Activation mechanisms of protein kinase B and protein kinase C
Ushio Kikkawa
Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
It is well established that the serine/threonine protein kinase families such as protein kinase B (PKB) and protein kinase C (PKC), each consists of multiple genes in mammals, play important roles in the receptor-mediated signaling processes. They have regulatory and catalytic domains in their amino- and carboxyl-terminal regions, respectively, and are activated through the association with the lipid messengers to their regulatory domains upon stimulation of the cells by biologically active substances. The carboxyl-terminal halves of PKB and PKC are closely related and have three phosphorylation motif sites: the activation loop site in the catalytic domain and the turn and hydrophobic sites in the carboxyl-terminal end region. In the case of PKB, the receptor stimulation induces not only the association of the phosphatidylinositol 3-kinase products to the regulatory domain but also phosphorylation at the activation loop and hydrophobic motif sites. These two mechanisms are both indispensable for PKB activation. We found that PKB is activated by heat shock in cultured cells to a certain level without the association of the lipid messengers to the regulatory domain. Furthermore, phosphorylation at the motif sitesis not essential for heat shock-induced activation, and a PKB protein complex is formed in heat-shocked cells, which is not detected in the control orreceptor-stimulated cells. On the other hand, PKC, one of the PKC isoforms, is regulated by the association of diacylglycerolto the regulatory domain, that is derived from the receptor-coupled hydrolysis of inositol phospholipids. In contrast to PKB, the motif sites of PKCare phosphorylated, constitutively. We revealed that the hydrogen peroxide treatment of the cells induces the formation of the active PKC, in a mechanism independent of the generation of the lipid messenger. The active PKC is phosphorylated at tyrosine residues and is recovered as an oligomer. These results indicate that PKB and PKC contribute to the stress responses of the cells presumably through manners distinct from those of the signal transduction of the biologically active substances.
Regulation of the metastasis suppressor gene RECK by oncogenes
Wen-Chun Hung 1, and Hui-Chiu Chang 2
1 School of Technology for Medical Sciences, and 2 Department of Physiology, Kaohsiung Medical University, Kaohsiung, Taiwan.
RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase (MMP) activity and inhibit tumor metastasis. Previous study demonstrated that oncogenic ras inhibited RECK expression via an Sp1 binding site in the RECK promoter. In this study,we investigated the molecular mechanism by which ras inhibited RECK expression. Co-transfection assay showed that Sp1 and Sp3 are transactivators, rather than repressors, for RECK gene. So, wetested whether ras activation induced the binding of histonedeacetylases (HDACs) to Sp1 to repress RECK expression. Indeed, Sp1-associated HDAC1 was increased after ras induction. DAPA assays also demonstrated that binding of HDAC1 to the DNA probe corresponding to the Sp1 region of RECK promoter was increased after ras induction. Interestingly, we also found that LMP1, the Epstein-Barr virus-encoded oncoprotein, induced down-regulation of RECKviathe same Sp1site. We next tested the effect of a HDAC inhibitor trichostatin A (TSA) on ras- and LMP1-induced inhibition of RECK and found that TSA potently antagonized the inhibitory action of ras and LMP1 on RECK. Additionally, our results showed that ras and LMP1 act via extracellular signal-regulated kinases (ERKs) to suppress RECK andinhibition of ERK activity by PD98059 antagonized ras and LMP1-induced down-regulation of RECK. Taken together, our resultssuggestthat oncogenic ras and LMP1represses RECK expression via a histone deacetylation mechanism.
Developmental regulation of focal adhesion complex proteins in kidney by rigidity
Yang-Kao Wang, Chin-I Tsai, Chau-Zen Wang and Ming-Jer Tang
Institute of Basic Medical Sciences and Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Our previous study has found that substratum rigidity of collagen gel down-regulated the expression of focal adhesion complex proteins through integrin in MDCK cells. In this study, we assessed whether the rigidity controlled expression of focal adhesion complex proteins in vivo. We found that focal adhesion kinase (FAK) was degraded in rat embryonic kidney from E17.5 to E19.5 and the intact FAK was completely absent after birth. Other focal adhesion complex proteins such as paxillin, p130cas and cytosolic protein c-src were also down-regulated during the developmental process, similar to the expression pattern of FAK. To assess whether substratum rigidity affected expression of focal adhesion complex proteins, we employed primary cultures of rat proximal tubule cells. Freshly isolated renal proximal tubule cells, initially exhibited little focal adhesion complex proteins, re-expressed focal adhesion complex proteins in primary cultures when they were cultured on collagen gel- or matrigel-coated dishes. Lowering substratum rigidity by culturing cells on collagen gel or matrigel prevented the re-expression of focal adhesion complex proteins in primary culture. Furthermore, in vivo studies demonstrated that induction of tissue hardening by 5/6 nephrectomy resulted in re-expression of focal adhesion complex proteins in kidney. Taken together, these data indicate that the substratum rigidity determines expression of focal adhesion complex proteins both in vitro and in vivo.
