Protein Shc Research Articles

Integrin β3 Phosphorylation Dictates Its Complex with the Shc Phosphotyrosine-binding (PTB) Domain

Adaptor protein Shc plays a key role in mitogen-activated protein kinase (MAPK) signaling pathway, which can be mediated through a number of different receptors including integrins. By specifically recognizing the tyrosine-phosphorylated integrin β(3), Shc has been shown to trigger integrin outside-in signaling, although the structural basis of this interaction remains nebulous. Here we present the detailed structural analysis of Shc phosphotyrosine-binding (PTB) domain in complex with the bi-phosphorylated β(3)integrin cytoplasmic tail (CT). We show that this complex is primarily defined by the phosphorylation state of the integrin C-terminal Tyr(759), which fits neatly into the classical PTB pocket of Shc. In addition, we have identified a novel binding interface which concurrently accommodates phosphorylated Tyr(747) of the highly conserved NPXY motif of β(3). The structure represents the first snapshot of an integrin cytoplasmic tail bound to a target for mediating the outside-in signaling. Detailed comparison with the known Shc PTB structure bound to a target TrkA peptide revealed some significant differences, which shed new light upon the PTB domain specificity.

The p66shc Gene Expression in Peripheral Blood Monocytes Is Increased in Patients With Coronary Artery Disease

The p66(shc) protein has been shown to control cellular responses to oxidative stress, being involved in atherosclerosis in animal models. However, the relationship between the p66(shc) gene expression levels and coronary artery disease (CAD) in humans remains unknown. In this study, we examined whether the p66(shc) gene expression in peripheral blood monocytes (PBMs) was increased in patients with CAD, compared with age- and sex-matched subjects without CAD. We hypothesize that the p66(shc) gene expression level in PBMs is increased in patients with CAD. Forty consecutive Japanese subjects who underwent coronary angiography for suspected CAD were enrolled in this study. The p66(shc) gene expression levels in PBMs were quantitatively measured by real-time reverse transcription-polymerase chain reactions. Uni- and multivariate analyses were applied for the correlates of CAD. CAD was diagnosed if there was > 75% obstruction of at least 1 major coronary artery or a history of percutaneous coronary intervention. There were no significant differences of blood chemistries and clinical characteristics between the patients with and without CAD, except the number of subjects who were on hypertension medication. The p66(shc) gene expression levels in PBMs were significantly higher in CAD patients compared with non-CAD subjects. Multiple stepwise regression analysis revealed that the p66(shc) gene expression levels and hypertension medication were independently related to CAD (R(2)=0.287). Further, the p66(shc) gene expres- sion levels were significantly increased (P < 0.05) in proportion to the number of diseased vessels. The present study is the first demonstration that increased the p66(shc) gene expression in PBMs is independently associated with CAD in Japanese subjects. The p66(shc) gene expression level in PBMs may be a novel biomarker of CAD in humans.

Protein kinase C-δ regulates the subcellular localization of Shc in H2O2-treated cardiomyocytes

Protein kinase C-δ (PKCδ) exerts important cardiac actions as a lipid-regulated kinase. There is limited evidence that PKCδ also might exert an additional kinase-independent action as a regulator of the subcellular compartmentalization of binding partners such as Shc (Src homologous and collagen), a family of adapter proteins that play key roles in growth regulation and oxidative stress responses. This study shows that native PKCδ forms complexes with endogenous Shc proteins in H(2)O(2)-treated cardiomyocytes; H(2)O(2) treatment also leads to the accumulation of PKCδ and Shc in a detergent-insoluble cytoskeletal fraction and in mitochondria. H(2)O(2)-dependent recruitment of Shc isoforms to cytoskeletal and mitochondrial fractions is amplified by wild-type-PKCδ overexpression, consistent with the notion that PKCδ acts as a signal-regulated scaffold to anchor Shc in specific subcellular compartments. However, overexpression studies with kinase-dead (KD)-PKCδ-K376R (an ATP-binding mutant of PKCδ that lacks catalytic activity) are less informative, since KD-PKCδ-K376R aberrantly localizes as a constitutively tyrosine-phosphorylated enzyme to detergent-insoluble and mitochondrial fractions of resting cardiomyocytes; relatively little KD-PKCδ-K376R remains in the cytosolic fraction. The aberrant localization and tyrosine phosphorylation patterns for KD-PKCδ-K376R do not phenocopy the properties of native PKCδ, even in cells chronically treated with GF109203X to inhibit PKCδ activity. Hence, while KD-PKCδ-K376R overexpression increases Shc localization to the detergent-insoluble and mitochondrial fractions, the significance of these results is uncertain. Our studies suggest that experiments using KD-PKCδ-K376R overexpression as a strategy to competitively inhibit the kinase-dependent actions of native PKCδ or to expose the kinase-independent scaffolding functions of PKCδ should be interpreted with caution.

