Stimulation Of Receptor Tyrosine Kinase Research Articles

Abstract A025: Investigating the molecular mechanisms of regulation of the RAS guanine nucleotide exchange factor, SOS1 by Grb2 and 14-3-3

Abstract The MAPK/ERK signaling pathway is one of the most heavily mutated in human cancers. In normal cells, the stimulation of receptor tyrosine kinases (e.g. EGFR) on the plasma membrane signals the GTP-loading and activation of RAS through a mechanism catalyzed by the guanine nucleotide exchange factor (GEF) Son of Sevenless 1 (SOS1). RAS activation, in turn, initiates the RAF-MEK-ERK kinase cascade which triggers many downstream signals that can affect gene transcription, protein translation, cell proliferation, and feedback regulation. Several factors regulate SOS1 GEF activity, including two autoinhibitory domains within its own multi-domain architecture. First, the N-terminal domains of SOS1 occlude the allosteric RAS-GTP binding site in the RAS exchanger motif—thereby precluding SOS1 GEF activity—and this inhibition is relieved upon membrane recruitment and PIP2 phospholipid binding. Second, the C-terminal Proline-Rich Region (PRR) of SOS1 also occludes allosteric RAS-GTP binding by an unknown mechanism and is relieved when the growth factor receptor-bound protein 2 (Grb2) binds to motifs in the PRR. While the independent functions of these domains and interactions to inhibition and activation of GEF activity of SOS1 are known, the molecular mechanisms are poorly understood. An additional negative feedback loop involves the phosphorylation of the SOS1 PRR at two sites by the ribosomal protein S6 kinase 1 (RSK1), a substrate of downstream ERK. These modifications create 14-3-3 binding sites on SOS1, where 14-3-3 binding downregulates GEF activity, possibly by obstructing Grb2 association with the SOS1 PRR and membrane localization of SOS1. This work reconstitutes the SOS1:Grb2 and the SOS1:14-3-3 protein complexes in vitro to study the molecular mechanisms underlying SOS1 activity regulation. Citation Format: Orlando E. Martinez, Jawahar Sudhamsu. Investigating the molecular mechanisms of regulation of the RAS guanine nucleotide exchange factor, SOS1 by Grb2 and 14-3-3 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A025.

Lack of PI 3-kinase isoform p110alpha impairs SMC differentiation and proliferation and promotes aortic aneurysm formation

Abstract Background Proliferation and phenotypic modulation of vascular smooth muscle cells (SMCs) significantly contribute to the functionality of the aortic wall. Dysregulation of underlying signal transduction pathways impairs the vessel wall structure and promote the development and progression of abdominal aortic aneurysms (AAA). The PI 3-kinase (PI3K) isoform p110α is activated downstream of receptor tyrosine kinases (RTKs) and represents the most relevant PI3K isoform in SMCs. Aim This project follows the hypothesis that p110α deficiency impairs proliferation and phenotypic modulation of SMCs as well as the structure of the extracellular matrix (ECM) and therefore promotes the development and progression of AAA. It was investigated how p110α deficiency affects the plasticity of SMCs, the production and structure of ECM components, and the formation of AAA. Methods and results Western blot analyses showed that SMCs isolated from smooth muscle specific p110α−/− (sm-p110α−/−) mice were characterized by decreased expression of the differentiation markers sm-α-actin, calponin and sm-MHC. Mechanistically, phosphorylation of key modulators of the SMC phenotype – AKT1, AKT2, FOXO1, -3 and -4 as well as GSK3β – was impaired in p110α−/− SMCs after RTK stimulation. These findings indicate that phenotypic modulation of p110α−/− SMCs is restricted. In addition, protein expression of elastin and fibrillin was reduced in p110α−/− SMCs. In silico analysis (MatLab macro CT-FIRE and Curvalign) of the ECM produced by SMCs in vitro revealed a significantly reduced elastin fiber length and width in p110α−/− SMCs compared to fibers produced by WT SMCs (p<0.05). Consistently, aortas from sm-p110α−/− mice showed a significantly higher number of elastic fiber breaks specifically in the thoracic section than WT controls (289±31 mmm–2 versus 190±9 mmm–2, n=5, p=0.015). Aortic aneurysms in sm-p110α−/− mice and wild-type littermates were analyzed using the established porcine pancreatic elastase (PPE) model. PPE was perfused into the infrarenal aorta to induce AAA formation. Ultrasound examination of the aorta revealed an enlarged aortic diameter in all PPE-treated mice. However, the increase in aortic diameter in sm-p110α−/− mice (70.16±10.82% mm, n=9) was significant more pronounced compared to wild-type animals (42.44±5.99%, n=10) (p<0.05). Three days after PPE perfusion, the number of elastic fiber breaks was significantly increased, and amount of proliferating SMCs were decreased in the infrarenal aorta of sm-p110α−/− mice compared to WT controls. Conclusion p110α deficiency in SMCs impairs aortic wall structure and promotes the development and progression of aortic aneurysms. Mechanistically, p110α activity maintains a differentiated SMC phenotype as well as the expression and assembly of ECM components. These data identify p110α signaling as a modifiable target for preventive and therapeutic strategies for aortic aneurysms. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD.

Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.

Targeted Degradation of the Oncogenic Phosphatase SHP2.

SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras-MAPK pathway upon stimulation of receptor tyrosine kinases, which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at submicromolar concentrations, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.

STEM-06. TISSUE FACTOR SIGNALING IN GLIOBLASTOMA TUMOR-INITIATING CELLS

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with few treatment options, characterized by over-activation of receptor tyrosine kinases (RTKs). Brain tumor-initiating cells (BTICs) is a subpopulation of cells in GBM which have the unique capacity to self-renew, facilitating recurrence. Tissue Factor (TF), normally involved in blood coagulation, is elevated in GBM and strongly correlates with more aggressive behavior. We hypothesize that TF promotes malignant behavior in glioblastoma by activating protease-activated receptor 2 (PAR2), leading to RTK stimulation and the maintenance of brain tumor-initiating cells. Therefore, our approaches are to determine: the mechanisms by which TF-PAR2 stimulates RTK signaling in GBM; the role of RTKs in mediating TF-induced BTIC maintenance in GBM, and the effect of blocking TF signaling on RTK-based therapies against GBM. Our data indicate that TF can activate multiple classes of tumor-promoting RTKs, and sustains cell proliferation through its main receptor, PAR2, even in the presence of an RTK inhibitor. We also found that TF suppression impairs the BTIC phenotype in high TF-expressing, RTK-driven GBM. This project will advance our understanding of how TF promotes malignant behavior, identify a powerful means by which cancers can resist RTK inhibitors currently in clinical use, and demonstrate the therapeutic potential of blocking TF-PAR2 signaling in cancer.

Tyrosine Phosphorylation of CD2AP Affects Stability of the Slit Diaphragm Complex

CD2-associated protein (CD2AP), a slit diaphragm-associated scaffolding protein involved in survival and regulation of the cytoskeleton in podocytes, is considered a "stabilizer" of the slit diaphragm complex that connects the slit diaphragm protein nephrin to the cytoskeleton of the cell. Tyrosine phosphorylation of slit diaphragm molecules can influence their surface expression, but it is unknown whether tyrosine phosphorylation events of CD2AP are also physiologically relevant to slit diaphragm stability. We used isoelectric focusing, western blot analysis, and immunofluorescence to investigate phosphorylation of CD2AP, and phospho-CD2AP antibodies and site-directed mutagenesis to define the specific phosphorylated tyrosine residues. We used cross-species rescue experiments in Cd2apKD zebrafish and in Drosophila cindrRNAi mutants to define the physiologic relevance of CD2AP phosphorylation of the tyrosine residues. We found that VEGF-A stimulation can induce a tyrosine phosphorylation response in CD2AP in podocytes, and that these phosphorylation events have an important effect on slit diaphragm protein localization and functionality in vivo. We demonstrated that tyrosine in position Y10 of the SH3-1 domain of CD2AP is indispensable for CD2AP function in vivo. We found that the binding affinity of nephrin to CD2AP is significantly enhanced in the absence of Y10; however, unexpectedly, this increased affinity leads not to stabilization but to functional impairment of the glomerular filtration barrier. Our findings provide insight into CD2AP and its phosphorylation in the context of slit diaphragm functionality, and indicate a fine-tuned affinity balance of CD2AP and nephrin that is influenced by receptor tyrosine kinase stimulation.

