Negative regulation of Lck by Cbl ubiquitin ligase

The Cbl-family ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent targeting to lysosomes. Cbl associates with the lymphoid-restricted nonreceptor tyrosine kinase Lck, but the functional relevance of this interaction remains unknown. Here, we demonstrate that T cell receptor and CD4 coligation on human T cells results in enhanced association between Cbl and Lck, together with Lck ubiquitination and degradation. A Cbl(-/-) T cell line showed a marked deficiency in Lck ubiquitination and increased levels of kinase-active Lck. Coexpression in 293T cells demonstrated that Lck kinase activity and Cbl ubiquitin ligase activity were essential for Lck ubiquitination and negative regulation of Lck-dependent serum response element-luciferase reporter activity. The Lck SH3 domain was pivotal for Cbl-Lck association and Cbl-mediated Lck degradation, with a smaller role for interactions mediated by the Cbl tyrosine kinase-binding domain. Finally, analysis of a ZAP-70-deficient T cell line revealed that Cbl inhibited Lck-dependent mitogen-activated protein kinase activation, and an intact Cbl RING finger domain was required for this functional effect. Our results demonstrate a direct, ubiquitination-dependent, negative regulatory role of Cbl for Lck in T cells, independent of Cbl-mediated regulation of ZAP-70.     

The fusion proteins TEL-PDGFRβ and FIP1L1-PDGFRα escape ubiquitination and degradation

Background

Chimeric oncogenes encoding constitutively active protein tyrosine kinases are associated with chronic myeloid neoplasms. TEL-PDGFRβ (TPβ, also called ETV6-PDGFRB) is a hybrid protein produced by the t(5;12) translocation, FIP1L1-PDGFRα (FPα) results from a deletion on chromosome 4q12 and ZNF198-FGFR1 is created by the t(8;13) translocation. These fusion proteins are found in patients with myeloid neoplasms associated with eosinophilia. Wild-type receptor tyrosine kinases are efficiently targeted for degradation upon activation, in a process that requires Cbl-mediated monoubiquitination of receptor lysines. Since protein degradation pathways have been identified as useful targets for cancer therapy, the aim of this study was to compare the degradation of hybrid and wild-type receptor tyrosine kinases.

Design and Methods

We used Ba/F3 as a model cell line, as well as leukocytes from two patients, to analyze hybrid protein degradation.

Results

In contrast to the corresponding wild-type receptors, which are quickly degraded upon activation, we observed that TPβ, FPα and the ZNF198-FGFR1 hybrids escaped down-regulation in Ba/F3 cells. The high stability of TPβ and FPα hybrid proteins was confirmed in leukocytes from leukemia patients. Ubiquitination of TPβ and FPα was much reduced compared to that of wild-type receptors, despite marked Cbl phosphorylation in cells expressing hybrid receptors. The fusion of a destabilizing domain to TPβ induced protein degradation. Instability was reverted by adding the destabilizing domain ligand, Shield1. The destabilization of this modified TPβ reduced cell transformation and STAT5 activation.

Conclusions

We have shown that chimeric receptor tyrosine kinases escape ubiquitination and down-regulation and that their stabilization is critical to efficient stimulation of cell proliferation.     

Structures of a platelet-derived growth factor/propeptide complex and a platelet-derived growth factor/receptor complex

