丝氨酸蛋白酶HtrA2/Omi可参与调节线粒体中的适配蛋白p66Shc

目的:p66Shc在线粒体内积累和HtrA2/Omi的功能缺陷都能导致线粒体损伤,诱导细胞凋亡.探讨在线粒体中HtrA2对p66Shc的调控作用.方法:构建p66Shc和成熟型HtrA2的真核表达质粒,共转染HEK293T细胞,免疫印迹法(Western blot)检测p66Shc蛋白;构建原核表达质粒,大肠杆菌纯化蛋白,体外切割实验,SDS-PAGE分离后考马斯亮蓝染色检测;提取HtrA2功能缺陷小鼠( mnd2)大脑组织的线粒体,检测线粒体内p66Shc的蛋白水平.结果:细胞实验和体外实验证明HtrA2可以切割p66Shc,且在mnd2小鼠大脑中,线粒体内p66Shc的蛋白水平明显升高(P＜0.05).结论:p66Shc是HtrA2的直接底物,且HtrA2参与调节线粒体中p66Shc的蛋白水平,揭示了HtrA2发挥神经保护功能新的可能机制.     

p66SHC promotes apoptosis and antagonizes mitogenic signaling in T cells

Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc(-/-) T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.     

Involvement of the p66Shc protein in glucose transport regulation in skeletal muscle myoblasts

The p66(Shc) protein isoform regulates MAP kinase activity and the actin cytoskeleton turnover, which are both required for normal glucose transport responses. To investigate the role of p66(Shc) in glucose transport regulation in skeletal muscle cells, L6 myoblasts with antisense-mediated reduction (L6/p66(Shc)as) or adenovirus-mediated overexpression (L6/p66(Shc)adv) of the p66(Shc) protein were examined. L6/(Shc)as myoblasts showed constitutive activation of ERK-1/2 and disruption of the actin network, associated with an 11-fold increase in basal glucose transport. GLUT1 and GLUT3 transporter proteins were sevenfold and fourfold more abundant, respectively, and were localized throughout the cytoplasm. Conversely, in L6 myoblasts overexpressing p66(Shc), basal glucose uptake rates were reduced by 30% in parallel with a approximately 50% reduction in total GLUT1 and GLUT3 transporter levels. Inhibition of the increased ERK-1/2 activity with PD98059 in L6/(Shc)as cells had a minimal effect on increased GLUT1 and GLUT3 protein levels, but restored the actin cytoskeleton, and reduced the abnormally high basal glucose uptake by 70%. In conclusion, p66(Shc) appears to regulate the glucose transport system in skeletal muscle myoblasts by controlling, via MAP kinase, the integrity of the actin cytoskeleton and by modulating cellular expression of GLUT1 and GLUT3 transporter proteins via ERK-independent pathways.     

The p66Shc Adaptor Protein Controls Oxidative Stress Response in Early Bovine Embryos

The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2–4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2–4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.     

A Drosophila shc gene product is implicated in signaling by the DER receptor tyrosine kinase.

Antibodies to the human Shc adaptor protein were used to isolate a cDNA encoding a Drosophila Shc protein (dShc) by screening an expression library. The dshc gene, which maps to position 67B-C on the third chromosome, encodes a 45-kDa protein that is widely expressed throughout the Drosophila life cycle. In flies, the dShc protein physically associates with the activated Drosophila epidermal growth factor receptor homolog (DER) and is inducibly phosphorylated on tyrosine by DER. The 45-kDa dShc protein is closely related both in overall organization and in amino acid sequence (46% identity) to the 52-kDa mammalian Shc isoform. In addition to a C-terminal Src homology 2 (SH2) domain, dShc contains an N-terminal phosphotyrosine-binding (PTB) domain, which associates in vitro with the autophosphorylated DER receptor tyrosine kinase and with phosphopeptides containing an Asn-Pro-X-pTyr motif, where pTyr stands for phosphotyrosine. A potential binding site for the dShc PTB domain is located at Tyr-1228 of DER. These results indicate that the shc gene has been conserved in evolution, as have the binding properties of the Shc PTB and SH2 domains. Despite the close relationship between the Drosophila and mammalian Shc proteins, dShc lacks the high-affinity Grb2-binding site found in mammalian Shc, suggesting that Shc proteins may have functions in addition to regulation of the Ras pathway.     

