Role of protein kinase C delta in reactivation of Kaposi's sarcoma-associated herpesvirus

TPA (12-O-tetradecanoylphorbol-13-acetate), a well-known activator of protein kinase C (PKC), can experimentally induce reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) in certain latently infected cells. We selectively blocked the activity of PKC isoforms by using GF 109203X or rottlerin and demonstrated that this inhibition largely decreased lytic KSHV reactivation by TPA. Translocation of the PKCdelta isoform was evident shortly after TPA stimulation. Overexpression of the dominant-negative PKCdelta mutant supported an essential role for the PKCdelta isoform in virus reactivation, yet overexpression of PKCdelta alone was not sufficient to induce lytic reactivation of KSHV, suggesting that additional signaling molecules participate in this pathway.     

Leucine zipper domain is required for Kaposi sarcoma-associated herpesvirus (KSHV) K-bZIP protein to interact with histone deacetylase and is important for KSHV replication

The Kaposi sarcoma-associated herpesvirus (KSHV; or human herpesvirus-8)-encoded protein called K-bZIP (also named K8) was found to be multifunctional. In this study, we discovered that K-bZIP interacts with histone deacetylase (HDAC) 1/2 in 12-O-tetradecanoylphorbol-13-acetate-stimulated BCBL-1 lymphocyte cells. K-bZIP appears to repress HDAC activity through this interaction, which we determined to be independent of K-bZIP SUMOylation. We dissected the domains of K-bZIP and found that the leucine zipper (LZ) domain is essential for the interaction of K-bZIP and HDAC. In addition, we constructed a KSHV bacterial artificial chromosome (BAC) with LZ domain-deleted K-bZIP (KSHVdLZ) and transfected this mutated KSHV BAC DNA into HEK 293T cells. As a result, it was consistently found that K-bZIP without its LZ domain failed to interact with HDAC2. We also showed that the interaction between K-bZIP and HDAC is necessary for the inhibition of the lytic gene promoters (ORF50 and OriLyt) of KSHV by K-bZIP. Furthermore, we found that the LZ domain is also important for the interaction of K-bZIP with the promoters of ORF50 and OriLyt. Most interestingly, although it was found to have suppressive effects on the promoters of ORF50 and OriLyt, KSHVdLZ replicates at a significantly lower level than its BAC-derived revertant (KSHVdLZRev) or KSHVWT (BAC36) in HEK 293T cells. The defectiveness of KSHVdLZ replication can be partially rescued by siRNA against HDAC2. Our results suggest that the function of K-bZIP interaction with HDAC is two-layered. 1) K-bZIP inhibits HDAC activity generally so that KSHVdLZ replicates at a lower level than does KSHVWT. 2) K-bZIP can recruit HDAC to the promoters of OriLyt and ORF50 through interaction with HDAC for K-bZIP to have a temporary repressive effect on the two promoters.     

Flavonoids, but not protein kinase C inhibitors, prevent stress protein synthesis during erythrophagocytosis.

Erythrophagocytosis induces in monocytes-macrophages the synthesis of stress proteins including the classical heat shock proteins (HSPs) and heme oxygenase (HO). To evaluate the role of oxygen radicals in this induction, we used the antioxidant flavonoids quercetin and kaempferol. These compounds inhibited HSP and HO synthesis, the latter being more sensitive. Quercetin and kaempferol also are inhibitors of protein kinase C (PKC). In order to determine whether inhibition of stress protein synthesis by flavonoids was mediated by their antioxidant properties or by PKC inhibition, we also tested more specific PKC antagonists, staurosporine and H-7. Staurosporine (SS) and H-7 decreased the synthesis of HSP70 and HSP83 but had no effect on HO. These data suggest that (1) erythrophagocytosis-related oxygen radicals are involved in the induction of the stress response in phagocytic cells, (2) the induction of HSPs and HO is differentially regulated, and (3) the effects of flavonoids on HO are linked to their scavenging activity rather than to PKC modulation.     

Induction of Epstein-Barr virus lytic cycle by tumor-promoting and non-tumor-promoting phorbol esters requires active protein kinase C.