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) are prototypic growth factors and receptor tyrosine kinases which have critical functions in development. We show that PDGFs share a conserved region in their prodomain sequences which can remain noncovalently associated with the mature cystine-knot growth factor domain after processing. The structure of the PDGF-A/propeptide complex reveals this conserved, hydrophobic association mode. We also present the structure of the complex between PDGF-B and the first three Ig domains of PDGFRβ, showing that two PDGF-B protomers clamp PDGFRβ at their dimerization seam. The PDGF-B:PDGFRβ interface is predominantly hydrophobic, and PDGFRs and the PDGF propeptides occupy overlapping positions on mature PDGFs, rationalizing the need of propeptides by PDGFs to cover functionally important hydrophobic surfaces during secretion. A large-scale structural organization and rearrangement is observed for PDGF-B upon receptor binding, in which the PDGF-B L1 loop, disordered in the structure of the free form, adopts a highly specific conformation to form hydrophobic interactions with the third Ig domain of PDGFRβ. Calorimetric data also shows that the membrane-proximal homotypic PDGFRα interaction, albeit required for activation, contributes negatively to ligand binding. The structural and biochemical data together offer insights into PDGF-PDGFR signaling, as well as strategies for PDGF-antagonism.     

Ubiquitination and degradation of Syk and ZAP-70 protein tyrosine kinases in human NK cells upon CD16 engagement

Syk and ZAP-70 nonreceptor protein tyrosine kinases (PTKs) are essential elements in several cascades coupling immune receptors to intracellular responses. The critical role of these kinases in promoting the propagation of intracellular signaling requires a tight regulation of their activity, thus the existence of a negative feedback loop regulating their expression can be hypothesized. Herein, we have investigated whether ubiquitin-dependent proteolysis could be a mechanism responsible for controlling the fate of Syk and ZAP-70 after their immunoreceptor-induced activation. We found that both Syk and ZAP-70 become ubiquitinated in response to aggregation of the low affinity Fc receptor for IgG (CD16) on human natural killer cells. We confirmed the identity of the major in vivo ubiquitinated kinase species by performing an in vitro ubiquitination assay. In addition, we found that after CD16 stimulation, ubiquitinated forms of Syk and ZAP-70 associate with the receptor complex. After CD16 engagement, we also observed a decrease in the stability of Syk and ZAP-70 PTKs that is counteracted by pretreatment with either proteasome or lysosomal inhibitors. Moreover, in the presence of the proteasome inhibitor, epoxomicin, we observed an accumulation of ubiquitinated forms of both kinases. Our findings provide evidence of ligand-induced ubiquitination of nonreceptor PTKs belonging to the Syk family and propose the ubiquitin-dependent proteasome-mediated degradation pathway as a mechanism for attenuating the propagation of intracellular signaling initiated by immune receptor engagement.     

PDGF-D−PDGFRβ signaling enhances IL-15–mediated human natural killer cell survival

The axis of platelet-derived growth factor (PDGF) and PDGF receptor-beta (PDGFRβ) plays prominent roles in cell growth and motility. In addition, PDGF-D enhances human natural killer (NK) cell effector functions when binding to the NKp44 receptor. Here, we report an additional but previously unknown role of PDGF-D, whereby it mediates interleukin-15 (IL-15)–induced human NK cell survival but not effector functions via its binding to PDGFRβ but independent of its binding to NKp44. Resting NK cells express no PDGFRβ and only a low level of PDGF-D, but both are significantly up-regulated by IL-15, via the nuclear factor κB signaling pathway, to promote cell survival in an autocrine manner. Both ectopic and IL-15–induced expression of PDGFRβ improves NK cell survival in response to treatment with PDGF-D. Our results suggest that the PDGF-D−PDGFRβ signaling pathway is a mechanism by which IL-15 selectively regulates the survival of human NK cells without modulating their effector functions.