Tarp regulates earlyChlamydia-induced host cell survival through interactions with the human adaptor protein SHC1

Tarp regulates early<i>Chlamydia</i>-induced host cell survival through interactions with the human adaptor protein SHC1

Involvement of Grb2 Adaptor Protein in Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK)-mediated Signaling and Anaplastic Large Cell Lymphoma Growth

Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr(160) phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr(152-156), Tyr(567), and a proline-rich region, Pro(415-417). Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.

Tarp regulates early Chlamydia-induced host cell survival through interactions with the human adaptor protein SHC1.

Many bacterial pathogens translocate effector proteins into host cells to manipulate host cell functions. Here, we used a protein microarray comprising virtually all human SRC homology 2 (SH2) and phosphotyrosine binding domains to comprehensively and quantitatively assess interactions between host cell proteins and the early phase Chlamydia trachomatis effector protein translocated actin-recruiting phosphoprotein (Tarp), which is rapidly tyrosine phosphorylated upon host cell entry. We discovered numerous novel interactions between human SH2 domains and phosphopeptides derived from Tarp. The adaptor protein SHC1 was among Tarp's strongest interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to tumor necrosis factor-induced apoptosis. Collectively, our findings reveal a critical role for SHC1 in early C. trachomatis-induced cell survival and suggest that Tarp functions as a multivalent phosphorylation-dependent signaling hub that is important during the early phase of chlamydial infection.

Ubiquitination of α5β1 Integrin Controls Fibroblast Migration through Lysosomal Degradation of Fibronectin-Integrin Complexes

Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction of the endocytosed fibronectin receptor, alpha 5 beta 1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the alpha 5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with alpha 5 beta 1 integrin. Importantly, we demonstrate that both alpha 5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of alpha 5 beta 1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and alpha 5 beta 1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.

Functional profiling of signal transduction pathway proteins in gastric cancer (GC) patients.

e14555 Background: With the advent of molecularly targeted therapy in cancer care, identification of target population for target drug is of utmost importance. Gastric cancer has not been extensively profiled for transduction pathway proteins. Here we report the prevalence of HER1, HER2, p95HER2, HER3, IGF1-R, c-MET, PI3K, Shc, VEGFR, panCK, and other signal transduction pathway protein expression and their levels of activation in GC patients. Methods: From 2004 to 2006, 500 GC surgical specimens were procured at Samsung Medical Center. All of the specimens were collected during gastrectomy and were appropriately frozen at – 80 °C. All of the specimens were analyzed at Prometheus, CA, USA. We applied COPIA to investigate the levels of expression and activation of signaling proteins in frozen tissues collected from GC patients. The Collaborative Proximity Immunoassay (COPIA) is a multiplexed protein microarray platform that utilizes the formation of a unique immuno-complex requiring co-localization of two detector-antibodies. The detector-antibodies are conjugated with corresponding channeling-enzymes, glucose oxidase (GO) and horseradish peroxidase (HRP). Once target proteins are bound by the capture antibodies, the channeling events between GO and HRP in proximity enables the profiling of the target proteins with extreme sensitivity. COPIA delivers extremely high analytical specificity as it requires multiple entities within target specific proximity for the signal generation/amplification. COPIA can also be configured for each specific target protein to allow differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., HER2). Results: We will be reporting on the prevalence of HER1, HER2, p95HER2, HER3, IGF1-R, c-MET, PI3K, Shc, VEGFR, panCK, and other signal transduction pathway protein expression and their levels of activation in 500 GC patients. Conclusions: The treatment of recurrent or metastatic GC may be optimized by functional profiling of GC. In addition, the incidence of alteration in transduction pathway proteins may guide oncologists for further clinical development of targeted therapies or combined therapies. No significant financial relationships to disclose.