Exposure to the tiglian 12-O-tetradecanoylphorbol-13-acetate (TPA) represents one of the most efficient and widely used protocols for inducing Epstein-Barr virus (EBV)-infected cells from latent into lytic cycle. Since TPA is both a potent tumor promoter and a potent activator of the cellular protein kinase C (PKC), we sought to determine whether either of these activities was closely linked to EBV lytic cycle induction. A panel of TPA structural analogs, encompassing tiglians with different spectra of biological activities, was assayed on a number of EBV-positive B-lymphoid cell lines. Lytic cycle induction correlated with the capacity to activate PKC, not with tumor promoter status; some nonpromoting tiglians were as efficient as TPA in inducing lytic cycle antigen expression. We then sought more direct evidence for an involvement of PKC in the induction process. In initial experiments, 1-(5-isoquinolinyl sulphonyl)-2-methylpiperazine (H-7), the best available pharmacological inhibitor of PKC, completely blocked the induction of the lytic cycle by TPA and its active analogs. This is consistent with, but does not prove, a requirement for active PKC in the induction process, since H-7 targets PKC preferentially but also has some effects on other kinases. We therefore turned to the synthetic pseudosubstrate peptide PKC(19-36) as a means of specific PKC inhibition and to the closely related but inactive peptide PKC(19-Ser-25-36) as a control. Using the technique of scrape loading to deliver the peptides into cells of an adherent EBV-positive target line, we found that the pseudosubstrate peptide PKC(19-36) completely and specifically blocked tiglian-induced entry of the cells into the lytic cycle. The evidence both from TPA analogs and from enzyme inhibition studies therefore indicates that the pathway linking TPA treatment to lytic cycle induction involves active PKC. Interestingly, inhibition of PKC had no effect upon the spontaneous entry into lytic cycle which occurs in naturally productive cell lines, suggesting that spontaneous entry is signalled by another route.     

Human fibroblast commitment to a senescence-like state in response to histone deacetylase inhibitors is cell cycle dependent.

Human diploid fibroblasts (HDF) complete a limited number of cell divisions before entering a growth arrest state that is termed replicative senescence. Two histone deacetylase inhibitors, sodium butyrate and trichostatin A, dramatically reduce the HDF proliferative life span in a manner that is dependent on one or more cell doublings in the presence of these agents. Cells arrested and subsequently released from histone deacetylase inhibitors display markers of senescence and exhibit a persistent G1 block but remain competent to initiate a round of DNA synthesis in response to simian virus 40 T antigen. Average telomere length in prematurely arrested cells is greater than in senescent cells, reflecting a lower number of population doublings completed by the former. Taken together, these results support the view that one component of HDF senescence mimics a cell cycle-dependent drift in differentiation state and that propagation of HDF in histone deacetylase inhibitors accentuates this component.     

De novo sphingolipid synthesis is essential for viability, but not for transport of glycosylphosphatidylinositol-anchored proteins, in African trypanosomes

De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate --> 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes.     

Endocytosis is regulated by protein kinase A, but not protein kinase C in a secretory epithelial cell line.

Endocytosis in the chloride secreting epithelial cell line T84 was monitored by uptake of the fluid-phase markers FITC-dextran and horseradish peroxidase (HRP). Uptake of marker was inhibited by incubation of cells at 4 degrees C, consistent with an endocytic uptake. Although activation of the cAMP-dependent second messenger pathway has been shown to stimulate exocytosis in this cell line, it caused a 63% reduction in endocytosis as measured by uptake of fluid-phase markers. In contrast, the presence of the protein kinase C activator phorbol-myristate acetate (PMA) caused no significant reduction in the level of endocytosis compared to control, nor did it reverse the inhibitory effect of PKA activation. The data thus suggest that endocytosis in T84 cells is regulated through activation of protein kinase A, but not through activation of protein kinase C.     

Vaccinia virus F9 virion membrane protein is required for entry but not virus assembly, in contrast to the related L1 protein

All sequenced poxviruses encode orthologs of the vaccinia virus L1 and F9 proteins, which are structurally similar and share about 20% amino acid identity. We found that F9 further resembles L1 as both proteins are membrane components of the mature virion with similar topologies and induce neutralizing antibodies. In addition, a recombinant vaccinia virus that inducibly expresses F9, like a previously described L1 mutant, had a conditional-lethal phenotype: plaque formation and replication of infectious virus were dependent on added inducer. However, only immature virus particles are made when L1 is repressed, whereas normal-looking intracellular and extracellular virions formed in the absence of F9. Except for the lack of F9, the polypeptide components of such virions were indistinguishable from those of wild-type virus. These F9-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, cells infected with F9-negative virions did not fuse after a brief low-pH treatment, as did cells infected with virus made in the presence of inducer. In these respects, the phenotype associated with F9 deficiency was identical to that produced by the lack of individual components of a previously described poxvirus entry/fusion complex. Moreover, F9 interacted with proteins of that complex, supporting a related role. Thus, despite the structural relationships of L1 and F9, the two proteins have distinct functions in assembly and entry, respectively.     