Natural killer (NK) cells—a distinct lymphocyte subset in the circulation—play critical roles in antiviral and antitumor immunity (1). One advantage of NK cells is that they recognize “nonself” cells without being activated by specific antigens, allowing a more rapid response than with T cells. This broad cytotoxicity and rapid killing make NK cells ideal for cancer immunotherapy (2). Of note, chimeric-antigen-receptor (CAR)-NK cells have several therapeutic advantages over CAR-T cells (2–4). However, NK cells’ shorter lifespan may limit their clinical efficacy. A better understanding of the mechanisms that regulate NK cell survival might therefore improve their clinical application for cancer immunotherapy.Platelet-derived growth factor (PDGF) is one of the main growth factors that regulate cell growth and division (5). The PDGF family consists of PDGF-A, PDGF-B, PDGF-C, and PDGF-D (5). These ligands bind to two tyrosine kinase receptors, PDGFRα and PDGFRβ (5). Upon activation by PDGFs, PDGF receptors dimerize and undergo autophosphorylation on tyrosine residues in the intracellular domain, which mediates the binding of cofactors and subsequently activates signal transduction, including Ras/Raf/MEK/Erk mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase/protein kinase B (P13K/AKT) (5). PDGFs play prominent roles in cell differentiation, cell growth, cell transformation, and cell migration (5). A recent study showed that PDGF-D is a ligand of NKp44, one of the natural cytotoxicity receptors expressed by activated human NK cells (6). PDGF-D binding to NKp44 prompted NK cells to secrete interferon (IFN)-γ and tumor necrosis factor (TNF)-α, arresting the growth of tumor cells (6). However, little is known about the role of PDGFR signaling in NK cell immunity.In this study, we report a previously unknown role of PDGF-D: regulation of interleukin 15 (IL-15)–mediated cell survival—not effector functions in human NK cells—that is dependent on PDGFRβ but independent of NKp44. Our findings suggest that the PDGF-D−PDGFRβ signaling pathway is a mechanism by which IL-15 selectively regulates the survival of human NK cells but not their effector functions.     

Herpes simplex virus 1 infected cell protein 0 forms a complex with CIN85 and Cbl and mediates the degradation of EGF receptor from cell surfaces

Infected cell protein 0 (ICP0) is a 775-residue multifunctional herpes simplex virus protein associated with numerous functions related to transactivation of gene expression and repression of host defenses to infection. We report that an uncharted domain of ICP0 located between residues 245 and 510 contains multiple SH3 domain binding motifs similar to those required for binding to CIN85, the M(r) 85,000 protein that interacts with Cbl. CIN85 and Cbl are involved in endocytosis and negative regulation of numerous receptor tyrosine kinases. We report that ICP0 binds CIN85 in a reciprocal manner and that the complexes pulled down by ICP0 also contain Cbl. We tested the role of ICP0 in the down-regulation of receptor tyrosine kinases by using epidermal growth factor receptor (EGFR) as a prototypic receptor. In transfection assays, ICP0, in the absence of other viral genes, down-regulated EGF-dependent expression of a reporter gene (luciferase). ICP0 also down-regulated both total and cell surface levels of EGFR in EGF-independent manner. In wild-type virus-infected cells, the surface levels of EGFR were also decreased in the absence of EGF stimulation. Stimulation by EGF enhanced the decrease in surface EGFR. We conclude that ICP0 encodes SH3 domain binding sites that function to down-regulate signaling pathways associated with receptor tyrosine kinases. The results suggest that ICP0 precludes signaling to the infected cells through the receptor tyrosine kinases.     

Requirement for an initial signal from the membrane-proximal region of the interleukin 2 receptor γc chain for Janus kinase activation leading to T cell proliferation

The interleukin 2 receptor (IL-2R) generates proliferative signals in T lymphocytes by ligand-induced heterodimerization of two chains, IL-2Rβ and γ_c, which associate with the tyrosine kinases Jak1 and Jak3, respectively. Genetic and molecular studies have demonstrated that Jak3 is essential for mitogenic signaling by the γ_c chain; because it is also the only molecule known to associate with γ_c, we speculated that Jak3 might be sufficient for signaling by this chain. Therefore, fusion proteins were constructed in which all or part of the cytoplasmic domain of γ_c was replaced by Jak3. Signaling was evaluated in the IL-2-dependent T cell line CTLL-2 using chimeric IL-2Rβ and γ_c chains that bind and are activated by the cytokine granulocyte–macrophage colony-stimulating factor. Chimeric γ_c chains containing only Jak3 in the cytoplasmic domain failed to mediate proliferation of CTLL-2 cells, but addition of a conserved membrane-proximal (PROX) domain of γ_c in tandem with Jak3 fully reconstituted γ_c function. The requirement for the PROX domain reflected an essential role in the activation of Jak3 in vivo. Despite lacking defined catalytic motifs, PROX induced an early Jak-independent signal, including tyrosine phosphorylation of IL-2Rβ and the tyrosine phosphatase SHP-2. The results define the minimal signaling components of γ_c and suggest a new mechanism by which the IL-2R initiates signaling in response to ligand.     

