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.     

Binding of Cbl to a phospholipase Cgamma1-docking site on platelet-derived growth factor receptor beta provides a dual mechanism of negative regulation

Ubiquitin conjugation to receptor tyrosine kinases is a critical biochemical step in attenuating their signaling through lysosomal degradation. Our previous studies have established Cbl as an E3 ubiquitin ligase for ubiquitinylation and degradation of platelet-derived growth factor receptor (PDGFR) alpha and PDGFRbeta. However, the role of endogenous Cbl in PDGFR regulation and the molecular mechanisms of this regulation remain unclear. Here, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and degradation of PDGFRbeta; this involves the Cbl TKB domain binding to PDGFRbeta phosphotyrosine 1021, a known phospholipase C (PLC) gamma1 SH2 domain-binding site. Lack of Cbl or ablation of the Cbl-binding site on PDGFRbeta impedes receptor sorting to the lysosome. Cbl-deficient cells also show more PDGF-induced PLCgamma1 association with PDGFRbeta and enhanced PLC-mediated cell migration. Thus, Cbl-dependent negative regulation of PDGFRbeta involves a dual mechanism that concurrently promotes ubiquitin-dependent lysosomal sorting of the receptor and competitively reduces the recruitment of a positive mediator of receptor signaling.     

Vascular endothelial growth factor can signal through platelet-derived growth factor receptors

Vascular endothelial growth factor (VEGF-A) is a crucial stimulator of vascular cell migration and proliferation. Using bone marrow-derived human adult mesenchymal stem cells (MSCs) that did not express VEGF receptors, we provide evidence that VEGF-A can stimulate platelet-derived growth factor receptors (PDGFRs), thereby regulating MSC migration and proliferation. VEGF-A binds to both PDGFRalpha and PDGFRbeta and induces tyrosine phosphorylation that, when inhibited, results in attenuation of VEGF-A-induced MSC migration and proliferation. This mechanism was also shown to mediate human dermal fibroblast (HDF) migration. VEGF-A/PDGFR signaling has the potential to regulate vascular cell recruitment and proliferation during tissue regeneration and disease.     

The Casitas B lineage lymphoma (Cbl) mutant G306E enhances osteogenic differentiation in human mesenchymal stromal cells in part by decreased Cbl-mediated platelet-derived growth factor receptor alpha and fibroblast growth factor receptor 2 ubiquitination

Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.     

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.     

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.     

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.     

Ubiquitin ligase Cbl-b acts as a negative regulator in discoidin domain receptor 2 signaling via modulation of its stability

Discoidin domain receptor 2 (DDR2), a collagen receptor tyrosine kinase, initiates signal transduction upon collagen binding, but little is known as to how DDR2 signaling is negatively regulated. Herein we demonstrate that Cbl family member Cbl-b predominantly promotes the ubiquitination of DDR2 upon collagen II stimulation. Cbl-b-mediated ubiquitination accelerates the degradation of activated DDR2. Finally, the production of MMP-13, a downstream target of DDR2, is enhanced in Cbl-b-knocked down MC3T3-E1 cells and Cbl-b-deficient mouse primary synovial fibroblasts. Thus, Cbl-b, by promoting the ubiquitination and degradation of DDR2, functions as a negative regulator in the DDR2 signaling pathway.     

A gain of function mutation in the activation loop of platelet-derived growth factor beta-receptor deregulates its kinase activity

The platelet-derived growth factor receptors (PDGFRs) are receptor tyrosine kinases implicated in multiple aspects of cell growth, differentiation, and survival. Recently, a gain of function mutation in the activation loop of the human PDGFRalpha has been found in patients with gastrointestinal stromal tumors. Here we show that a mutation in the corresponding codon in the activation loop of the murine PDGFRbeta, namely an exchange of asparagine for aspartic acid at amino acid position 849 (D849N), confers transforming characteristics to embryonic fibroblasts from mutant mice, generated by a knock-in strategy. By comparing the enzymatic properties of the wild-type versus the mutant receptor protein, we demonstrate that the D849N mutation lowers the threshold for kinase activation, causes a dramatic alteration in the pattern of tyrosine phosphorylation kinetics following ligand stimulation, and induces a ligand-independent phosphorylation of several tyrosine residues. These changes result in deregulated recruitment of specific signal transducers. The GTPase-activating protein for Ras (RasGAP), a negative regulator of the Ras mitogenic pathway, displayed a delayed binding to the mutant receptor. Moreover, we have observed enhanced ligand-independent ERK1/2 activation and an increased proliferation of mutant cells. The p85 regulatory subunit of the phosphatidylinositol 3 '-kinase was constitutively associated with the mutant receptor, and this ligand-independent activation of the phosphatidylinositol 3'-kinase pathway may explain the observed strong protection against apoptosis and increased motility in cellular wounding assays. Our findings support a model whereby an activating point mutation results in a deregulated PDGFRbeta with oncogenic predisposition.     

