Critical role for endocytosis in the regulation of signaling by the Kaposi's sarcoma-associated herpesvirus K1 protein

Kaposi's sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family. KSHV is the etiologic agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The first open reading frame of the KSHV genome encodes a type 1 transmembrane glycoprotein named K1. K1 is structurally similar to the B-cell receptor (BCR), and its cytoplasmic tail contains an immunoreceptor tyrosine-based activation motif that can activate Syk kinase and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Recent evidence suggests that receptor signaling occurs not only at the cell membrane, but from intracellular compartments as well. We have found that K1 is internalized in a clathrin-dependent manner, and efficient internalization is coupled to its signaling function. Once internalized, K1 traffics from the early endosome to the recycling endosome. Interestingly, blocking K1's activation of Syk and PI3K prevents K1 from internalizing. We have also found that blocking clathrin-mediated endocytosis prevents downstream signaling by K1. These results strongly suggest that internalization of K1 is intimately associated with normal signaling. When K1 internalization was examined in B lymphocytes, we found that K1 cointernalized with the BCR. Altogether, these results suggest that K1's signaling function is tightly coupled to its internalization.     

Cdk1 inhibition induces mutually inhibitory apoptosis and reactivation of Kaposi's sarcoma-associated herpesvirus

Primary effusion lymphoma (PEL) cells are predominantly infected by the latent form of Kaposi's sarcoma-associated herpesvirus (KSHV), with virus reactivation occurring in a small percentage of cells. Latency enables KSHV to persist in the host cell and promotes tumorigenesis through viral gene expression, thus presenting a major barrier to the elimination of KSHV and the treatment of PEL. Therefore, it is important to identify cellular genes that are essential for PEL cell survival or the maintenance of KSHV latency. Here we report that cyclin-dependent kinase 1 (Cdk1) inhibition can induce both apoptosis and KSHV reactivation in a population of PEL cells. Caspases, but not p53, are required for PEL cell apoptosis induced by Cdk1 inhibition. p38 kinase is activated by Cdk1 inhibition and mediates KSHV reactivation. Interestingly, upon Cdk1 inhibition, KSHV is reactivated predominantly in the nonapoptotic subpopulation of PEL cells. We provide evidence that this is due to mutual inhibition between apoptosis and KSHV reactivation. In addition, we found that KSHV reactivation activates protein kinase B (AKT/PKB), which promotes cell survival and facilitates KSHV reactivation. Our study thus establishes a key role for Cdk1 in PEL cell survival and the maintenance of KSHV latency and reveals a multifaceted relationship between KSHV reactivation and PEL cell apoptosis.     

Modulation of Kaposi's sarcoma-associated herpesvirus infection and replication by MEK/ERK, JNK, and p38 multiple mitogen-activated protein kinase pathways during primary infection

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma, a dominant AIDS-related tumor of endothelial cells, and several other lymphoproliferative malignancies. While activation of the phosphatidylinositol 3-kinase-protein kinase C-MEK-ERK pathway is essential for KSHV infection, we have recently shown that KSHV also activates JNK and p38 mitogen-activated protein kinase (MAPK) pathways during primary infection (J. Xie, H. Y. Pan, S. Yoo, and S.-J. Gao, J. Virol. 79:15027-15037, 2005). Here, we found that activation of both JNK and p38 pathways was also essential for KSHV infection. Inhibitors of all three MAPK pathways reduced KSHV infectivity in both human umbilical vein endothelial cells (HUVEC) and 293 cells. These inhibitory effects were dose dependent and occurred at the virus entry stage of infection. Consistently, inhibition of all three MAPK pathways with dominant-negative constructs reduced KSHV infectivity whereas activation of the ERK pathway but not the JNK and p38 pathways enhanced KSHV infectivity. Importantly, inhibition of all three MAPK pathways also reduced the yield of infectious virions during KSHV productive infection of HUVEC. While the reduction of infectious virions was in part due to the reduced infectivity, it was also the result of direct modulation of KSHV lytic replication by the MAPK pathways. Accordingly, KSHV upregulated the expression of RTA (Orf50), a master transactivator of KSHV lytic replication, and activated its promoter during primary infection. Furthermore, KSHV activation of RTA promoter during primary infection was modulated by all three MAPK pathways, predominantly through their downstream target AP-1. Together, these results indicate that, by modulating multiple MAPK pathways, KSHV manipulates the host cells to facilitate its entry into the cells and postentry productive lytic replication during primary infection.     