Epidermal growth factor receptor-mediated cell motility: phospholipase C activity is required,but mitogen-activated protein kinase activity is not sufficient for induced cell movement

We recently have demonstrated that EGF receptor (EGFR)-induced cell motility requires receptor kinase activity and autophosphorylation (P. Chen, K. Gupta, and A. Wells. 1994. J. Cell Biol. 124:547-555). This suggests that the immediate downstream effector molecule contains a src homology-2 domain. Phospholipase C gamma (PLC gamma) is among the candidate transducers of this signal because of its potential roles in modulating cytoskeletal dynamics. We utilized signaling-restricted EGFR mutants expressed in receptor devoid NR6 cells to determine if PLC activation is necessary for EGFR-mediated cell movement. Exposure to EGF (25 nM) augmented PLC activity in all five EGFR mutant cell lines which also responded by increased cell movement. Basal phosphoinositide turnover was not affected by EGF in the lines which do not present the enhanced motility response. The correlation between EGFR-mediated cell motility and PLC activity suggested, but did not prove, a causal link. A specific inhibitor of PLC, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1 microM) diminished both the EGF- induced motility and PLC responses, while its inactive analogue {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 had no effect on these responses. Both the PLC and motility responses were decreased by expression of a dominant-negative PLC gamma-1 fragment in EGF-responsive infectant lines. Lastly, anti-sense oligonucleotides (20 microM) to PLC gamma-1 reduced both responses in NR6 cells expressing wild-type EGFR. These findings strongly support PLC gamma as the immediate post receptor effector in this motogenic pathway. We have demonstrated previously that EGFR-mediated cell motility and mitogenic signaling pathways are separable. The point of divergence is undefined. All kinase-active EGFR mutants induced the mitogenic response while only those which are autophosphorylated induced PLC activity. {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 did not affect EGF-induced thymidine incorporation in these motility-responsive infectant cell lines. In addition, the dominant-negative PLC gamma-1 fragment did not diminish EGF-induced thymidine incorporation. All kinase active EGFR stimulated mitogen-activated protein (MAP) kinase activity, regardless of whether the receptors induced cell movement; this EGF-induced MAP kinase activity was not affected by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 at concentrations that depressed the motility response. Thus, the signaling pathways which lead to motility and cell proliferation diverge at the immediate post-receptor stage, and we suggest that this is accomplished by differential activation of effector molecules.     

The retinoblastoma protein, RB, is required for gastrointestinal endocrine cells to exit the cell cycle, but not for hormone expression

Important functions of the RB family proteins include inhibition of cell cycle progression and regulation of terminal differentiation. We have examined the role of RB and the related protein, p107, in regulating cell cycle activity and differentiation of gastrointestinal endocrine cells, a relatively quiescent cell population, by conditionally disrupting the RB gene in neurogenin3 (Ngn3)-expressing cells in both p107^+/+ and p107^−/− mice. Endocrine cells in the small intestine, colon, pancreas, and stomach were present in normal numbers in RB and RB-p107 mutants except for an increase in serotonin cells and decrease in ghrelin cells in the antral stomach. Deletion of RB resulted in a dramatic increase in proliferating serotonin cells in the antral stomach and intestine, whereas other enteroendocrine cell types exhibited much lower cell cycle activity or remained quiescent. The related p107 protein appears dispensable for enteroendocrine differentiation and does not functionally compensate for the loss of RB. Our results suggest that RB is required for enteroendocrine cells, particularly serotonin cells, to undergo cell cycle arrest as they terminally differentiate. RB has relatively subtle effects on enteroendocrine cell differentiation and is not required for the expression of the normal repertoire of hormones in the gastrointestinal tract.     

A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle

Probes derived from cDNAs encoding isozymes of rat protein kinase C (PKC) were used to screen the genome of the budding yeast S. cerevisiae. A single gene (PKC1) was isolated that encodes a putative protein kinase closely related to the alpha, beta, and gamma subspecies of mammalian PKC. Deletion of PKC1 resulted in recessive lethality. Cells depleted of the PKC1 gene product displayed a uniform phenotype, a characteristic of cell division cycle (cdc) mutants, and arrested cell division at a point subsequent to DNA replication, but prior to mitosis. Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrested growth with small buds. PKC1 may regulate a previously unrecognized checkpoint in the cell cycle.     

Protein kinase C is required for responses to T cell receptor ligands but not to interleukin-2 in T cells

We have tested the role of protein kinase C in mRNA expression and T cell proliferation mediated through the T cell receptor and through the interleukin-2 (IL-2) receptor. Chronic treatment of a mouse T cell clone with phorbol esters caused a complete loss of protein kinase C activity and a concomitant loss of proliferation to T cell receptor ligands (antigen, lectins, antireceptor antibodies). In contrast, kinase C-depleted T cells retained the ability to proliferate to IL-2. Loss of the T cell receptor response was not due to decreased cell surface expression of receptor or impairment of early receptor function (phosphatidylinositol turnover, calcium mobilization). Kinase C-depleted T cells showed no induction of mRNAs for activation-associated genes on exposure to the T cell receptor ligand Concanavalin A; expression of a subset of the same mRNAs in response to IL-2 was unaffected. We conclude that kinase C is required for mRNA expression and subsequent proliferation mediated through the T cell receptor pathway but is not involved in mRNA expression and proliferation in response to IL-2.