Epidermal growth factor receptor is a cellular receptor for human cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread opportunistic herpesvirus that causes severe and fatal diseases in immune-compromised individuals, including organ transplant recipients and individuals with AIDS. It is also a leading cause of virus-associated birth defects and is associated with atherosclerosis and coronary restenosis. HCMV initiates infection and intracellular signalling by binding to its cognate cellular receptors and by activating several signalling pathways including those mediated by mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, interferons, and G proteins. But a cellular receptor responsible for viral entry and HCMV-induced signalling has yet to be identified. Here we show that HCMV infects cells by interacting with epidermal growth factor receptor (EGFR) and inducing signalling. Transfecting EGFR-negative cells with an EGFR complementary DNA renders non-susceptible cells susceptible to HCMV. Ligand displacement and crosslinking analyses show that HCMV interacts with EGFR through gB, its principal envelope glycoprotein. gB preferentially binds EGFR and EGFR-ErbB3 oligomeric molecules in Chinese hamster ovary cells transfected with erbB family cDNAs. Taken together, these data indicate that EGFR is a necessary component for HCMV-triggered signalling and viral entry.     

Human immunodeficiency virus induces phosphorylation of its cell surface receptor

AIDS is an immunoregulatory disorder characterized by depletion of the CD4+, helper/inducer lymphocyte population. The causative agent of this disease is the human immunodeficiency virus, HIV, which infects CD4+ cells and leads to cytopathic effects characterized by syncytia formation and cell death. Recent studies have demonstrated that binding of HIV to its cellular receptor CD4 is necessary for viral entry. We find that binding of HIV to CD4 induces rapid and sustained phosphorylation of CD4 which could involve protein kinase C. HIV-induced CD4 phosphorylation can be blocked by antibody against CD4 and monoclonal antibody against the HIV envelope glycoprotein gp120, indicating that a specific interaction between CD4 and gp120 is required for phosphorylation. Electron microscopy shows that a protein kinase C inhibitor does not impair binding of HIV to CD4+ cells, but causes an apparent accumulation of virus particles at the cell surface, at the same time inhibiting viral infectivity. These results indicate a possible role for HIV-induced CD4 phosphorylation in viral entry and identify a potential target for antiviral therapy.     

MAPK信号转导通路与HCMV感染

MAPK信号转导通路参与了HCMV的感染过程.MAPK通路中的ERK和p38通路在HCMV复制周期中起重要作用,它通过磷酸化转录因子引起病毒及宿主相关基因的转录,从而调控HCMV的复制.HCMV的包膜糖蛋白及其他多种基因表达产物可通过不同的机制以一定时序激活MAPK通路,调节自身及宿主细胞相应基因表达,以利于病毒完成其生活周期,并参与病毒的致病过程.深入研究MAPK信号转导通路与HCMV感染的关系可为治疗HCMV感染引起的疾病提供新的治疗靶点.     

Internalization of the human immunodeficiency virus does not require the cytoplasmic domain of CD4

Binding of the human immunodeficiency virus (HIV) to infectable host cells, such as B and T lymphocytes, monocytes and colorectal cells, is mediated by a high-affinity interaction between the gp120 component of the viral envelope glycoprotein and the CD4 receptor. Upon binding, it is thought that the second component of the envelope, gp41, mediates fusion between the viral envelope and host cell membranes. However, the early steps of HIV infection have not yet been thoroughly elucidated. Viral entry was first reported to be mediated by pH-dependent receptor-mediated endocytosis; subsequent studies have shown entry to be pH-independent. Although direct fusion of virus to plasma membranes of infected cells has been observed by electron microscopy, it is still formally possible that the infectious path of the virus involves receptor-mediated endocytosis. To gain a better understanding of receptor function in viral entry, we have analysed the ability of several altered or truncated forms of CD4 to serve as effective viral receptors. Our results indicate that domains beyond the HIV-binding region of CD4 are not required for viral infection. Some of the altered forms of CD4 that serve as effective HIV receptors are severely impaired in their ability to be endocytosed. These experiments therefore support the notion that viral fusion to the plasma membrane is sufficient for infection.     

Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane

The receptor for epidermal growth factor (EGF) is a 170,000-180,000 molecular weight single-chain glycoprotein of 1,186 amino acids. Its sequence suggests that it has an external EGF-binding domain, formed by the NH2-terminal 621 amino acids, linked to a cytoplasmic region by a single membrane-spanning segment. In the cytoplasmic portion, starting 50 residues from the membrane, there is a 250-residue stretch similar to the catalytic domain of the src gene family of retroviral tyrosine protein kinases, and, indeed, a tyrosine-specific protein kinase activity intrinsic to the receptor is stimulated when EGF is bound. Increased tyrosine phosphorylation of cellular proteins, detected in A431 cells following EGF binding, may be important in the mitogenic signal pathway. Tumour promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), counteract this increase, as well as causing loss of a high affinity class of EGF binding sites. The major receptor for TPA has been identified as the serine/threonine-specific Ca2+/phospholipid-dependent diacylglycerol-activated protein kinase, protein kinase C. By substituting for diacylglycerol, TPA stimulates protein kinase C. Protein kinase C phosphorylates purified EGF receptor at specific sites, and this reduces EGF-stimulated tyrosine protein kinase activity. TPA treatment of A431 cells increases serine and threonine phosphorylation of the EGF receptor at the same sites, which suggests that the reduction of EGF receptor kinase activity in TPA-treated cells is a consequence of the receptor's phosphorylation by the kinase. We have attempted to identify these phosphorylation sites and show here that protein kinase C phosphorylates threonine 654 in the human EGF receptor. This threonine is in a very basic sequence nine residues from the cytoplasmic face of the plasma membrane in the region before the protein kinase domain; it is thus in a position to modulate signalling between this internal domain and the external EGF-binding domain.     

Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck

The CD4 T-cell surface antigen is an integral membrane glycoprotein of relative molecular mass 55,000 which binds class II major histocompatibility complex (MHC) molecules expressed on antigen presenting cells (APCs). It is thought to stabilize physical interactions between T cells and APCs (for a review, see ref. 1). Evidence is accumulating that suggests that CD4 can transduce an independent signal during T-cell activation. It has recently been shown that CD4 expressed on human and murine T cells is physically associated with the Src-related tyrosine protein kinase p56lck (refs 7, 8). These results indicate that CD4 can function as a signal transducer and suggest that tyrosine phosphorylation events may be important in CD4-mediated signalling. Here, we present evidence that cross-linking of the CD4 receptor induces a rapid increase in the tyrosine-specific protein kinase activity of p56lck and is associated with the rapid phosphorylation of one of the subunits (zeta) of the T-cell receptor complex on tyrosine residues. These data provide direct evidence for a specific CD4 signal transduction pathway that is mediated through p56lck and suggest that some of the tyrosine phosphorylation events detected during antigen-mediated T-cell activation may result from signalling through this surface molecule.     

Essential role for Gab2 in the allergic response.

Dos/Gab family scaffolding adapters (Dos, Gab1, Gab2) bind several signal relay molecules, including the protein-tyrosine phosphatase Shp-2 and phosphatidylinositol-3-OH kinase (PI(3)K); they are also implicated in growth factor, cytokine and antigen receptor signal transduction. Mice lacking Gab1 die during embryogenesis and show defective responses to several stimuli. Here we report that Gab2-/- mice are viable and generally healthy; however, the response (for example, degranulation and cytokine gene expression) of Gab2-/- mast cells to stimulation of the high affinity immunoglobulin-epsilon (IgE) receptor Fc(epsilon)RI is defective. Accordingly, allergic reactions such as passive cutaneous and systemic anaphylaxis are markedly impaired in Gab2-/- mice. Biochemical analyses reveal that signalling pathways dependent on PI(3)K, a critical component of Fc(epsilon)RI signalling, are defective in Gab2-/- mast cells. Our data identify Gab2 as the principal activator of PI(3)K in response to Fc(epsilon)RI activation, thereby providing genetic evidence that Dos/Gab family scaffolds regulate the PI(3)K pathway in vivo. Gab2 and/or its associated signalling molecules may be new targets for developing drugs to treat allergy.     

Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3

Oncogenic tyrosine kinases have proved to be promising targets for the development of highly effective anticancer drugs. However, tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (HER) family show only limited activity against HER2-driven breast cancers, despite effective inhibition of epidermal growth factor receptor (EGFR) and HER2 in vivo. The reasons for this are unclear. Signalling in trans is a key feature of this multimember family and the critically important phosphatidylinositol-3-OH kinase (PI(3)K)/Akt pathway is driven predominantly through transphosphorylation of the kinase-inactive HER3 (refs 9, 10). Here we show that HER3 and consequently PI(3)K/Akt signalling evade inhibition by current HER-family TKIs in vitro and in tumours in vivo. This is due to a compensatory shift in the HER3 phosphorylation-dephosphorylation equilibrium, driven by increased membrane HER3 expression driving the phosphorylation reaction and by reduced HER3 phosphatase activity impeding the dephosphorylation reaction. These compensatory changes are driven by Akt-mediated negative-feedback signalling. Although HER3 is not a direct target of TKIs, HER3 substrate resistance undermines their efficacy and has thus far gone undetected. The experimental abrogation of HER3 resistance by small interfering RNA knockdown restores potent pro-apoptotic activity to otherwise cytostatic HER TKIs, re-affirming the oncogene-addicted nature of HER2-driven tumours and the therapeutic promise of this oncoprotein target. However, because HER3 signalling is buffered against an incomplete inhibition of HER2 kinase, much more potent TKIs or combination strategies are required to silence oncogenic HER2 signalling effectively. The biologic marker with which to assess the efficacy of HER TKIs should be the transphosphorylation of HER3 rather than autophosphorylation.     

No T-cell tyrosine protein kinase signalling or calcium mobilization after CD4 association with HIV-1 or HIV-1 gp120.

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.     

Myc rescue of a mutant CSF-1 receptor impaired in mitogenic signalling.

The colony-stimulating factor-1 receptor (CSF-1R) mediates its pleiotropic effects through the coupling of its ligand-activated tyrosine kinase to multiple intracellular effector proteins, whose combined actions determine the magnitude and specificity of the biological response. The interaction of cytoplasmic signalling molecules with CSF-1R is mediated in part by sequence motifs flanking sites of receptor tyrosine phosphorylation. Mutation of an autophosphorylation site at tyrosine 809 in the cytoplasmic domain of human CSF-1R does not significantly reduce its ligand-stimulated tyrosine kinase activity, binding to phosphatidylinositol 3-kinase, or induction of the immediate early response genes, c-fos and junB (ref.2). Unlike cells bearing wild-type receptors, mouse NIH3T3 cells expressing mutant CSF-1R(Phe 809) were unable to grow in serum-free medium containing human recombinant CSF-1 and did not form colonies in semi-solid medium in its presence. CSF-1 induction of c-myc messenger RNA in these cells was impaired, but enforced expression of an exogenous c-myc gene restored their ability to proliferate in response to the growth factor. These studies demonstrate a receptor-mediated bifurcation of intracellular signal transduction pathways during the immediate early response and assign a central role for c-myc in CSF-1-induced mitogenesis.     

Tumour maintenance is mediated by eNOS

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.     