Characterization of HER2 functional profiling in fine needle aspirates (FNA) in patients with metastatic breast cancer (MBC).

e21029 Background: The Collaborative Proximity Immunoassay (COPIA) is a multiplexed microarray platform that utilizes the formation of a unique immuno-complex requiring co-localization of two detector-antibodies. The detector-antibodies are conjugated with corresponding channeling-enzymes for proximity mediated signal generation/amplification for the expression and activation profiling of HER1, HER2, p95HER2, HER3, IGF1-R, c-MET, PI3K, Shc, VEGFR, CK, and other signal transduction proteins. Here we report HER2 expression and activation profiling in fine needle aspirate (FNA) biopsy samples from MBC patients in an ongoing investigation with this novel technology. Methods: FNA samples were collected from metastatic sites from female patients with progressive, Stage IIIB/IV breast cancer (n = 25, baseline age 42 ± 13yr). Patients with various clinical primary ER/PR/HER2 statuses by IHC staining were allowed in the study. The ER/PR/HER2 status of the primary tumor sample was obtained by the local sites. Expression and activation status for HER2 and the other signal transduction Proteins (STP) were measured using COPIA on FNA samples. Results: Data from the FNA samples of the first 10 patients showed that 5 (50%) were HER2 activated (pHER2+), 2 (20%) were HER2 over-expressed (tHER2+), pHER2+ and p95HER2 over-expressed. 7 of 10 FNA samples expressed weak to moderate levels of tHER2 with different pHER2 levels. HER2 status (expression/activation) was negative in 1 patient. The concordance between the COPIA-derived data from the FNA and the primary tumor IHC will be evaluated. In addition, over-expression and/or activation of other STP including HER1, HER3, IGF1- R, c-MET will be measured in the entire cohort using COPIA. Conclusions: COPIA assays can be used to quantitate expression and activation of STPs in FNA specimens. These results need to be further analyzed for their potential value to inform treatment decisions in relapsed breast cancer patients. No significant financial relationships to disclose.

Abstract 4019: Functional profiling of signal transduction pathway proteins in gastric cancer patients

Abstract Although gastrectomy is the only curative treatment in gastric cancer (GCA) patients, a high recurrence rate ranging from 40 ∼ 60% following curative surgery still accounts for poor overall survival. In order to further improve survival, there is an urgent need to identify reliable molecular prognostic markers for survival or recurrence following adjuvant chemoradiation therapy, which will evolve to the development of patient-tailored treatment strategies. The improvement of gastric cancer therapy will eventually depend on novel therapeutic approaches targeting molecules critical for cancer proliferation. As the transduction pathway proteins function in cell signaling and transmit signals regulating growth, differentiation, adhesion, migration, and apoptosis, they have become targets of various therapeutic agents. Hence, we have developed a multiplexed immunoassay platform for functional profiling of the transduction pathway proteins. The COllaborative Proximity ImmunoAssay (COPIA) is a multiplexed protein microarray platform that utilizes the formation of a unique immuno-complex requiring co-localization of two detector-antibodies. The detector-antibodies are conjugated with corresponding channeling-enzymes, glucose oxidase (GO) and horseradish peroxidase (HRP). Once target proteins are bound by the capture antibodies, the channeling events between GO and HRP in proximity enables the profiling of the target proteins with extreme sensitivity. COPIA delivers extremely high analytical specificity as it requires multiple entities within target specific proximity for the signal generation/amplification. COPIA can also be configured for each specific target protein to allow differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., HER2). We applied COPIA to investigate the levels of expression and activation of signaling proteins in frozen tissues collected from GCA patients. Here we report the prevalence of HER1, HER2, p95HER2, HER3, IGF1-R, c-MET, PI3K, Shc, VEGFR, panCK, and other signal transduction pathway protein expression and their levels of activation in GCA patients. The improvement of gastric cancer therapy will eventually depend on novel therapeutic approaches targeting specific markers identified by functional profiling. As the disease profile often shifts in recurrent cancers and under different therapeutic pressure, clinical information obtained by analyzing samples (often with limited availability) obtained from ‘evolving / heterogeneous disease’ can help clinicians adjust their disease treatment options for each patient according to ‘personal’ cancer profile-shift. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4019.

