 3-kinase, which is required to activate atypical protein kinase C (aPKC) and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes [Miura et al. (2003) Biochemistry 42, 14335-14341]. Presently, we have examined the importance of Cbl in activating PI 3-kinase and aPKC during insulin action in 3T3/L1 adipocytes by expressing Y371F and Y731F Cbl mutants, which nullify pYXXM binding of Cbl to SH2 domains of downstream effectors. Interestingly, these mutants inhibited insulin-induced increases in (a) binding of Cbl to both Crk and the p85 subunit of PI 3-kinase, (b) activation of Cbl-dependent PI 3-kinase, (c) activation and translocation of aPKC to the plasma membrane, (d) translocation of Glut4 to the plasma membrane, (e) and glucose transport. Importantly, coexpression of wild-type Cbl reversed the inhibitory effects of Cbl mutants. In contrast to Cbl-dependent PI 3-kinase, Cbl mutants did not significantly inhibit the activation of PI 3-kinase by IRS-1, which is also required during insulin action. Our findings suggest that (a) Cbl uses pYXXM motifs to simultaneously activate PI 3-kinase and Crk/C3G/TC10 pathways and (b) Cbl, along with IRS-1, functions upstream of PI 3-kinase and aPKCs during insulin-stimulated glucose transport in 3T3/L1 adipocytes.     

A phosphatidylinositol-3-kinase-dependent signal transition regulates ARF1 and ARF6 during Fcgamma receptor-mediated phagocytosis

Fcgamma receptor (FcgammaR)-mediated phagocytosis of IgG-coated particles is regulated by 3'-phosphoinositides (3'PIs) and several classes of small GTPases, including ARF6 from the ADP Ribosylation Factor subfamily. The insensitivity of phagocytosis to brefeldin A (BFA), an inhibitor of certain ARF guanine nucleotide exchange factors (GEFs), previously indicated that ARF1 did not participate in phagocytosis. In this study, we show that ARF1 was activated during FcgammaR-mediated phagocytosis and that blocking normal ARF1 cycling inhibited phagosome closure. We examined the distributions and activation patterns of ARF6 and ARF1 during FcgammaR-mediated phagocytosis using fluorescence resonance energy transfer (FRET) stoichiometric microscopy of macrophages expressing CFP- or YFP-chimeras of ARF1, ARF6, and a GTP-ARF-binding protein domain. Both GTPases were activated by BFA-insensitive factors at sites of phagocytosis. ARF6 activation was restricted to the leading edge of the phagocytic cup, while ARF1 activation was delayed and delocalized over the phagosome. Phagocytic cups formed after inhibition of PI 3-kinase (PI-3K) contained persistently activated ARF6 and minimally activated ARF1. This indicates that a PI-3K-dependent signal transition defines the sequence of ARF GTPase activation during phagocytosis and that ARF6 and ARF1 coordinate different functions at the forming phagosome.     

Negative regulation of class IA phosphoinositide 3-kinase by protein kinase Cdelta Limits Fcgamma receptor-mediated phagocytosis in macrophages

Stimulation of macrophages by various ligands results in the activation of both phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC). Here, we showed that PKCdelta selectively inhibits class IA PI3K. Prior exposure of macrophages to a PKC activator, phorbol 12-myristate 13-acetate (PMA) inhibited the PI3K activation induced by the Fcgamma receptor (FcgammaR) ligation but not that induced by C5a. Prolonged PKC inhibition by GF109203X increased the basal PI3K activity of quiescent macrophages. The effect of the PKC inhibitor can be observed in macrophages from mice lacking class IB PI3K (p110gamma). Thus PKC was suggested to selectively attenuate the class IA activity. Chronic PKC activation by PMA induced PKCdelta degradation and Akt activation. Enhancement of the basal Akt actvity was also observed in cells stably deficient in PKCdelta prepared by shRNA technique. FcgammaR-mediated phagocytosis was dramatically increased in these cells. Thus it is suggested that inactivation of class IA PI3K by PKCdelta is functioning in regulation of FcgammaR-mediated phagocytosis.     

Cbl PYXXM motifs activate the P85 subunit of phosphatidylinositol 3-kinase, Crk, atypical protein kinase C, and glucose transport during thiazolidinedione action in 3T3/L1 and human adipocytes

The thiazolidinedione (TZD), rosiglitazone, has previously been found to tyrosine-phosphorylate Cbl and activate Cbl-dependent phosphatidylinositol (PI) 3-kinase and atypical protein kinase Cs (aPKCs) while stimulating glucose transport in 3T3/L1 adipocytes. Presently, the role of Cbl in rosiglitazone action was further assessed in both 3T3/L1 and human adipocytes by expressing Y371F and/or Y731F mutant forms of Cbl that nullified the functionality of canonical pYXXM motifs in Cbl. These mutants diminished the interaction of Cbl with the p85 subunit of PI 3-kinase and inhibited subsequent increases in Cbl-dependent PI 3-kinase activity, aPKC activity, and glucose transport. These mutants also inhibited the interaction of Cbl with Crk, which has been implicated in the activation of other PI 3-kinase-independent signaling factors that have been found to be required during activation of glucose transport by insulin and other agonists. We conclude that pYXXM motifs in Cbl serve to activate PI 3-kinase-dependent and possibly PI 3-kinase-independent pathways that are required for TZD-dependent glucose transport in adipocytes.