From the Cover: PNAS Plus: Functional malignant cell heterogeneity in pancreatic neuroendocrine tumors revealed by targeting of PDGF-DD

Intratumoral heterogeneity is an inherent feature of most human cancers and has profound implications for cancer therapy. As a result, there is an emergent need to explore previously unmapped mechanisms regulating distinct subpopulations of tumor cells and to understand their contribution to tumor progression and treatment response. Aberrant platelet-derived growth factor receptor beta (PDGFRβ) signaling in cancer has motivated the development of several antagonists currently in clinical use, including imatinib, sunitinib, and sorafenib. The discovery of a novel ligand for PDGFRβ, platelet-derived growth factor (PDGF)-DD, opened the possibility of a previously unidentified signaling pathway involved in tumor development. However, the precise function of PDGF-DD in tumor growth and invasion remains elusive. Here, making use of a newly generated Pdgfd knockout mouse, we reveal a functionally important malignant cell heterogeneity modulated by PDGF-DD signaling in pancreatic neuroendocrine tumors (PanNET). Our analyses demonstrate that tumor growth was delayed in the absence of signaling by PDGF-DD. Surprisingly, ablation of PDGF-DD did not affect the vasculature or stroma of PanNET; instead, we found that PDGF-DD stimulated bulk tumor cell proliferation by induction of paracrine mitogenic signaling between heterogeneous malignant cell clones, some of which expressed PDGFRβ. The presence of a subclonal population of tumor cells characterized by PDGFRβ expression was further validated in a cohort of human PanNET. In conclusion, we demonstrate a previously unrecognized heterogeneity in PanNET characterized by signaling through the PDGF-DD/PDGFRβ axis.Undeniably, cancer progression is the consequence of dynamic, and yet poorly understood, cell–cell interactions driven by frequently deregulated signaling pathways (1). Further complexity arises from the notion that tumors are composed of phenotypically and functionally distinct subsets of both malignant and stromal cells (2, 3). Therefore, accounting for intratumoral heterogeneity poses an additional challenge when designing therapies that can efficiently control or eliminate tumors. An improved understanding of the functional contribution of different signaling pathways to genetic and phenotypic variation within tumors is therefore highly warranted.Members of the platelet-derived growth factor (PDGF) family and their receptors (PDGFRs) have been extensively investigated and shown to be critical for cellular processes such as proliferation, survival, and motility during tumor growth and invasion (4). The roles of PDGF isoforms and their target cells in tumor development have been charted in different tumor types (5), and as a result, pharmacological blockade of PDGF signaling is now routinely used for the treatment of diverse malignancies, such as gastrointestinal stromal tumors and chronic myelomonocytic leukemia, among others (6, 7). The PDGF family is composed of four polypeptide chains that assemble into five dimeric isoforms (PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and PDGF-DD) that bind and activate two receptor tyrosine kinases (PDGFRα and PDGFRβ) expressed mainly by cells of mesenchymal origin (8). PDGF-DD is the most recently identified member of the family (9, 10), and unlike the other ligands, the role of PDGF-DD in normal development and pathology is largely a conundrum.Herein, we report the use of a Pdgfd knockout mouse to explore the specific role of PDGF-DD in malignant growth. By monitoring tumorigenesis in the RIP1-TAg2 mouse model of pancreatic neuroendocrine tumors (PanNET), we found that disruption of PDGF-DD signaling significantly delayed tumor growth. In the absence of PDGF-DD, functional compensation by PDGF-BB was apparent in the stromal compartment. Unexpectedly, however, we identified a subpopulation of malignant cells expressing PDGFRβ with accompanying responsiveness to PDGF-DD. By modulating PDGFRβ⁺ malignant cells, PDGF-DD contributes to the maintenance of functional malignant cell heterogeneity in experimental PanNET.     