364 THE TUMORSUPPRESSOR PTEN INFLUENCES METASTASES OF CLEAR CELL RENAL CELL CARCINOMA (CCRCC) BY DEPHOSPHORYLATION OF SHC

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2010364 THE TUMORSUPPRESSOR PTEN INFLUENCES METASTASES OF CLEAR CELL RENAL CELL CARCINOMA (CCRCC) BY DEPHOSPHORYLATION OF SHC Walburgis Brenner, Elke Schneider, Dirk Prawitt, Frederik C. Roos, Ekkehart Lausch, Christian Hampel, and Joachim W. Thüroff Walburgis BrennerWalburgis Brenner Mainz, Germany More articles by this author , Elke SchneiderElke Schneider Mainz, Germany More articles by this author , Dirk PrawittDirk Prawitt Mainz, Germany More articles by this author , Frederik C. RoosFrederik C. Roos Mainz, Germany More articles by this author , Ekkehart LauschEkkehart Lausch Freiburg, Germany More articles by this author , Christian HampelChristian Hampel Mainz, Germany More articles by this author , and Joachim W. ThüroffJoachim W. Thüroff Mainz, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.431AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Metastasis is regulated by intracellular processes. Among others, the tumor suppressor PTEN (phosphatase and tensin homologue deleted from chromosome 10) is involved in regulation of cell migration. PTEN is a dual specific phosphatase using the phospholipid PIP3 and the proteins FAK and Shc as its substrate. These signaling mediators cause enhanced cell motility, although the role of the different PTEN activities in this process is unknown. In this study we analyzed the role of PTEN in the migration of renal cancer cells. METHODS We quantified PTEN in 135 ccRCC by Western blot. The renal carcinoma cell line 786-0, which is inherent PTEN negative, was transfected by the functional PTEN gene. For analyses of the PTEN activities, transfections with mutated PTEN genes were performed. This led to a loss of the lipid phosphatase activity (G129E) and/or the protein phosphatase activity (C124S or delta exon 3), respectively. The activity of PTEN depends on its ability to bind to the cell membrane by the PDZ binding domain. Therefore, a PTEN gene construct was also created using an inactivated PDZ binding domain (delta PDZ). Cell proliferation was measured by a BrdU incorporation assay. Cell migration was analyzed in a Boyden chemotaxis chamber with fibronectin (10 μg/mL) as chemotaxin. The molecular effect of transfected PTEN mutants was analyzed by quantifying the phosphorylation of PTEN downstream targets Akt, FAK and Shc by Western blot. RESULTS In renal tumor tissue, we found a statistically significant lower PTEN expression in patients dying within 5 years after surgery, compared to those surviving this time period. Renal cancer cells transfected with the functional PTEN gene showed a profoundly diminished migration. This effect depended on the protein phosphatase activity and PDZ binding, since transfection with a mutated PTEN gene leading to a loss of protein phosphatase activity or of the PDZ binding domain, but not of lipid phosphatase activity, abolished this effect. Involved in this effect is Shc, since the protein phosphatase reduced PTEN mutant abolished the PTEN dependent reduction of phospho-Shc, but not of phospho-FAK. CONCLUSIONS PTEN plays a critical role in metastasis of RCC, depending on protein phosphatase activity via Shc and the PDZ binding domain. This new insight could be the basis of additional approaches complementing current cancer therapy in metastasing RCC. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e144 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Walburgis Brenner Mainz, Germany More articles by this author Elke Schneider Mainz, Germany More articles by this author Dirk Prawitt Mainz, Germany More articles by this author Frederik C. Roos Mainz, Germany More articles by this author Ekkehart Lausch Freiburg, Germany More articles by this author Christian Hampel Mainz, Germany More articles by this author Joachim W. Thüroff Mainz, Germany More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