Molecular virology of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV), the most recently discovered human tumour virus, is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and some forms of Castleman's disease. KSHV is a rhadinovirus, and like other rhadinoviruses, it has an extensive array of regulatory genes obtained from the host cell genome. These pirated KSHV proteins include homologues to cellular CD21, three different beta-chemokines, IL-6, BCL-2, several different interferon regulatory factor homologues, Fas-ligand ICE inhibitory protein (FLIP), cyclin D and a G-protein-coupled receptor, as well as DNA synthetic enzymes including thymidylate synthase, dihydrofolate reductase, DNA polymerase, thymidine kinase and ribonucleotide reductases. Despite marked differences between KSHV and Epstein-Barr virus, both viruses target many of the same cellular pathways, but use different strategies to achieve the same effects. KSHV proteins have been identified which inhibit cell-cycle regulation checkpoints, apoptosis control mechanisms and the immune response regulatory machinery. Inhibition of these cellular regulatory networks app ears to be a defensive means of allowing the virus to escape from innate antiviral immune responses. However, due to the overlapping nature of innate immune and tumour-suppressor pathways, inhibition of these regulatory networks can lead to unregulated cell proliferation and may contribute to virus-induced tumorigenesis.     

Caveolin-1 maintains activated Akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A

Previously it has been reported that caveolin-1 (cav-1) has antiapoptotic activities in prostate cancer cells and functions downstream of androgenic stimulation. In this study, we demonstrate that cav-1 overexpression significantly reduced thapsigargin (Tg)-stimulated apoptosis. Examination of the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling cascade revealed higher activities of PDK1 and Akt but not PI3-K in cav-1-stimulated cells compared to control cells. We subsequently found that cav-1 interacts with and inhibits serine/threonine protein phosphatases PP1 and PP2A through scaffolding domain binding site interactions. Deletion of the cav-1 scaffolding domain significantly reduces phosphorylated Akt and cell viability compared with wild-type cav-1. Analysis of potential substrates for PP1 and PP2A revealed that cav-1-mediated inhibition of PP1 and PP2A leads to increased PDK1, Akt, and ERK1/2 activities. We demonstrate that increased Akt activities are largely responsible for cav-1-mediated cell survival using dominant-negative Akt mutants and specific inhibitors to MEK1/MEK and show that cav-1 increases the half-life of phosphorylated PDK1 and Akt after inhibition of PI3-K by LY294002. We further demonstrate that cav-1-stimulated Akt activities lead to increased phosphorylation of multiple Akt substrates, including GSK3, FKHR, and MDM2. In addition, overexpression of cav-1 significantly increases translocation of phosphorylated androgen receptor to nucleus. Our studies therefore reveal a novel mechanism of Akt activation in prostate cancer and potentially other malignancies.     

Signaling cascades triggered by bacterial metabolic end products during reactivation of Kaposi's sarcoma-associated herpesvirus

The present studies explore the role of polymicrobial infection in the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) and analyze signaling pathways activated upon this induction. We hypothesized that activation of the cellular stress-activated mitogen-activated protein kinase (MAPK) p38 pathway would play a key role in the bacterium-mediated disruption of viral latency similar to that of previously reported results obtained with other inducers of gammaherpesvirus lytic replication. KSHV within infected BCBL-1 cells was induced to replicate following exposure to metabolic end products from gram-negative or -positive bacteria that were then simultaneously exposed to specific inhibitors of signal transduction pathways. We have determined that bacterium-mediated induction of lytic KSHV infection is significantly reduced by the inhibition of the p38 MAPK pathway. In contrast, inhibition of the phosphatidylinositol 3-kinase pathway did not impair induction of lytic replication or p38 phosphorylation. Protein kinase C, though activated, was not the major pathway used for bacterium-induced viral reactivation. Furthermore, hyperacetylation of histones 3 and 4 was detected. Collectively, our results show that metabolic end products from these pathogens induce lytic replication of KSHV in BCBL-1 cells primarily via the activation of a stress-activated MAPK pathway. Importantly, we demonstrate for the first time a mechanism by which polymicrobial bacterial infections result in KSHV reactivation and pathogenesis.     