Regulation of stem cell factor receptor signaling by Cbl family proteins (Cbl-b/c-Cbl)

Activation of the KIT receptor tyrosine kinase contributes to the pathogenesis of several human diseases, but the mechanisms regulating KIT signaling have not been fully characterized. Here, we show that stem cell factor (SCF), the ligand for KIT, induces the interaction between KIT and Cbl proteins and their mutual degradation. Upon SCF stimulation, KIT binds to and induces the phosphorylation of Cbl proteins, which in turn act as E3 ligases, mediating the ubiquitination and degradation of KIT and themselves. Tyrosine kinase binding and RING finger domains of Cbl are essential for Cbl-mediated ubiquitination and degradation of KIT. We propose a negative feedback loop controlling the SCF-KIT signaling pathway, in which SCF activates KIT. The activated KIT in turn induces phosphorylation and activation of Cbl proteins. The Cbl proteins then bind and direct the degradation of activated KIT, leading to down-regulation of KIT signaling.     

Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor β receptor

The bovine papillomavirus E5 protein is a 44-aa transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) β receptor and induces constitutive tyrosine phosphorylation and activation of the receptor, resulting in cell transformation. The E5 protein does not resemble PDGF, but rather activates the receptor in a ligand-independent fashion, thus providing a unique system to examine activation of receptor tyrosine kinases. Here, we used a variety of approaches to explore the mechanism of receptor activation by the E5 protein. Chemical cross-linking experiments revealed that the E5 protein activated only a small fraction of the endogenous PDGF β receptor in transformed fibroblasts and suggested that this fraction was constitutively dimerized. Coimmunoprecipitation experiments using extracts of cells engineered to coexpress full-length and truncated PDGF β receptors confirmed that the E5 protein induced oligomerization of the receptor. Furthermore, in cells expressing the E5 protein, a kinase-active receptor was able to trans-phosphorylate a kinase-negative mutant receptor but was unable to catalyze intramolecular autophosphorylation. These results indicated that the E5 protein induced PDGF β receptor activation by forming a stable complex with the receptor, resulting in receptor dimerization and trans-phosphorylation.     

Selection of Human Cytomegalovirus Mutants with Resistance against PDGFRα-Derived Entry Inhibitors

The human cytomegalovirus (HCMV) infects fibroblasts via an interaction of its envelope glycoprotein gO with the cellular platelet-derived growth factor receptor alpha (PDGFRα), and soluble derivatives of this receptor can inhibit viral entry. We aimed to select mutants with resistance against PDGFRα-Fc and the PDGFRα-derived peptides GT40 and IK40 to gain insight into the underlying mechanisms and determine the genetic barrier to resistance. An error-prone variant of strain AD169 was propagated in the presence of inhibitors, cell cultures were monitored weekly for signs of increased viral growth, and selected viruses were tested regarding their sensitivity to the inhibitor. Resistant virus was analyzed by DNA sequencing, candidate mutations were transferred into AD169 clone pHB5 by seamless mutagenesis, and reconstituted virus was again tested for loss of sensitivity by dose-response analyses. An S48Y mutation in gO was identified that conferred a three-fold loss of sensitivity against PDGFRα-Fc, a combination of mutations in gO, gH, gB and gN reduced sensitivity to GT40 by factor 4, and no loss of sensitivity occurred with IK40. The resistance-conferring mutations support the notion that PDGFRα-Fc and GT40 perturb the interaction of gO with its receptor, but the relatively weak effect indicates a high genetic barrier to resistance.     