The PTB domain of ShcA couples receptor activation to the cytoskeletal regulator IQGAP1

Adaptor proteins respond to stimuli and recruit downstream complexes using interactions conferred by associated protein domains and linear motifs. The ShcA adaptor contains two phosphotyrosine recognition modules responsible for binding activated receptors, resulting in the subsequent recruitment of Grb2 and activation of Ras/MAPK. However, there is evidence that Grb2-independent signalling from ShcA has an important role in development. Using mass spectrometry, we identified the multidomain scaffold IQGAP1 as a ShcA-interacting protein. IQGAP1 and ShcA co-precipitate and are co-recruited to membrane ruffles induced by activated receptors of the ErbB family, and a reduction in ShcA protein levels inhibits the formation of lamellipodia. We used NMR to characterize a direct, non-canonical ShcA PTB domain interaction with a helical fragment from the IQGAP1 N-terminal region that is pTyr-independent. This interaction is mutually exclusive with binding to a more conventional PTB domain peptide ligand from PTP-PEST. ShcA-mediated recruitment of IQGAP1 may have an important role in cytoskeletal reorganization downstream of activated receptors at the cell surface.

Combined therapy with disintegrin and melphalan as a new strategy in inhibition of endometrial cancer cell line (Ishikawa) growth.

Endometrial cancer is one of the most frequently diagnosed cancer in females with prevalence of 22 in 100,000 women. The etiology of the cancer remains unclear. Despite significant progress towards understanding the patho-mechanism of the disease, effective treatment is still lacking. The results of the study suggest that combined treatment of Ishikawa cells for 24 h with disintegrin and then for 24 h with melphalan severely inhibits important biological functions of the cells. We showed that such strategy have a potent cytotoxic effect. The mechanism of process undergoes probably through inhibition of integrin - dependent signaling. In this study we shown down regulation of Shc and FAK proteins in cells treated with echistatin and melphalan. It suggests that signaling pathways that involve Shc and FAK participation may represent target for antineoplastic strategy. The functional significance of the combined treatment of Ishikwa cells with echistatin and melphalan was found at the level of collagen biosynthesis. Decreased biosynthesis of collagen in extracellular matrix may suppress cell growth and induce apoptosis. The treatment with echistatin and melphalan also showed decreased expression of IGF receptor in comparison to the cells treated with both compounds separately. The data presented suggest that combined therapy with disintegrin - echistatin and alkyalting drug - mephalan may represent a new approach to more effective and safe cancer therapy.

Insulin receptor substrates-5 and -6 are poor substrates for the insulin receptor

Insulin receptor substrates (IRS)-5 and -6 are two recently identified members of the IRS family. We investigated their roles as insulin receptor substrates and compared them with Src-homology-2-containing (Shc) protein, a well-established substrate. Bioluminescence resonance energy transfer (BRET) experiments showed no interaction between the receptor and IRS-5, while interaction with IRS-6 was not enhanced by insulin. By contrast, Shc showed an insulin-induced BRET response, as did a truncated form of IRS-1 (1-262). While Shc was heavily phosphorylated after stimulation of the insulin receptor, IRS-5 and -6 showed very weak phosphorylation levels. These results suggest that, although these two adaptors have previously been proposed as substrates for the insulin receptor, they are poor substrates for the insulin receptor. This calls into question their relevance to insulin signalling.