p66SHC promotes T cell apoptosis by inducing mitochondrial dysfunction and impaired Ca2+ homeostasis

p66Shc, a redox enzyme that enhances reactive oxygen species (ROS) production by mitochondria, promotes T cell apoptosis. We have addressed the mechanisms regulating p66Shc-dependent apoptosis in T cells exposed to supraphysiological increases in [Ca2+]c. p66Shc expression resulted in profound mitochondrial dysfunction in response to the Ca2+ ionophore A23187, as revealed by dissipation of mitochondrial transmembrane potential, cytochrome c release and decreased ATP levels. p66Shc expression also caused a dramatic alteration in the cells' Ca2+-handling ability, which resulted in Ca2+ overload after A23187 treatment. The impairment in Ca2+ homeostasis was ROS dependent and caused by defective Ca2+ extrusion due at least in part to decreased plasma membrane ATPase (PMCA) expression. Both effects of p66Shc required Ca2+-dependent serine-36 phosphorylation. The mitochondrial effects of p66Shc were potentiated by but not strictly dependent on the rise in [Ca2+]c. Thus, Ca2+-dependent p66Shc phosphorylation causes both mitochondrial dysfunction and impaired Ca2+ homeostasis, which synergize in promoting T cell apoptosis.     

The life span determinant p66Shc localizes to mitochondria where it associates with mitochondrial heat shock protein 70 and regulates trans-membrane potential

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.     

Reactive oxygen species induced by p66Shc longevity protein mediate nongenomic androgen action via tyrosine phosphorylation signaling to enhance tumorigenicity of prostate cancer cells

Steroid hormones exhibit diverse biological activities. Despite intensive studies on steroid function at the genomic level, their nongenomic actions remain an enigma. In this study, we investigated the role of reactive oxygen species (ROS) in androgen-stimulated prostate cancer (PCa) cell proliferation. In androgen-treated PCa cells, increased cell growth and ROS production correlated with elevated p66Shc protein, an authentic oxidase. This growth stimulation was blocked by antioxidants. Further, elevated expression of p66Shc protein by cDNA transfection encoding wild-type protein, but not a redox-deficient (W134F) mutant, was associated with increased PCa cell proliferation. Conversely, knockdown of p66Shc expression by shRNA resulted in diminished cell growth. Increased p66Shc expression in PCa cells enhanced their tumorigenicity in xenograft animals. Importantly, p66Shc protein level is higher in clinical prostate adenocarcinomas than in adjacent noncancerous cells. Expression of redox-deficient p66Shc mutant protein abolished androgen-stimulated cell growth. In androgen-treated, H(2)O(2)-treated, and p66Shc cDNA-transfected PCa cells, cellular prostatic acid phosphatase, an authentic tyrosine phosphatase, was inactivated by reversible oxidation; subsequently, ErbB-2 was activated by phosphorylation at tyrosine-1221/1222. These results together support the notion that androgens induce ROS production through the elevation of p66Shc protein, which inactivates tyrosine phosphatase activity for the activation of interacting tyrosine kinase, leading to increased cell proliferation and enhanced tumorigenicity. Our results thus suggest that p66Shc protein functions at the critical junction point between androgens and tyrosine phosphorylation signaling in human PCa cells.     

Distinct signaling functions for Shc isoforms in the heart

Thrombin activates protease-activated receptor-1 (PAR-1) and engages signaling pathways that influence the growth and survival of cardiomyocytes as well as extracellular matrix remodeling by cardiac fibroblasts. This study examines the role of Shc proteins in PAR-1-dependent signaling pathways that influence ventricular remodeling. We show that thrombin increases p46Shc/p52Shc phosphorylation at Tyr(239)/Tyr(240) and Tyr(317) (and p66Shc-Ser(36) phosphorylation) via a pertussis toxin-insensitive epidermal growth factor receptor (EGFR) transactivation pathway in cardiac fibroblasts; p66Shc-Ser(36) phosphorylation is via a MEK-dependent mechanism. In contrast, cardiac fibroblasts express beta(2)-adrenergic receptors that activate ERK through a pertussis toxin-sensitive EGFR transactivation pathway that does not involve Shc isoforms or lead to p66Shc-Ser(36) phosphorylation. In cardiomyocytes, thrombin triggers MEK-dependent p66Shc-Ser(36) phosphorylation, but this is not via EGFR transactivation (or associated with Shc-Tyr(239)/Tyr(240) and/or Tyr(317) phosphorylation). Importantly, p66Shc protein expression is detected in neonatal, but not adult, cardiomyocytes; p66Shc expression is induced (via a mechanism that requires protein kinase C and MEK activity) by Pasteurella multocida toxin, a Galpha(q) agonist that promotes cardiomyocyte hypertrophy. These results identify novel regulation of individual Shc isoforms in receptor-dependent pathways leading to cardiac hypertrophy and the transition to heart failure. The observations that p66Shc expression is induced by a Galpha(q) agonist and that PAR-1 activation leads to p66Shc-Ser(36) phosphorylation identifies p66Shc as a novel candidate hypertrophy-induced mediator of cardiomyocyte apoptosis and heart failure.     