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.     

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.     

The Cbl RING finger C-terminal flank controls epidermal growth factor receptor fate downstream of receptor ubiquitination

Evolutionarily conserved sequences of the E3/protein-ubiquitin ligase Cbl regulate epidermal growth factor receptor (EGF-R) signaling and degradation. These sequences encompass Cbl's tyrosine kinase-binding domain, linker region, RING finger (RF), and an uncharacterized flank C-terminal to the RF (residues 420-436). The latter domain, designated the RF tail, extends beyond Cbl's ubiquitin-conjugating enzyme (Ubc)-binding domain and has no known function. We report structure-function studies evaluating the impact of Cbl RF tail truncations on EGF-R fate in HEK 293 cells. All of the truncation mutants exhibit greatly reduced binding to activated EGF-R and lack proline-rich sequences that mediate direct Cbl association with SH3 proteins such as Grb2, yet a subset of mutants collectively enhances EGF-R ubiquitination, downregulation, and degradation. Significantly, EGF-R degradation correlates better with RF tail-dependent degradation of the Cbl substrate Sprouty2 than with EGF-R ubiquitination: expression of the RF tail truncation mutant Cbl 1-433 enhanced EGF-R ubiquitination while impeding Sprouty2 degradation, and Cbl 1-433 failed to enhance EGF-R downregulation or degradation. Our results suggest that EGF-R fate is controlled by a checkpoint downstream of receptor ubiquitination whose regulation by the Cbl RF tail may require Sprouty2 degradation.     

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.     

Enhancement of fcgamma receptor-mediated phagocytosis by transforming mutants of Cbl

Phagocytosis mediated by FcgammaR plays an important role in host defense. The molecular events involved in this process have not been completely defined. The adapter protein Cbl has been implicated in FcgammaR signaling, but the function of Cbl in phagocytosis is unknown. Here we show that overexpression of the transforming mutants of Cbl, Cbl-70Z, and v-Cbl, but not wild-type (wt) Cbl, enhance phagocytosis mediated by FcgammaR in COS cells. Cbl-70Z, but not Cbl-wt, also enhanced FcgammaR-mediated phagocytosis in P388D1 murine macrophage cells. Cbl-70Z did not affect tyrosine phosphorylation or in vitro kinase activity of Syk, indicating that Syk may not be the direct target of Cbl-70Z in the enhancement of phagocytosis. A point mutation (G306E) in the phosphotyrosine domain of Cbl-70Z, as well as a C-terminal 67-aa deletion, partially abolished the enhancing effect on FcgammaR-mediated phagocytosis. A double mutant of Cbl-70Z containing both the G306E mutation and the C-terminal deletion completely lacked the ability to enhance phagocytosis. Thus, both the phosphotyrosine binding domain and the carboxyl-terminal tail were required for optimal enhancement of phagocytosis by Cbl-70Z. Functional phosphatidylinositol 3-kinase was required for Cbl-70Z to enhance phagocytosis, since wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FcgammaR-mediated phagocytosis in the presence of Cbl-70Z. These studies demonstrate that mutants of Cbl can modulate the phagocytic pathway mediated by FcgammaR and imply a functional involvement of c-Cbl in Fcgamma receptor-mediated phagocytosis.     

Vascular endothelial growth factor-dependent down-regulation of Flk-1/KDR involves Cbl-mediated ubiquitination. Consequences on nitric oxide production from endothelial cells

Ligand-stimulated degradation of receptor tyrosine kinase (RTK) is an important regulatory step of signal transduction. The vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is responsible for the VEGF-stimulated nitric oxide (NO) production from endothelial cells. Cellular mechanisms mediating the negative regulation of Flk-1 signaling in endothelial cells have not been investigated. Here we show that Flk-1 is rapidly down-regulated following VEGF stimulation of bovine aortic endothelial cells (BAECs). Consequently, VEGF pretreatment of endothelial cells prevents any further stimulation of Flk-1, resulting in decreased NO production from subsequent VEGF challenges. Ubiquitination of RTKs targets them for degradation; we demonstrate that activation of Flk-1 by VEGF leads to its polyubiquitination in BAECs. Furthermore, VEGF stimulation of BAECs or COS-7 cells transiently transfected with Flk-1 results in the phosphorylation of the ubiquitin ligase Cbl, the enhanced association of Cbl with Flk-1, and the relocalization of Cbl to vesicular structures in BAECs. Overexpression of Cbl in COS-7 cells enhances VEGF-induced ubiquitination of Flk-1, whereas a Cbl mutant lacking the ubiquitin ligase RING finger domain, 70Z/3-Cbl, does not. Moreover, expression of Cbl in contrast to 70Z/3-Cbl inhibits the Flk-1-dependent activation of eNOS and, thus, NO release. In BAEC overexpressing Cbl, the degradation of Flk-1 upon VEGF stimulation is accelerated compared with cells transfected with a control vector (green fluorescent protein). Our findings demonstrate that Flk-1 is rapidly down-regulated following sustained VEGF stimulation and identify Cbl as a negative regulator of Flk-1 signaling to eNOS. Cbl thus plays a role in the regulation of VEGF signaling by mediating the stimulated ubiquitination and, consequently, degradation of Flk-1 in endothelial cells.     

Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination

Spred proteins modulate growth factor receptor signaling by inhibiting the Ras-MAPK cascade. Here, we show that Spred-1, Spred-2, and Spred-3 are ubiquitinated in HEK293T cells stimulated with epidermal growth factor (EGF) or pervanadate. Spred-2 tyrosines Y228 and/or Y231 in the Kit binding domain were identified as putative phosphorylation site(s) critical for Spred-2 ubiquitination. Depletion of Cbl and Cbl-b E3 ubiquitin ligases by RNA interference, or overexpression of a Cbl dominant inhibitory mutant (Cbl-N), inhibited Spred-2 ubiquitination, while conversely, wild type Cbl enhanced Spred-2 ubiquitination. Interaction of Spred-2 with Cbl-N was detectable by co-immunoprecipitation and required the Cbl SH2 domain and Spred-2 Y228 and Y231 residues. Studies on endogenous Spred-2 in ME4405 melanoma cells showed that pervanadate induced Spred-2 ubiquitination and a marked reduction in Spred-2 steady-state levels that was partially blocked by the proteasomal inhibitor, MG-132. These results suggest a role for Spred-2 tyrosine phosphorylation and ubiquitination in controlling Spred-2 expression levels.     

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.     

Phosphorylation of the platelet-derived growth factor receptor-beta and epidermal growth factor receptor by G protein-coupled receptor kinase-2. Mechanisms for selectivity of desensitization

Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-beta (PDGFRbeta) and epidermal growth factor receptor (EGFR), may be desensitized by serine/threonine kinases. One such kinase, G protein-coupled receptor kinase-2 (GRK2), is known to mediate agonist-dependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFRbeta, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFRbeta in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFRbeta but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFRbeta and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFRbeta mutant, GRK2-mediated PDGFRbeta phosphorylation required receptor tyrosine kinase activity, as does PDGFRbeta ubiquitination. Agonist-induced ubiquitination of the PDGFRbeta, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFRbeta down-regulation. Like the vast majority of GRK2 substrates, the PDGFRbeta, but not the EGFR, activated heterotrimeric G proteins allosterically in membranes from cells expressing physiologic protein levels. We conclude that GRK2 can phosphorylate and desensitize the PDGFRbeta, perhaps through mechanisms related to receptor ubiquitination. Specificity of GRK2 for receptor protein-tyrosine kinases, expressed at physiologic levels, may be determined by the ability of these receptors to activate heterotrimeric G proteins, among other factors.     

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.     

Catalytically inactive SHIP2 inhibits proliferation by attenuating PDGF signaling in 3T3-L1 preadipocytes

Inadequate proliferation and/or differentiation of preadipocytes may lead to adipose tissue dysfunction characterized by hypertrophied, insulin-resistant adipocytes. Platelet-derived growth factor (PDGF) may alter adipose tissue function by promoting proliferation of preadipocytes. Two principal signaling pathways that regulate proliferation are PI3K/PI(3,4,5)P3/Akt and Shc/Ras/ERK1/2. SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) dephosphorylates PI(3,4,5)P3, and also binds to Shc. Our goal was to determine how SHIP2 affects these PDGF signaling routes. To assess the role of the 5-phosphatase domain, we expressed wild-type or catalytically inactive dominant-negative SHIP2 (P686A-D690A-R691A; PDR/AAA) in 3T3-L1 preadipocytes. Surprisingly, SHIP2 PDR/AAA inhibited proliferation more potently than wild-type SHIP2. After three days of proliferation, phospho-Akt, phospho-ERK1/2, and PDGF receptor (PDGFR) levels were reduced in PDR/AAA-expressing preadipocytes. SHIP2 PDR/AAA interference with PDGFR signaling was demonstrated using imatinib, an inhibitor of PDGFR tyrosine kinase. The anti-proliferative effect of imatinib observed in control preadipocytes was not significant in SHIP2 PDR/AAA-expressing preadipocytes, indicating a pre-existing impairment of PDGFR-dependent mitogenesis in these cells. The inhibition of PDGF-activated mitogenic pathways by SHIP2 PDR/AAA was consistent with a decrease in PDGFR phosphorylation caused by a drop in receptor levels in SHIP2 PDR/AAA-expressing cells. SHIP2 PDR/AAA promoted ubiquitination of the PDGFR and its degradation via the lysosomal pathway independently of the association between the E3 ubiquitin ligase c-Cbl and PDGFR. Overall, our findings indicate that SHIP2 PDR/AAA reduces preadipocyte proliferation by attenuating PDGFR signaling.