Positive and negative regulation of antigen receptor signaling by the Shc family of protein adapters

The Shc adapter family includes four members that are expressed as multiple isoforms and participate in signaling by a variety of cell-surface receptors. The biological relevance of Shc proteins as well as their variegated function, which relies on their highly conserved modular structure, is underscored by the distinct and dramatic phenotypic alterations resulting from deletion of individual Shc isoforms both in the mouse and in two model organisms, Drosophila melanogaster and Caenorhabditis elegans. The p52 isoform of ShcA couples antigen and cytokine receptors to Ras activation in both lymphoid and myeloid cells. However, the recognition of the spectrum of activities of p52ShcA in the immune system has been steadily expanding in recent years to other fundamental processes both at the cell and organism levels. Two other Shc family members, p66ShcA and p52ShcC/Rai, have been identified recently in T and B lymphocytes, where they antagonize survival and attenuate antigen receptor signaling. These developments reveal an unexpected and complex interplay of multiple Shc proteins in lymphocytes.

The Adaptor Protein Shc Plays a Key Role during Early B Cell Development

The adaptor protein Shc is phosphorylated downstream of many cell surface receptors, including Ag and cytokine receptors. However, the role of Shc in B cell development has not been addressed. Here, through conditional expression of a dominant negative Shc mutant and conditional loss of Shc protein expression, we tested a role for Shc during early B lymphopoiesis. We identified a requirement for Shc beginning at the transition from the pre-pro-B to pro-B stage, with a strong reduction in the number of pre-B cells. This developmental defect is due to increased cell death rather than impaired proliferation or commitment to the B lineage. Additional studies suggest a role for Shc in IL-7-dependent signaling in pro-B cells. Shc is phosphorylated in response to IL-7 stimulation in pro-B cells, and pro-B cells from mice with impaired Shc signaling display increased apoptosis. Together, these data demonstrate a critical role for Shc in early B lymphopoiesis with a requirement in early B cell survival. In addition, we also identify Shc as a required player in signaling downstream of the IL-7R in early B cells.

Cellular BRET assay suggests a conformational rearrangement of preformed TrkB/Shc complexes following BDNF-dependent activation

We developed a cellular Bioluminescent Resonance Energy Transfer (BRET) assay based on the interaction of TrkB fused to Renilla luciferase with the intracellular adaptor protein Shc fused to Enhanced Yellow Fluorescent Protein (EYFP). The TrkB agonist Brain Derived Neurotrophic Factor (BDNF) induced a maximum BRET signal as of 10min with an EC50 value of 1.4nM, similar to the other endogenous agonists NT-3 and NT-4/5, 1.5nM and 0.34nM, respectively. Interestingly, measure of the BRET signal with increasing expression of Shc-EYFP, in the presence or absence of BDNF, suggested a conformational change of preformed TrkB/Shc complexes rather than Shc recruitment. Furthermore, the Y516F TrkB mutant deficient to bind Shc as well as the kinase-dead K572R TrkB mutant was unable to respond to BDNF and exhibited a lower basal BRET signal than that of the wild-type TrkB receptor, again suggesting a preformed complex with constitutive activity. The double YY706/707FF TrkB mutant in the kinase activation loop also showed reduced basal activity but surprisingly kept its capacity to enhance BDNF-induced interaction with Shc, though with less efficacy. The Trk selective kinase inhibitors K252a and BMS-9 blocked BDNF-induced BRET signal with similar potency (100–150nM), the preferential c-Met inhibitor PF-2341006 being one order of magnitude less potent. Remarkably, in the absence of BDNF, K252a and BMS-9 also reduced basal activity to the level of the Y516F TrkB mutant, suggesting that these compounds were able to reduce the TrkB constitutive activity. BRET responses of mutants and to kinase inhibitors thus reveal a complex level of interaction between TrkB and Shc and suggest that this BRET assay could be of great utility to test blockers of TrkB signalling in a physiologically relevant context.