The Adaptor Proteins p66Shc and Grb2 Regulate the Activation of the GTPases ARF1 and ARF6 in Invasive Breast Cancer Cells

Signals downstream of growth factor receptors play an important role in mammary carcinogenesis. Recently, we demonstrated that the small GTPases ARF1 and ARF6 were shown to be activated downstream of the epidermal growth factor receptor (EGFR) and act as a key regulator of growth, migration, and invasion of breast cancer cells. However, the mechanism via which the EGFR recruits and activates ARF1 and ARF6 to transmit signals has yet to be fully elucidated. Here, we identify adaptor proteins Grb2 and p66Shc as important regulators mediating ARF activation. We demonstrate that ARF1 can be found in complex with Grb2 and p66Shc upon EGF stimulation of the basal-like breast cancer MDA-MB-231 cell line. However, we report that these two adaptors regulate ARF1 activation differently, with Grb2 promoting ARF1 activation and p66Shc blocking this response. Furthermore, we show that Grb2 is essential for the recruitment of ARF1 to the EGFR, whereas p66Shc hindered ARF1 receptor recruitment. We demonstrate that the negative regulatory role of p66Shc stemmed from its ability to block the recruitment of Grb2/ARF1 to the EGFR. Conversely, p66Shc potentiates ARF6 activation as well as the recruitment of this ARF isoform to the EGFR. Interestingly, we demonstrate that Grb2 is also required for the activation and receptor recruitment of ARF6. Additionally, we show an important role for p66Shc in modulating ARF activation, cell growth, and migration in HER2-positive breast cancer cells. Together, our results highlight a central role for adaptor proteins p66Shc and Grb2 in the regulation of ARF1 and ARF6 activation in invasive breast cancer cells.     

p66Shc regulates migration of castration-resistant prostate cancer cells

Metastatic castration-resistant (CR) prostate cancer (PCa) is a lethal disease for which no effective treatment is currently available. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species (ROS) and is elevated in clinical PCa and multiple CR PCa cell lines. We hypothesize p66Shc also increases the migratory activity of PCa cells through ROS and investigate the associated mechanism. Using the transwell assay, our study reveals that the level of p66Shc protein correlates with cell migratory ability across several PCa cell lines. Furthermore, we show hydrogen peroxide treatment induces migration of PCa cells that express low levels of p66Shc in a dose-dependent manner, while antioxidants inhibit migration. Conversely, PCa cells that express high levels of endogenous p66Shc or by cDNA transfection possess increased cell migration which is mitigated upon p66Shc shRNA transfection or expression of oxidase-deficient dominant-negative p66Shc W134F mutant. Protein microarray and immunoblot analyses reveal multiple proteins, including ErbB-2, AKT, mTOR, ERK, FOXM1, PYK2 and Rac1, are activated in p66Shc-elevated cells. Their involvement in PCa migration was examined using respective small-molecule inhibitors. The role of Rac1 was further validated using cDNA transfection and, significantly, p66Shc is found to promote lamellipodia formation through Rac1 activation. In summary, the results of our current studies clearly indicate p66Shc also regulates PCa cell migration through ROS-mediated activation of migration-associated proteins, notably Rac1.     

Epidermal growth factor receptor phosphorylates protein kinase C {delta} at Tyr332 to form a trimeric complex with p66Shc in the H2O2-stimulated cells

Protein kinase C (PKC) delta is phosphorylated at Tyr311 and Tyr332 and its catalytic activity is enhanced in the H(2)O(2)-stimulated cells, but the enzymes that recognize these tyrosine residues, especially Tyr332, have been remained to be clarified. The analysis of the endogenous proteins in COS-7 cells revealed that PKCdelta binds to p66Shc, an adaptor protein containing two phosphotyrosine-binding domains, in a manner dependent on its tyrosine phosphorylation upon H(2)O(2) stimulation. The studies using the mutated PKCdelta clarified that PKCdelta associates with p66Shc through the phosphorylated Tyr332 residue. Epidermal growth factor (EGF) receptor was detected in the anti-p66Shc immunoprecipitate prepared from the H(2)O(2)-stimulated cells, and this receptor-type tyrosine kinase phosphorylated PKCdelta at Tyr332 in vitro. PKCdelta was, however, not tyrosine phosphorylated in the EGF-stimulated cells, whereas H(2)O(2)-induced tyrosine phosphorylation of PKCdelta and its association with p66Shc were strongly suppressed by EGF receptor kinase inhibitors such as AG1478 and PD153035. These results indicate that EGF receptor phosphorylates PKCdelta at Tyr332 in the H(2)O(2)-stimulated but not in the growth-factor treated cells, and suggest that PKCdelta in the complex with p66Shc and EGF receptor may play a role in the stress-signalling pathway.     

Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells

p66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. However, a role of p66Shc in VEGF signaling and physiological responses in ECs is unknown. Here we show that VEGF promotes p66Shc phosphorylation at Ser36 through the JNK/ERK or PKC pathway as well as Rac1 binding to a nonphosphorylated form of p66Shc in ECs. Depletion of endogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role in VEGFR2 phosphorylation in caveolae/lipid rafts as well as downstream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube formation. These studies uncover a novel role of p66Shc as a positive regulator for ROS-dependent VEGFR2 signaling linked to angiogenesis in ECs and suggest p66Shc as a potential therapeutic target for various angiogenesis-dependent diseases.     

Steroids up-regulate p66Shc longevity protein in growth regulation by inhibiting its ubiquitination

Background

p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism.

Methods and Findings

In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist. Further, actinomycin D and cyclohexamide could only partially block the elevated p66Shc protein level by steroids. Treatment with proteasomal inhibitors, but not lysosomal protease inhibitor, resulted in elevated p66Shc protein levels, even higher than that by steroids. Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins.

Conclusions

The data collectively indicate that functional steroid receptors are required in steroid up-regulation of p66Shc protein levels in prostate and ovarian cancer cells, correlating with cell proliferation. In these steroid-treated cells, elevated p66Shc protein level is apparently in part due to inhibiting its ubiquitination. The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway.     

Phosphorylation of Shc proteins in human sperm in response to capacitation and progesterone treatment

Several authors have demonstrated the involvement of tyrosine kinases during sperm capacitation and acrosome reaction. Shc proteins (p46^Shc, p52^Shc, and p66^Shc) are cytoplasmic substrates of activated tyrosine kinases and are widely expressed in mammalian somatic tissues. Experiments were designed to demonstrate the presence of Shc in spermatozoa and to study its involvement in the signal transduction events leading to acrosome reaction. Anti-Shc antibodies strongly reacted with the acrosomal region of methanol-fixed human sperm. Only one Shc isoform (p52^Shc) was detected on Western blot. To study the degree of phosphorylation of Shc during capacitation and acrosome reaction, sperm samples were divided into two groups: noncapacitated and capacitated/progesterone treated. Lysates from both groups were immunoprecipitated with anti-phosphotyrosine antibodies and the precipitated (ie, phosphorylated) proteins were tested with anti-Shc antibodies. The intensity of p52^Shc was clearly increased in capacitated/progesterone-stimulated cells. Mol. Reprod. Dev. 50:113–120, 1998. © 1998 Wiley-Liss, Inc.     

Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells.

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is an essential element of the Ras signaling pathway in multiple systems. The SH2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several proline-rich domain-containing proteins such as Sos1. To define the difference in Grb2-associated proteins in hepatocarcinoma cells, we performed coprecipitation analysis using recombinant GST-Grb2 fusion proteins and found that several protein components (p170, p125, p100, and p80) differently associated with GST-Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cells. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang liver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST-Grb2 SH2 proteins exclusively bound to the p46(Shc), p52(Shc), and p66(Shc) are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N-SH3 and C-SH3 domains of Grb2 fusion proteins coprecipitated with dynamin II besides Sos1. These results suggest that dynamin II may be a functional molecule involved in Grb2-mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells.     

The Shc locus regulates insulin signaling and adiposity in mammals

Longevity of a p66Shc knockout strain (ShcP) was previously attributed to increased stress resistance and altered mitochondria. Microarrays of ShcP tissues indicated alterations in insulin signaling. Consistent with this observation, ShcP mice were more insulin sensitive and glucose tolerant at organismal and tissue levels, as was a novel p66Shc knockout (ShcL). Increasing and decreasing Shc expression in cell lines decreased and increased insulin sensitivity, respectively - consistent with p66Shc's function as a repressor of insulin signaling. However, differences between the two p66Shc knockout strains were also observed. ShcL mice were fatter and susceptible to fatty diets, and their fat was more insulin sensitive than controls. On the other hand, ShcP mice were leaner and resisted fatty diets, and their adipose was less insulin sensitive than controls. ShcL and ShcP strains are both highly inbred on the C57Bl/6 background, so we investigated gene expression at the Shc locus, which encodes three isoforms, p66, p52, and p46. Isoform p66 is absent in both strains; thus, the remaining difference to which to attribute the 'lean' phenotype is expression of the other two isoforms. ShcL mice have a precise deletion of p66Shc and normal expression of p52 and p46Shc isoforms in all tissues; thus, a simple deletion of p66Shc results in a 'fat' phenotype. However, ShcP mice in addition to p66Shc deletion have a fourfold increase in p46Shc expression in white fat. Thus, p46Shc overexpression in fat, rather than p66Shc deletion, is the likely cause of decreased adiposity and reduced insulin sensitivity in the fat of ShcP mice, which has implications for the longevity of the strain.