Transcript-level regulation of MALAT1-mediated cell cycle and apoptosis genes using dual MEK/Aurora kinase inhibitor "BI-847325" on anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is the most lethal malignancy in thyroid carcinomas. Long non-coding RNAs (lncRNAs) are a member of non-coding RNAs, regulating the expression of gene. Metastasis-associated lung adenocarcinoma transcript1 (MALAT1) is an onco-lncRNA that is overexpressed in several carcinomas including ATC. Evidence showed that MALAT1 has a crucial function in apoptosis, and cell cycle progression. In order to take advantage of 3D cell culture system in cancer investigation, we have used a 3D in vitro ATC model to determine the effect of dual MEK/Aurora kinase inhibitor BI-847325 anticancer drug on the fundamental molecular mechanisms of MALAT1-mediated gene regulation in ATC. In this study, ATC cell lines (C643 and SW1736) were grown in alginate scaffold. Encapsulated cells were treated by BI-847325. Changes in expression of MALAT1, Mcl1, miR-363-3p, and cyclinD1 were measured by qRT-PCR. MALAT1 gene expression following BI-847325 treatment was significantly downregulated in C643 and SW1736 cell lines. Reversely, miR-363-3p expression was significantly upregulated by BI-847325 in both ATC cell lines. Mcl1 expression was significantly downregulated after treatment in C643 cell lines. Moreover, the expression of this gene was not significantly reduced following BI-847325 treatment in SW1736 cell line. Additionally, cyclin D1 expression was significantly downregulated after treatment in both ATC cell lines. Altogether, the result of this study was the first report of MALAT1's molecular function in ATC and suggested that BI-847325 which inhibits both MEK and Aurora kinase family could be effective against ATC by regulating the genes involved in cell cycle and apoptosis including MALAT1and its downstream genes. Graphical abstract Schematic representation of the biological role of MALAT1 in cyclin D1, miR-363-3p and Mcl1 gene regulations. Stimulation of receptor tyrosine kinase (RTK) by growth factors (GFs) phosphorylates RAS that subsequently activates RAF. Then, RAF phosphorylates MEK. Consequently, activated MEK phosphorylates ERK downstream effector, leading to the MALAT1 gene expression. MALAT1 is a negative regulator of Mcl1 mRNA by sponging of miR-363-3p. In addition, MALAT1 leads to Axin1 and APC downregulation and Wnt/β-catenin signaling pathway activation. Stable β-catenin translocates from the cytoplasm to the nucleus and promotes cyclin D1 gene expression.

Methylation-dependent Tissue Factor Suppression Contributes to the Reduced Malignancy of IDH1-mutant Gliomas.

Gliomas with isocitrate dehydrogenase 1 mutations (IDH1mut) are less aggressive than IDH1 wild-type (IDH1wt) gliomas and have global genomic hypermethylation. Yet it is unclear how specific hypermethylation events contribute to the IDH1mut phenotype. Previously, we showed that the gene encoding the procoagulant tissue factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas, correlating with greatly reduced thrombosis in patients with IDH1mut glioma. Because TF also increases the aggressiveness of many cancers, the current study explored the contribution of TF suppression to the reduced malignancy of IDH1mut gliomas.Experimental Design: TF expression was manipulated in patient-derived IDH1mut and IDH1wt glioma cells, followed by evaluation of in vitro and in vivo behavior and analyses of cell signaling pathways. A demethylating agent, decitabine, increased F3 transcription and TF-dependent coagulative activity in IDH1mut cells, but not in IDH1wt cells. TF induction enhanced the proliferation, invasion, and colony formation of IDH1mut cells, and increased the intracranial engraftment of IDH1mut GBM164 from 0% to 100% (P = 0.0001). Conversely, TF knockdown doubled the median survival of mice engrafted with IDH1wt/EGFRvIIIamp GBM6, and caused complete regression of IDH1wt/EGFRamp GBM12 (P = 0.001). In vitro and in vivo effects were linked to activation of receptor tyrosine kinases (RTK) by TF through a Src-dependent intracellular pathway, even when extracellular RTK stimulation was blocked. TF stimulated invasion predominately through upregulation of β-catenin. These data show that TF suppression is a component of IDH1mut glioma behavior, and that it may therefore be an attractive target against IDH1wt gliomas.

PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs)

Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells.

Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.