The amino-terminal region of Tyk2 sustains the level of interferon α receptor 1, a component of the interferon α/β receptor

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-α (IFN-α) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-α. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-α receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-α. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.     

The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor signaling

An early stage in thymocyte development, after rearrangement of the β chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of CD4⁻CD8⁻ double negative (DN) cells to CD4⁺CD8⁺ double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo β chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.     

From the Cover: Phosphorylation of protocadherin proteins by the receptor tyrosine kinase Ret

The clustered protocadherins (Pcdhs) are a large family of cadherin-like transmembrane proteins expressed in the nervous system. Stochastic expression of Pcdh genes and alternative splicing of their pre-mRNAs have the potential to generate enormous protein diversity at the cell surface of neurons. At present, the regulation and function of Pcdh proteins are largely unknown. Here, we show that Pcdhs form a heteromeric signaling complex(es), consisting of multiple Pcdh isoforms, receptor tyrosine kinases, phosphatases, and cell adhesion molecules. In particular, we find that the receptor tyrosine kinase rearranged during transformation (Ret) binds to Pcdhs in differentiated neuroblastoma cells and is required for stabilization and differentiation-induced phosphorylation of Pcdh proteins. In addition, the Ret ligand glial cell line-derived neurotrophic factor induces phosphorylation of Pcdhγ in motor neurons and phosphorylation of Pcdhα and Pcdhγ in sympathetic neurons. Conversely, Pcdh proteins are also required for the stabilization of activated Ret in neuroblastoma cells and sympathetic ganglia. Thus, Pcdhs and Ret are functional components of a phosphorylation-dependent signaling complex.     

Calcineurin regulates the stability and activity of estrogen receptor α

Estrogen receptor α (ER-α) mediates estrogen-dependent cancer progression and is expressed in most breast cancer cells. However, the molecular mechanisms underlying the regulation of the cellular abundance and activity of ER-α remain unclear. We here show that the protein phosphatase calcineurin regulates both ER-α stability and activity in human breast cancer cells. Calcineurin depletion or inhibition down-regulated the abundance of ER-α by promoting its polyubiquitination and degradation. Calcineurin inhibition also promoted the binding of ER-α to the E3 ubiquitin ligase E6AP, and calcineurin mediated the dephosphorylation of ER-α at Ser²⁹⁴ in vitro. Moreover, the ER-α (S294A) mutant was more stable and activated the expression of ER-α target genes to a greater extent compared with the wild-type protein, whereas the extents of its interaction with E6AP and polyubiquitination were attenuated. These results suggest that the phosphorylation of ER-α at Ser²⁹⁴ promotes its binding to E6AP and consequent degradation. Calcineurin was also found to be required for the phosphorylation of ER-α at Ser¹¹⁸ by mechanistic target of rapamycin complex 1 and the consequent activation of ER-α in response to β-estradiol treatment. Our study thus indicates that calcineurin controls both the stability and activity of ER-α by regulating its phosphorylation at Ser²⁹⁴ and Ser¹¹⁸. Finally, the expression of the calcineurin A–α gene (PPP3CA) was associated with poor prognosis in ER-α–positive breast cancer patients treated with tamoxifen or other endocrine therapeutic agents. Calcineurin is thus a promising target for the development of therapies for ER-α–positive breast cancer.