Hopanoids Play a Role in Membrane Integrity and pH Homeostasis in Rhodopseudomonas palustris TIE-1

Sedimentary hopanes are pentacyclic triterpenoids that serve as biomarker proxies for bacteria and certain bacterial metabolisms, such as oxygenic photosynthesis and aerobic methanotrophy. Their parent molecules, the bacteriohopanepolyols (BHPs), have been hypothesized to be the bacterial equivalent of sterols. However, the actual function of BHPs in bacterial cells is poorly understood. Here, we report the physiological study of a mutant in Rhodopseudomonas palustris TIE-1 that is unable to produce any hopanoids. The deletion of the gene encoding the squalene-hopene cyclase protein (Shc), which cyclizes squalene to the basic hopene structure, resulted in a strain that no longer produced any polycyclic triterpenoids. This strain was able to grow chemoheterotrophically, photoheterotrophically, and photoautotrophically, demonstrating that hopanoids are not required for growth under normal conditions. A severe growth defect, as well as significant morphological damage, was observed when cells were grown under acidic and alkaline conditions. Although minimal changes in shc transcript expression were observed under certain conditions of pH shock, the total amount of hopanoid production was unaffected; however, the abundance of methylated hopanoids significantly increased. This suggests that hopanoids may play an indirect role in pH homeostasis, with certain hopanoid derivatives being of particular importance.

Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells

Tyrosine phosphatase non-receptor type substrate-1 (SHPS-1), a transmembrane protein, plays a vital role in cell migration and proliferation. Our previous studies have shown that insulin-like growth factor-I (IGF-I) stimulates SHPS-1 phosphorylation, leading to recruitment of SHP-2, c-Src, Shc, and Grb2.p85 to phosphorylated SHPS-1. Assembly of this signaling complex is required for optimal stimulation of both mitogen-activated protein and phosphatidylinositol 3-kinase pathways. The main aim of the present study was to identify novel proteins that interacted with the cytoplasmic domain of SHPS-1 (SHPS-1/CD) in response to IGF-I stimulation and define the role of these interactions in mediating specific biological functions. We performed a functional proteomic screening to identify SHPS-1 binding partners using combination of mRNA display and the tandem affinity purification-tag methods. Screening identified a number of proteins not previously known to interact with phosphorylated SHPS-1/CD. These novel SHPS-1 binding partners represent several functional categories including heat shock proteins, protein kinases and phosphatases, and proteins that regulate transcription or translation. In Vivo and in vitro studies suggested that most of the proteins bound to SHPS-1 via binding to one of the four SH2 domain containing proteins, SHP-2, CTK, SUPT6H, and STAT1, that directly bound to SHPS-1. Although the binding of most of these proteins to SHPS-1 was positively regulated by IGF-I, a few were negatively regulated, suggesting differential regulation of protein complexes assembled on SHPS-1/CD in response to IGF-I. Further studies showed that truncation of SHPS-1/CD significantly impaired IGF-I-dependent AKT signal transduction and subsequent biological functions including cell survival, protein synthesis, protein aggregation, and prevention of apoptosis. The results emphasize the importance of formation of SHPS-1 signaling complex induced by IGF-I and provide novel insights into our knowledge of the role of this molecular scaffold in regulation of IGF-I-stimulated signal transduction and biological actions.

Molecular Mechanism for SHP2 in Promoting HER2-induced Signaling and Transformation

The Src homology phosphotyrosyl phosphatase 2 (SHP2) plays a positive role in HER2-induced signaling and transformation, but its mechanism of action is poorly understood. Given the significance of HER2 in breast cancer, defining a mechanism for SHP2 in the HER2 signaling pathway is of paramount importance. In the current report we show that SHP2 positively modulates the Ras-extracellular signal-regulated kinase 1 and 2 and the phospoinositide-3-kinase-Akt pathways downstream of HER2 by increasing the half-life the activated form of Ras. This is accomplished by dephosphorylating an autophosphorylation site on HER2 that serves as a docking platform for the SH2 domains of the Ras GTPase-activating protein (RasGAP). The net effect is an increase in the intensity and duration of GTP-Ras levels with the overall impact of enhanced HER2 signaling and cell transformation. In conformity to these findings, the HER2 mutant that lacks the SHP2 target site exhibits an enhanced signaling and cell transformation potential. Therefore, SHP2 promotes HER2-induced signaling and transformation at least in part by dephosphorylating a negative regulatory autophosphorylation site. These results suggest that SHP2 might serve as a therapeutic target against breast cancer and other cancers characterized by HER2 overexpression.