Interaction with Shc prevents aberrant Erk activation in the absence of extracellular stimuli

Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a new mechanism by which the adaptor protein Shc directly binds the MAP kinase Erk, thus preventing its activation in the absence of extracellular stimuli. The Shc-Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex forms through unique binding sites on both the Shc PTB domain and the N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc-induced through interaction with the phosphorylated receptor-releases Erk, allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered a tumor suppressor in human cells.

Frs2α enhances fibroblast growth factor-mediated survival and differentiation in lens development

Most growth factor receptor tyrosine kinases (RTKs) signal through similar intracellular pathways, but they often have divergent biological effects. Therefore, elucidating the mechanism of channeling the intracellular effect of RTK stimulation to facilitate specific biological responses represents a fundamental biological challenge. Lens epithelial cells express numerous RTKs with the ability to initiate the phosphorylation (activation) of Erk1/2 and PI3-K/Akt signaling. However, only Fgfr stimulation leads to lens fiber cell differentiation in the developing mammalian embryo. Additionally, within the lens, only Fgfrs activate the signal transduction molecule Frs2α. Loss of Frs2α in the lens significantly increases apoptosis and decreases phosphorylation of both Erk1/2 and Akt. Also, Frs2α deficiency decreases the expression of several proteins characteristic of lens fiber cell differentiation, including Prox1, p57(KIP2), aquaporin 0 and β-crystallins. Although not normally expressed in the lens, the RTK TrkC phosphorylates Frs2α in response to binding the ligand NT3. Transgenic lens epithelial cells expressing both TrkC and NT3 exhibit several features characteristic of lens fiber cells. These include elongation, increased Erk1/2 and Akt phosphorylation, and the expression of β-crystallins. All these characteristics of NT3-TrkC transgenic lens epithelial cells depend on Frs2α. Therefore, tyrosine phosphorylation of Frs2α mediates Fgfr-dependent lens cell survival and provides a mechanistic basis for the unique fiber-differentiating capacity of Fgfs on mammalian lens epithelial cells.

Dynamic Reprogramming of the Kinome in Response to Targeted MEK Inhibition in Triple-Negative Breast Cancer

Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combinationtherapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

Abstract CN03-04: PI3K pathway inhibitors: How deep should we go?

Abstract PI3K pathway activation is commonly observed in human cancer and is critical for tumor progression and resistance to other antineoplastic drugs, both cytotoxic chemotherapy and targeted agents. Several molecular aberrations affecting key components of this pathway have been described, including genetic mutations and amplifications (PIK3CA, AKT) and loss of function of negative regulators (PTEN). Rapalogs were the first inhibitors of downstream effectors of the PI3K pathway to enter the clinic, although with limited antitumor activity. Preclinical studies show that inhibition of mTOR relieves feedback inhibition of receptor tyrosine kinases (RTK), leading to subsequent PI3K/AKT activation. Second generation PI3K pathway inhibitors are still in early phases of clinical development and include: (a) pure pan-PI3K inhibitors; (b) dual PI3K/mTOR inhibitors; (c) AKT inhibitors; (d) mTOR complex 1 and 2 inhibitors; and (d) isoform-specific PI3K inhibitors. Partial responses and prolonged disease stabilization have been reported in multiple tumor types, such as breast, gynecological, prostate, lung, mesotheliomas, sarcomas and lymphomas, although in a lower than expected rate. Pharmacodynamic (PD) studies showed reduction of activation of key pathway readouts both in tumor and surrogate tissues, significant decline in proliferation markers and reduction of glucose avidity on FDG-PET scans, confirming that the targets are being hit. Clinical benefit has not been restricted to patients whose tumor harbor PI3K pathway activation and the PD markers have not shown a clear predictive value. Importantly, the antitumor activity of isoform-specific PI3K inhibitors in PIK3CA mutants is still unknown. Nevertheless, it is now clear that PI3K pathway inhibition increases the activity of MEK/ERK signaling as a result of negative feedback and compensatory RTK stimulation, confirming the adaptive capabilities of oncogenic networks. Therefore, the next generation of trials with PI3K pathway inhibitors should focus in combination strategies, including other targeted therapies, such as BRAF, MEK, EGFR and HER2 inhibitors. For a clear definition of the future role of PI3K pathway inhibitors in the clinic, additional PD studies in these combination trials are of paramount importance. In the case of lack of clinical activity, if the pathway is not effectively inhibited, investigators should go for improved dosing and schedules, provided that toxicity is acceptable. On the other hand, if lack of clinical activity is seen despite effective pathway inhibition, a greater understanding of tumor biology is needed. Results of these studies are eagerly anticipated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr CN03-04.