Estrogen receptor α (ER-α) plays a central role in the proliferation of breast cancer cells by increasing the expression of oncogenes, such as those encoding cyclin D1 and c-Myc (1). The expression and activity of ER-α are increased in >70% of breast cancer cases, and the receptor is targeted by drugs such as tamoxifen (2, 3). A substantial proportion of ER-α–positive breast cancer cells become resistant to anti‐estrogens, however, resulting in the progression of the disease. The mechanisms by which the cancer cells acquire resistance to these agents include the generation of splice variants of ER-α, the mutation of the ER-α gene (ESR1), and changes in stability of the ER-α protein (4).Increased protein stability appears to be a key contributor to the up-regulation of ER-α in breast cancer. The ubiquitination of ER-α is one mechanism responsible for ER-α degradation. Several E3 ligases that mediate the degradation of ER-α have been identified and include E6-associated protein (E6AP) (5), carboxyl terminus of Hsp70-interacting protein (CHIP) (6), breast cancer type 1 (BRCA1) (7), BRCA1-associated RING domain 1 (8), S phase kinase–associated protein 2 (SKP2) (9), and mouse double minute 2 homolog (10). On the other hand, other E3 ligases—such as RING finger protein (RNF) 31, shank-associated RH domain–interacting protein, and RNF8 (11–13)—have been shown to promote ER-α signaling by stabilizing ER-α protein.The residues Lys³⁰² and Lys³⁰³ of ER-α are targeted for ubiquitination (14). The ubiquitination of ER-α is associated with its phosphorylation, with several kinases such as cyclin-dependent kinase (CDK) 11 (15), Src (5), protein kinase C (16), p38 mitogen-activated protein kinase (9), and extracellular signal–regulated kinase 7 (17) having been shown to phosphorylate the protein. The phosphorylation of ER-α at Ser²⁹⁴ has thus been related to its ubiquitination by SKP2 (9), with the Ser²⁹⁴-phosphorylated form of ER-α being a preferred substrate for ubiquitination by SKP2 in vitro. However, the expression level of ER-α was found to be unaltered in cells depleted of SKP2, suggesting that other E3 ligases may contribute to the degradation of ER-α subsequent to its phosphorylation at Ser²⁹⁴.Calcium is an important regulator of signaling pathways that control oncogenesis and cancer progression, and Ca²⁺ signaling has been linked to signaling by ER-α. β-estradiol (E2) has been shown to induce rapid Ca²⁺ influx in cells, and the Ca²⁺-binding protein calmodulin interacts with ER-α, increases its stability, and modulates E2-regulated gene expression (18). Calcineurin is a Ca²⁺/calmodulin-activated serine–threonine phosphatase that plays a major role in the regulation of immediate cellular responses and gene expression by Ca²⁺ signaling (19). It is also a target of immunosuppressive drugs administered in clinical practice, such as cyclosporine A and FK506. Calcineurin is composed of two subunits: a catalytic subunit, designated calcineurin A, that is encoded by three genes (PPP3CA, PPP3CB, and PPP3CC), and a regulatory subunit, designated calcineurin B, that is encoded by two genes (PPP3R1 and PPP3R2).In the present study, we found that calcineurin plays a previously unrecognized role as a positive regulator of the stability and activity of ER-α in breast cancer cells by mediating its dephosphorylation at Ser²⁹⁴, as well as the activation of mechanistic target of rapamycin complex 1 (mTORC1) and the consequent phosphorylation of ER-α at Ser¹¹⁸, respectively. Furthermore, a high-expression level of PPP3CA was associated with poor prognosis in a subset of breast cancer patients, suggesting that the selective inhibition of calcineurin might be an effective approach to the treatment of ER-α–positive breast cancer.     

FRS2 alpha attenuates FGF receptor signaling by Grb2-mediated recruitment of the ubiquitin ligase Cbl

Attenuation of growth factor signaling is essential for the regulation of developmental processes and tissue homeostasis in most organisms. The product of Cbl protooncogene is one such regulator, which functions as an ubiquitin ligase that ubiquitinates and promotes the degradation of a variety of cell signaling proteins. Here, we demonstrate that Grb2 bound to tyrosine-phosphorylated FRS2 alpha forms a ternary complex with Cbl by means of its Src homology 3 domains resulting in the ubiquitination of fibroblast growth factor (FGF) receptor and FRS2 alpha in response to FGF stimulation. These observations highlight the importance of FRS2 alpha in the assembly of both positive (i.e., Sos, phosphatidylinositol 3-kinase) and negative (i.e., Cbl) signaling proteins to mediate a balanced FGF signal transduction. However, the partial inhibition of FGF receptor down-regulation in FRS2 alpha-/- cells indicates that the attenuation of signaling by FGF receptor is regulated by redundant or multiple mechanisms.     