Inhibition of v-Src-induced transformation by trichostatin A: implication of p97eps8 as a trichostatin A target
Tzeng-Horng Leu1, Shiuh-Hwa Yeh2, Ya-Chun Chuang1, Shu Li Su2 andMing-Chei Maa2
1 Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, and 2 Institute of Biochemistry, Chung Shan Medical University, Taichung, Taiwan
Histone acetylase (HAT) and histone deacetylase (HDAC) are two crucial enzymes determining the structure of chromatin that regulates gene expression. In this study, we observed that TSA, a specific HDAC inhibitor, could effectively inhibit the growth of v-Src transformed cells (IV5) and abrogate their ability to form colonies in soft agar. Further analysis demonstrated that while TSA reduced the expression of Eps8 in a dose- and time-dependent manner, the kinase activity and expression level of v-Src were not altered. Notably, removal of TSA restored not only the expression of Eps8 but also the cellular growth. Northern and RT-PCR analysis revealed the significant reduction of eps8 transcripts in TSA-treated IV5 relative to control. Furthermore, when active Src expressing CE cells were forced to overexpress p97Eps8, they became resistant to TSA-mediated antiproliferation. Our results highlighted a critical role for p97Eps8 in TSA exerted growth inhibition in v-Src transformed cells.
Bi-directional signals transduced by DAPK-ERK interaction promotes the apoptotic effect of DAPK
Chun-Hau Chen, Won-Jing Wang, Jean-Cheng Kuo, Hsiao-Chien Tsai, and Ruey-Hwa Chen
Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
Death-associated protein kinase (DAPK) is a death domain-containing serine/threonine kinase, and participates in various apoptotic paradigms. Here, we identify extracellular signal-regulated kinase (ERK) as a DAPK-interacting protein. DAPK interacts with ERK through a docking sequence within its death domain and is a substrate of ERK. Phosphorylation of DAPK at Ser735 by ERK increases the catalytic activity of DAPK both in vitro and in vivo. Conversely, DAPK promotes the cytoplasmic retention of ERK, thereby inhibiting ERK signaling in the nucleus. This reciprocal regulation between DAPK and ERK constitutes a positive feedback loop that ultimately promotes the apoptotic activity of DAPK. Disruption of this regulatory circuit by blocking ERK-DAPK interaction attenuates DAPK-mediated apoptosis. These results indicate that bi-directional signaling between DAPK and ERK may contribute to the apoptosis-promoting function of the death domain of DAPK.
Regulation of PKC targeting
Yasuhito Shirai and Naoaki Saito
Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases, plays an important role in various cellular events including differentiation, proliferation, neurotransmission and gene expression etc. How can PKC give distinct roles in such large number of cellular functions. One of the reasons is that the PKC family contains at least 10 subtypes. However, they show similar substrate-specificity in vitro and multiple subtypes are expressed in the same cell. The subtype-specificity should be controlled by well-organized mechanism.
We have so far investigated “PKC targeting” in living cells using GFP-tagged PKC, and found that lipid messengers such as ceramide and fatty acids induce subtype-specific targeting of PKC and each subtype of PKC is distinctly regulated by the lipid messengers. For example, epsilon subtype is translocated from the cytoplasm to the Golgi complex by arachidonic acid, but to the plasma membrane by saturated fatty acids. In contrast, delta subtype doesn’t respond to any fatty acids although it translocates to the Golgi complex in response to ceramide. The differential sensitivity to arachidonic acid of epsilon and delta is determined by the subtle differences in their C1B domain and different intracellular domains of epsilon PKC are involved in its targeting to the Golgi and the plasma membrane.
These results indicate that the PKC targeting regulated by the lipid messengersregulates not only subtype-specific function of PKC but also temporally and spatially distinct activation of PKC, thereby contributing multipul PKC functions. In addition, the diversity of PKC targeting is controlled by their intramolecular domains. Recently, we found that diacylglycerol kinase (DGK) is also important for PKC targeting. The regulation of PKC targeting by DGK is also discussed.