PI(3)K/PTEN/AKT pathway

PI(3)K/PTEN/AKT pathway

Spatial organization of transmembrane receptor signalling

The spatial organization of transmembrane receptors is a critical step in signal transduction and receptor trafficking in cells. Transmembrane receptors engage in lateral homotypic and heterotypic cis-interactions as well as intercellular trans-interactions that result in the formation of signalling foci for the initiation of different signalling networks. Several aspects of ligand-induced receptor clustering and association with signalling proteins are also influenced by the lipid composition of membranes. Thus, lipid microdomains have a function in tuning the activity of many transmembrane receptors by positively or negatively affecting receptor clustering and signal transduction. We review the current knowledge about the functions of clustering of transmembrane receptors and lipid-protein interactions important for the spatial organization of signalling at the membrane.

Phosphoinositide 3-Kinase Pathway Activation in Phosphate and Tensin Homolog (PTEN)-deficient Prostate Cancer Cells Is Independent of Receptor Tyrosine Kinases and Mediated by the p110β and p110δ Catalytic Subunits

Class IA phosphoinositide 3-kinase (PI3K) p110 catalytic subunits are activated upon Src homology 2 domain-mediated binding of their p85 regulatory subunits to tyrosine-phosphorylated pYXXM motifs in receptor tyrosine kinases (RTKs) or adaptor proteins. The PI3K pathway is activated by phosphate and tensin homolog (PTEN) loss in most prostate cancers (PCa), but the contribution of upstream RTKs that may be targeted therapeutically has not been assessed. Immunoblotting of p85-associated proteins in serum-starved PTEN-deficient LNCaP and C4-2 PCa cells showed a small set of discrete tyrosine-phosphorylated proteins, but these proteins were not recognized by an anti-pYXXM motif antibody and were not found in PTEN-deficient PC3 PCa cells. LC/MS/MS using label-free proteomics and immunoblotting showed that p85 was associated primarily with p110beta and p110delta. An interaction with ErbB3 was also detected but was independent of ErbB3 tyrosine phosphorylation and was not required for basal PI3K activity. Basal tyrosine phosphorylation of p110beta and p110delta could be blocked by c-Src inhibitors, but this did not suppress PI3K activity, which was similarly independent of Ras. Basal PI3K activity was mediated by p110beta in PC3 cells and by both p110beta and p110delta in LNCaP cells, whereas p110alpha was required for PI3K activation in response to RTK stimulation by heregulin-beta1. These findings show that basal PI3K activity in PTEN-deficient PCa cells is RTK-independent and can be mediated by p110beta and p110delta. Increased p110beta expression in PCa may be required for RTK-independent PI3K pathway activation in adult prostate epithelium with genetic or epigenetic PTEN down-regulation.

Deubiquitinase Activities Required for Hepatocyte Growth Factor-Induced Scattering of Epithelial Cells

SummaryThe scattering response of epithelial cells to activation of the Met receptor tyrosine kinase represents one facet of an “invasive growth” program [1, 2]. It is a complex event that incorporates loss of cell-cell adhesion, morphological changes, and cell motility. Ubiquitination is a reversible posttranslational modification that may target proteins for degradation or coordinate signal transduction pathways [3, 4]. There are ∼79 active deubiquitinating enzymes (DUBs) predicted in the human genome [5, 6]. Here, via a small interfering RNA (siRNA) library approach, we have identified 12 DUBs that are necessary for aspects of the hepatocyte growth factor (HGF)-dependent scattering response of A549 cells. Different phenotypes are evident that range from full loss of scattering, similar to receptor knockdown (e.g., USP30, USP33, USP47), to loss of cell-cell contacts even in the absence of HGF but defective motility (e.g., USP3, ATXN3L). The knockdowns do not incur defective receptor, phosphatidylinositol 3-kinase, or MAP kinase activation. Our data suggest widespread involvement of the ubiquitin system at multiple stages of the Met activation response, implying significant crosstalk with phosphorylation-based transduction pathways. Development of small-molecule inhibitors of particular DUBs may offer a therapeutic approach to contain metastasis.