PI3K/Akt signaling pathway involved in regulation of T lymphocyte activation and apoptosis mediated by CD3ε

To study the expression and kinase activity of phosphatidylinositol 3′ -kinase (PI3K) and protein kinase B (PKB or Akt) during activation and apoptosis of human Jurkat T lymphocytes (TJK) with stable expression of CD8ε chimera fused human CD8α extracellular and transmembrane domains to intracellular domain of mouse CD3ε, Western blot, kinase activities detection and immunoprecipitation were carried out. It was shown that Jurkat cells with expression of wild type chimera CD8ε died by apoptosis after continuous stimulation of anti-CD8 monoclonal antibody. The expressions of PI3K and Akt, and the kinase activity of Akt remarkably increased during the process. However, this phenomenon did not occur in the Jurkat cells (T1JK) with expression of the mutant of CD8ε chimera (Y170F), suggesting that PI3K/Akt signaling pathway is involved in activation and apoptosis of T lymphocyte mediated by CD3ε.     

Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation

Polyoma middle-T antigen is required for viral transformation of cultured cells and for tumorigenesis in animals. Like many other transforming gene products, middle-T is bound to the membrane and has an associated tyrosine kinase activity in vitro. This activity seems to result from the interaction of middle-T with pp60c-src, the cellular homologue of the transforming gene product of the Rous sarcoma virus, pp60v-src (refs 3-5). Both pp60v-src (ref. 6) and another retrovirus transforming gene product, pp68v-ros (ref. 7) were shown recently to have an associated phosphatidylinositol (PI) kinase activity in vitro and to increase PI turnover in vivo. These results suggest that viral transformation may be directly connected to a complex network of second messengers generated from PI turnover. Here, we assayed for PI kinase activity in immunoprecipitates made with middle-T- or pp60c-src-specific antisera of cells infected with polyoma virus. A PI kinase activity was detected in those immunoprecipitates which contained middle-T. Studies of mutants of middle-T defective in transformation indicate a close correlation between PI kinase activity and transformation.     

A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV) is a major pathogen in immunosuppressed individuals, including patients with acquired immune deficiency syndrome. The nucleoside analogue ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)-guanine) is one of the few drugs available to treat HCMV infections, but resistant virus is a growing problem in the clinic and there is a critical need for new drugs. The study of ganciclovir-resistant mutants has indicated that the selective action of ganciclovir depends largely on virus-controlled phosphorylation in HCMV-infected cells. The enzyme(s) responsible have not been identified. Here we report that the HCMV gene UL97, whose predicted product shares regions of homology with protein kinases, guanylyl cyclase and bacterial phosphotransferases, controls phosphorylation of ganciclovir in HCMV-infected cells. A four-amino-acid deletion of UL97 in a conserved region, which in cyclic AMP-dependent protein kinase participates in substrate recognition, causes impaired ganciclovir phosphorylation. The implications of these results for antiviral drug development and drug resistance are discussed.     

Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK). Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-'addicted' human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases.     

Transforming potential of the c-fms proto-oncogene (CSF-1 receptor)

The c-fms proto-oncogene encodes a transmembrane glycoprotein that is probably identical to the receptor for the macrophage colony stimulating factor, CSF-1. Forty C-terminal amino acids of the normal receptor are replaced by 11 unrelated residues in the feline v-fms oncogene product, deleting a C-terminal tyrosine residue (Tyr969) whose phosphorylation might negatively regulate the receptor kinase activity. We show that the human c-fms gene stimulates growth of mouse NIH 3T3 cells in agar in response to human recombinant CSF-1, indicating that receptor transduction is sufficient to induce a CSF-1 responsive phenotype. Although cells transfected with c-fms genes containing either Tyr969 or Phe969 were not transformed, cotransfection of these genes with CSF-1 complementary DNA induced transformation, with c-fms(Phe969) showing significantly more activity than c-fms(Tyr969). In the absence of CSF-1, chimaeric v-fms/c-fms genes encoding the wild-type c-fms C terminus were poorly transforming, whereas chimaeras bearing Phe969 were as transforming as v-fms. Thus, the Phe969 mutation, although not in itself sufficient to induce transformation, activates the oncogenic potential of c-fms in association with an endogenous ligand or in conjunction with mutations elsewhere in the c-fms gene that confer ligand-independent signals for growth.