Involvement of protein kinase C in hepatocyte growth factor signaling
Hong-Chen Chen
Graduate Institute of Biomedical Science, National Chung Hsing University, Taichung, Taiwan
Protein kinase C (PKC), a novel type of PKC, has been shown to play critical roles in the control of cell behavior, including cell proliferation, differentiation, and apoptosis. In this study, we found that the selective PKC inhibitor rottlerin, but not the selective classical type PKC inhibitor Gö6976, blocked scatter response of Madin-Darby canine kidney cells to hepatocyte growth factor (HGF). Whereas phorbol ester stimulated a transient activation of PKC, HGF induced a delayed but sustained activation of PKC whose activity was increased 3 h after HGF treatment, gradually reached a plateau (10-fold increase) at 12 h, and lasted for at least another 12 h. Expression of green fluorescent protein (GFP)-tagged PKC in Madin-Darby canine kidney cells revealed that PKCcould localize in different subcellular compartments, including the perinuclear region, vesicle-like organelles, and cell-cell junctions. Upon HGF stimulation, GFP-PKC became evident at the polarized Golgi complexes and membrane ruffles, which correlated with its ability to enhance cell scattering. Taken together, our results establish a new role for PKC in HGF signaling and implicate its role in cell motility.
Induction of the disease-associated keratin 16 gene expression by epidermal growth factor is regulated through cooperation of transcription factors Sp1 and c-Jun
Ying-Nai Wang and Wen-Chang Chang
Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Overexpression of keratin 16 has been observed in keratinocytes in those skin diseases characterized by hyperproliferation such as psoriasis. Therefore, keratin 16 is usually referred to as a disease-associated keratin. In the present study, we found that EGF increasedthe expression of keratin 16 mRNA and protein synthesis in a time-dependent manner in HaCaT cells.Reporter assays revealed that EGF response region was at the range from -162 to -114 bp.Disruption of the Sp1 site (-127 to -122 bp) and the AP1 site (-148 to -142 bp) of the keratin 16 promoter by site-directed mutagenesis significantly inhibited keratin 16 promoter activity induced by EGF. Furthermore, keratin 16 gene expression induced by Ras activation was also regulatedin the same manner as EGF response. By using theDNA affinity precipitation assay in HaCaT and SL2 cells, Sp1directly interacted with the Sp1 site of promoter, and c-Jun and c-Fosprecipitated with the Sp1 oligonucleotide was majorly due to the interaction between Sp1 and AP1 proteins. Moreover, cotransfection assays revealed that Sp1 acted synergistically with c-Jun to activatethe keratin 16. The coactivators p300/CBP could collaborate with Sp1 and c-Jun in the activation of keratin 16 promoter,and EGF-induced promoter activation was blocked by the viral oncoprotein E1A. Taken together, these results suggested that Sp1 and AP1 sites in the essential promoter regionwere critical for EGF response,and Sp1 showed a functional cooperation with c-Jun and coactivators p300/CBP in driving the transcriptional regulation of EGF-induced keratin 16 gene expression.
Opposite effects of HMG-CoA reductase inhibitors on inducible nitric oxide synthase and cyclooxygenase-2gene expression in murine macrophages: acting on signaling cascades of NF-B, STAT1, CREB and C/EBP
Wan-Wan Lin, Jui-Ching Chen,and Ching-Wen Chen
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan
The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors,statins, are potent inhibitors of cholesterol synthesis and have wide therapeutic use in cardiovascular diseases. Recent evidences, however, suggest that the beneficial effects of statins may extend beyond their action on serum cholesterol levels, and are mediated by the impairment of isoprenylation and function of small G proteins. In our study, we investigated the effects of lovastatin, pravastatin, atorvastatin, and fluvastatin on formation of NO and PGE2in murine RAW 264.7 cells. Within 0.1-30 μM, statins can inhibit LPS- and IFN-- induced NO formation and iNOS induction to different extents. This inhibition occurs at the transcriptional level, and displays potency in the order of lovastatin > atorvastatin > fluvastatin pravastatin. In contrast, we observed a stimulatory effect of statin alone on COX-2 induction and PGE2 production, displaying potencies as lovastatin > fluvastatin > atorvastatin > pravastatin. We found that LPS-induced IKK and NF-κB activation, and IFN-γ-induced STAT1 phosphorylation were reduced by lovastatin. Transfection experiments with COX-2 promoter construct showed the necessity of c/EBPand CRE promoter sites, but not NF-B promoter site. Results indicated the requirement of MAPKs in the COX-2 induction. Moreover, statin-induced inhibition of NO production, κB activation, as well as activation of COX-2 promoter, COX-2 protein induction and PGE2 production wereall reversed by mevalonate, geranylgeranyl-PP and farnesyl-PP, suggesting these actions are resulting from impairment of the isoprenylation of small G proteins. The differential effects of statins on iNOS and COX-2 gene expression further indicate the complexity played by small G proteins in regulating signaling pathways.