The luteinizing hormone-releasing hormone inhibits the anti-apoptotic activity of insulin-like growth factor-1 in pituitary alphaT3 cells by protein kinase Calpha-mediated negative regulation of Akt

The luteinizing hormone-releasing hormone (LHRH) receptor is a G protein-coupled receptor involved in the synthesis and release of pituitary gonadotropins and in the proliferation and apoptosis of pituitary cells. Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that has a mitogenic effect on pituitary cells. In this study, we used the alphaT3 gonadotrope cell line as a model to characterize the IGF-1R signaling pathways and to investigate whether this receptor interacts with the LHRH cascade. We found that IGF-1 activated the IGF-1R, insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase, and Akt in a time-dependent manner in alphaT3 cells. The MAPK (ERK1/2, p38, and JNK) pathways were only weakly activated by IGF-1. In contrast, LHRH strongly stimulated the MAPK pathways but had no effect on Akt activation. Cotreatment with IGF-1 and LHRH had various effects on these signaling pathways. 1) It strongly increased IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated phosphatidylinositol 3-kinase through activation of the epidermal growth factor receptor. 2) It had an additive effect on ERK1/2 activation without modifying the phosphorylation of p38 and JNK1/2. 3) It strongly reduced IGF-1 activation of Akt. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and cell cycle analysis revealed that, in addition to having an additive effect on ERK1/2 activation, cotreatment with IGF-1 and LHRH also had an additive effect on cell proliferation. The LHRH-induced inhibition of Akt stimulated by IGF-1 was completely blocked by Safingol, a protein kinase C (PKC) alpha-specific inhibitor, and by a dominant negative form of PKCalpha. Finally, we showed that the inhibitory effect of LHRH on IGF-1-induced PKCalpha-mediated Akt activation was associated with a marked reduction in Bad phosphorylation and a substantial decrease in the ability of IGF-1 to rescue alphaT3 cells from apoptosis induced by serum starvation. Our results demonstrate for the first time that several interactions take place between IGF-1 and LHRH receptors in gonadotrope cells.     

Differential activation of murine herpesvirus 68- and Kaposi's sarcoma-associated herpesvirus-encoded ORF74 G protein-coupled receptors by human and murine chemokines

Infection of mice with murine gammaherpesvirus 68 (MHV-68) is a well-characterized small animal model for the study of gammaherpesvirus infection. MHV-68 belongs to the same herpesvirus family as herpesvirus saimiri (HVS) of New World squirrel monkeys and human herpesvirus 8 (HHV-8) (also referred to as Kaposi's sarcoma-associated herpesvirus [KSHV]). The open reading frame ORF74 of HVS, KSHV, and MHV-68 encodes a protein with homology to G protein-coupled receptors and chemokine receptors in particular. ORF74 of KSHV (human ORF74 [hORF74]) is highly constitutively active and has been implicated in the pathogenesis of Kaposi's sarcoma. MHV-68-encoded ORF74 (mORF74) is oncogenic and has been implicated in viral replication and reactivation from latency. Here, we show that mORF74 is a functional chemokine receptor. Chemokines with an N-terminal glutamic acid-leucine-arginine (ELR) motif (e.g., KC and macrophage inflammatory protein 2) act as agonists on mORF74, activating phospholipase C, NF-kappaB, p44/p42 mitogen-activated protein kinase, and Akt signaling pathways and inhibiting formation of cyclic AMP. Using (125)I-labeled CXCL1/growth-related oncogene alpha as a tracer, we show that murine CXCL10/gamma interferon-inducible protein 10 binds mORF74, and functional assays show that it behaves as an antagonist for this virally encoded G protein-coupled receptor. Profound differences in the upstream activation of signal transduction pathways between mORF74 and hORF74 were found. Moreover, in contrast to hORF74, no constitutive activity of mORF74 could be detected.     

Survival signaling in type II pneumocytes activated by surfactant protein-A

Surfactant-associated protein-A (SP-A) is a component of pulmonary surfactant that acts as a cytokine through interaction with a cell-surface receptor (SPAR) on lung epithelial cells. SP-A regulates important physiological processes including surfactant secretion, gene expression, and protection against apoptosis. Tyrosine kinase and PI3K inhibitors block effects of SP-A, suggesting that SPAR may be a receptor tyrosine kinase and activate the PI3K-PKB/Akt pathway. Here we report that SP-A treatment leads to rapid tyrosine-specific phosphorylation of several important proteins in lung epithelial cells including insulin receptor substrate-1 (IRS-1), an upstream activator of PI3K. Analysis of anti-apoptotic signaling species downstream of IRS-1 showed activation of PKB/Akt but not of MAPK. Phosphorylation of IkappaB was minimally affected by SP-A as was NFkappaB gel shift activity. However, FKHR was rapidly phosphorylated in response to SP-A and its DNA-binding activity was significantly reduced. Since FKHR is pro-apoptotic, this may play an important role in signaling the anti-apoptotic effects of SP-A. Therefore, we have characterized survival-enhancing signaling activated by SP-A leading from SPAR through IRS-1, PI3K, PKB/Akt, and FKHR. The activity of this pathway may explain, in part, the resilience of type II cells to lung injury and their survival to repopulate alveolar epithelium after peripheral lung damage.