Membrane protein targeting to the MVB/lysosome.

Cells sense and respond to their environment largely through the function of receptors, transporters, and channels within the plasma membrane. For cells to appropriately respond to environmental cues, they must maintain the proper protein complement at the cell surface. This involves both delivering proteins to the cell surface and removing them when necessary. The multivesicular body (MVB) sorting reaction within the endocytic pathway provides an important cellular mechanism for terminating the function of integral membrane proteins destined for degradation within the lysosome (reviewed in 1–3). The MVB sorting machinery recognizes a subset of endocytic cargoes and concentrates them into regions within the endosomal membrane. These sections then bud as small vesicles into the lumen of the endosome, giving the endosome a multivesicular appearance by electron microscopy. Subsequent fusion of the MVB with the lysosome delivers these intralumenal vesicles to the hydrolytic environment of the lysosome, where the lipid and protein contents of the vesicles are degraded (reviewed in 4). The topology of membrane invagination into the endosomal lumen (exvagination from the cytosol) during MVB sorting is similar to the budding process whereby enveloped viruses, including HIV-1 and Ebola virus, egress from the cell (Figure 1). Viral structural proteins, such as Gag from HIV-1 and VP40 from Ebola virus, utilize the MVB sorting machinery to bud either from the plasma membrane or into an intracellular compartment for subsequent release from the cell (reviewed in 5,6). Therefore, the MVB sorting machinery is important both for viral replication as well as for lysosomal delivery of endocytosed proteins. In addition, a number of recent studies have shown a role for the MVB sorting machinery in late steps of cytokinesis 7–13, emphasizing the importance of this machinery for diverse cellular processes of similar membrane topology. Figure 1 Roles for cellular machinery in MVB sorting and viral particle formation MVB sorting is conserved throughout eukaryotes, and studies in both yeast and mammalian systems have identified a series of trans-acting factors that mediate this reaction (reviewed in 2,14,15). The endosomal sorting complexes required for transport (ESCRTs) and associated proteins constitute the majority of this machinery. Cargo recognition is mediated by interactions with the Vps27/Hrs-Hse1/STAM complex as well as with ESCRT-I (Vps23/Tsg101, Vps28, Vps37 and Mvb12) 16–20. ESCRT-II (Vps22, Vps25, Vps36) appears to function downstream or in parallel to ESCRT-I, and is also believed to interact with cargo based on ubiquitin-binding domain activity within Vps36 21–25. ESCRT-II additionally serves to facilitate the recruitment and assembly of ESCRT-III subunits (Vps20/CHMP6, Snf7/CHMP4, Vps2/CHMP2, Vps24/CHMP3, Did2/CHMP1, Vps60/CHMP5) into a polymer 25–28. Interactions between ESCRT-III and the Vps4-Vta1 complex then promote disassembly of ESCRT-III and may be linked to the completion of intralumenal vesicle (ILV) formation 29–35. The concerted actions of these multimeric complexes serve to concentrate and deliver MVB cargoes into ILVs of the endosome for their eventual degradation within the lysosome. While lysosomal degradation provides a mechanism for the long-term attenuation of transmembrane proteins (such as cell surface receptors, transporters, and channels), sorting into the MVB appears to abrogate their functions prior to degradation. Sorting activated receptors into the MVB sequesters their cytoplasmic kinase domains into the endosomal lumen and away from cytoplasmic substrates, effectively terminating signaling from activated receptors. A well-studied example illustrating the importance of this reaction is the sorting of activated epidermal growth factor receptor (EGFR) (reviewed in 36,37). Defects that prevent EGFR sorting into intralumenal vesicles are oncogenic due to receptor hyperactivity, and this oncogenic activity is attributed to deficiencies in both receptor sequestration and lysosomal degradation (reviewed in 38,39). While MVB sorting can have an important impact on signaling, signaling can also impact MVB sorting. EGFR delivery into the MVB pathway is accelerated in response to EGF stimulation; moreover, EGF stimulation actually promotes MVB biogenesis 40,41. The Hrs-STAM complex is phosphorylated after stimulation of receptor tyrosine kinases 42–44, and this phosphorylation impacts machinery levels and receptor degradation 45. Therefore, it is important to understand the mechanisms by which signaling and MVB sorting impact each other and how flux through the MVB pathway is regulated.