Clinical isolates of Helicobacter pylori from cancer patients induce COX-2 gene expression and
cell invasion: involvement of TLR2 and c-Src-dependent tyrosine phosphorylation of IKK
Ya-Jen Chang1, Ming-Shiang Wu2, Jaw-Town Lin2 and Ching-Chow Chen1
Department of Pharmacology1 and Internal Medicine2, College of Medicine, National Taiwan University, and Primary Care Medicine2, National Taiwan University Hospital, Taipei, Taiwan
Clinical outcomes associated with H pylori infection include duodenal ulcer, gastric ulcer, gastric adenocarcinoma and gastric mucosa associated lymphoid tissue (MALT) lymphoma.H. pylori infection on gastric epithelial cells has been shown to induce transcriptional factors, such as NF-B, activation. The aim of this study was to elucidate the mechanism of COX-2 gene expression induced by H. pylori isolates from gastric cancer patients andto examine the existence of a novel NF-B activation pathway recently demonstrated from this lab.Induction of COX-2 protein and mRNA expression and COX-2 promoter activity in AGS cells were found. The mutant or dominant negative mutants of TLR2, TLR9, PLCγ2, PKCα, c-Src, NIK or IKK, but not TLR4, inhibited H. pylori-induced COX-2 promoter activiy. Overexpression of wt TLR2, but not wt TLR4 or wt TLR9 increased COX-2 promoter activity. Phosphorylation of IKKβ at Tyr188 and Tyr199via PKC dependent c-Src activation was demonstrated by overexpression of constitutive active PKCα (A/E), or wt c-Src with the dominant negative IKKβ (Y188F), IKKβ (Y199F) or IKK(FF) mutants, and the in vitro c-Src dependent tyrosine phosphorylation of IKKβ. H. pylori isolate from gastric cancer, but not from gastritis induced Matrigel invasion and this effect was inhibited by NS-398 and celecoxib. These results suggest that H. pylori acts through TLR2 /TLR9 then activates both PI-PLCγ /PKC/c-Src/IKKα/β and NIK/IKKα/β, resulting in phosphorylation and degradation of IBα, and leading to stimulation of NF-B and COX-2 gene expression. This COX-2 expression may contribute to the carcinogenesis in cancer patients elicited by these strains.
Chromatin remodeling and transcriptional regulation of steroidogenic acute regulatory protein by prostaglandin E2
Shaw-Jenq Tsai, Kuei-Yang Hsiao, and Chih-Chao Hsu
Department of Physiology, College of Medicine, National Cheng Kung University, Tainan, Taiwan
Aberrant expression of steroidogenic acute regulatory protein (StAR) in ectopic endometriotic stromal cells is believed to play critical roles in the etiology of endometriosis. The expression of StAR is stimulated by prostaglandin (PG) E2, which is elevated in peritoneal fluid due to retrograde menstruation-induced inflammation. Three of four PGE2 receptor (EP) isoforms, namely EP2, EP3 and EP4 were identified in endometriotic stromal cell. PGE2 as well as butaprost (a selective EP2 agonist) triggered PKA-dependent ERK phosphorylation, CREB phosphorylation, histone H3 acetylation, and StAR expression. Treatment of endometriotic stromal cell with pharmacological activators for PKA but not PKC or calcium-calmodulin dependent kinase mimics actions of PGE2. Promoter activity assay using luciferase-fused StAR promoter demonstrated that PGE2-induced StAR expression is mediated by PKA-dependent binding of c/EBPs as well as CREB. Co-immunoprecipitation demonstrated that phosphorylated CREB was physically associated with CREB-binding protein (CBP), a co-activator possess intrinsic histone acetyltransferase activity, and histone H3, a substrate of CBP. By using chromatin immunoprecipitation in conjunction with real-time PCR techniques, we showed StAR promoter bound histone H3 acetylation was increased at 30 min and reached the maximal level at 60 min. Nascent StAR RNA expression was found to elevate at 30 min, peak at 60 min, and return to basal level at 240 min after PGE2. Inactivation of PKA but not ERK activity by selective inhibitors abrogated PGE2-induced CREB phosphorylation, histone H3 acetylation, nascent StAR RNA synthesis, and StAR protein expression. We conclude that induction of StAR by PGE2 is mediated via EP2-coupled, PKA-dependent CREB phosphorylation and histone H3 acetylation leading to chromatin remodeling and thus increase the accessibility of transcription factors to the StAR promoter.