β2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced β₂-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating β₂-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.     

Disruption of angiogenesis and tumor growth with an orally active drug that stabilizes the inactive state of PDGFRβ/B-RAF

Kinases are known to regulate fundamental processes in cancer including tumor proliferation, metastasis, neovascularization, and chemoresistance. Accordingly, kinase inhibitors have been a major focus of drug development, and several kinase inhibitors are now approved for various cancer indications. Typically, kinase inhibitors are selected via high-throughput screening using catalytic kinase domains at low ATP concentration, and this process often yields ATP mimetics that lack specificity and/or function poorly in cells where ATP levels are high. Molecules targeting the allosteric site in the inactive kinase conformation (type II inhibitors) provide an alternative for developing selective inhibitors that are physiologically active. By applying a rational design approach using a constrained amino-triazole scaffold predicted to stabilize kinases in the inactive state, we generated a series of selective type II inhibitors of PDGFRβ and B-RAF, important targets for pericyte recruitment and endothelial cell survival, respectively. These molecules were designed in silico and screened for antivascular activity in both cell-based models and a Tg(fli1-EGFP) zebrafish embryogenesis model. Dual inhibition of PDGFRβ and B-RAF cellular signaling demonstrated synergistic antiangiogenic activity in both zebrafish and murine models of angiogenesis, and a combination of previously characterized PDGFRβ and RAF inhibitors validated the synergy. Our lead compound was selected as an orally active molecule with favorable pharmacokinetic properties which demonstrated target inhibition in vivo leading to suppression of murine orthotopic tumors in both the kidney and pancreas.     

Protein kinase C-theta (PKCθ) phosphorylates and inhibits the guanine exchange factor,GIV/Girdin

Gα-interacting, vesicle-associated protein (GIV/Girdin) is a multidomain signal transducer that enhances PI3K-Akt signals downstream of both G-protein–coupled receptors and growth factor receptor tyrosine kinases during diverse biological processes and cancer metastasis. Mechanistically, GIV serves as a non-receptor guanine nucleotide exchange factor (GEF) that enhances PI3K signals by activating trimeric G proteins, Gαi1/2/3. Site-directed mutations in GIV’s GEF motif disrupt its ability to bind or activate Gi and abrogate PI3K-Akt signals; however, nothing is known about how GIV’s GEF function is regulated. Here we report that PKCθ, a novel protein kinase C, down-regulates GIV’s GEF function by phosphorylating Ser(S)1689 located within GIV’s GEF motif. We demonstrate that PKCθ specifically binds and phosphorylates GIV at S1689, and this phosphoevent abolishes GIV’s ability to bind and activate Gαi. HeLa cells stably expressing the phosphomimetic mutant of GIV, GIV-S1689→D, are phenotypically identical to those expressing the GEF-deficient F1685A mutant: Actin stress fibers are decreased and cell migration is inhibited whereas cell proliferation is triggered, and Akt (a.k.a. protein kinase B, PKB) activation is impaired downstream of both the lysophosphatidic acid receptor, a G-protein–coupled receptor, and the insulin receptor, a receptor tyrosine kinase. These findings indicate that phosphorylation of GIV by PKCθ inhibits GIV''s GEF function and generates a unique negative feedback loop for downregulating the GIV-Gi axis of prometastatic signaling downstream of multiple ligand-activated receptors. This phosphoevent constitutes the only regulatory pathway described for terminating signaling by any of the growing family of nonreceptor GEFs that modulate G-protein activity.