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Background Calcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool. Objectives Here, we investigated whether PKC-β controls intracellular calcium dynamics through Stim1. Methods Several biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope. Results Our data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors. Conclusion In sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.

Cyclophilin A (CypA) is a ubiquitously expressed and highly conserved protein with peptidyl-prolyl cis-trans isomerase activity that is involved in various biological activities by regulating protein folding and trafficking. Although CypA has been reported to positively regulate osteoblast differentiation, the mechanistic details remain largely unknown. In this study, we aimed to elucidate the mechanism of CypA-mediated regulation of osteoblast differentiation. Overexpression of CypA promoted osteoblast differentiation in bone morphogenic protein 4 (BMP4)-treated C2C12 cells, while knockdown of CypA inhibited osteoblast differentiation in BMP4-treated C2C12. CypA and Runx2 were shown to interact based on immunoprecipitation experiments and CypA increased Runx2 transcriptional activity in a dose-dependent manner. Our results indicate that this may be because CypA can increase the DNA binding affinity of Runx2 to Runx2 binding sites such as osteoblast-specific cis-acting element 2. Furthermore, to identify factors upstream of CypA in the regulation of osteoblast differentiation, various kinase inhibitors known to affect osteoblast differentiation were applied during osteogenesis. Akt inhibition resulted in the most significant suppression of osteogenesis in BMP4-induced C2C12 cells overexpressing CypA. Taken together, our results show that CypA positively regulates osteoblast differentiation by increasing the DNA binding affinity of Runx2, and Akt signaling is upstream of CypA.

Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation.

Calcium mobilization is necessary for cell movement during embryonic development, lymphocyte synapse formation, wound healing, and cancer cell metastasis. Depletion of calcium in the lumen of the endoplasmic reticulum using inositol triphosphate (IP3) or thapsigargin (TG) is known to induce oligomerization and cytoskeleton-mediated translocation of stromal interaction molecule 1 (STIM1) to the plasma membrane, where it interacts with the calcium release-activated calcium channel Orai1 to mediate calcium influx; this process is referred to as store-operated calcium entry (SOCE). Furthermore, aberrant STIM1 or SOCE regulation is associated with cancer cell motility and metastasis. The p21-activated kinases (PAKs), which are downstream effectors of GTPases, reportedly regulate cytoskeletal organization, protrusive activity, and cell migration. Although cytoskeletal remodeling apparently contributes to calcium mobilization via SOCE, and vice versa, the mechanisms by which they regulate each other remain unclear. In this study, we aimed to characterize whether PAK1 modulates calcium mobilization and STIM1 localization. Our data demonstrate that PAK1 interacts with STIM1 in vitro and that this interaction was enhanced by treatment with a nascent adhesion inducer, such as phorbol 12,13-dibutyrate (PDBu). Under basal conditions, both proteins appeared to primarily colocalize in the cytosol, whereas treatment with PDBu induced their colocalization to vinculin-positive peripheral adhesions. Downregulation of PAK1 activity via chemical inhibitors or by PAK1 shDNA expression impaired STIM1-mediated calcium mobilization via SOCE. Based on these findings, we propose that PAK1 interacts with STIM1 to regulate calcium mobilization and the formation of cellular adhesions., Cancer: Mediating metastatic migration A molecular mechanism underlying cell movement may contribute to the aggressive migration of metastatic tumor cells. A team led by Ki-Duk Song at Chonbuk National University, Jeonju-si, and Joong-Kook Choi at Chungbuk National University, Cheongju in South Korea investigated the function of a protein called p21-activated kinase 1 (PAK1). PAK1 is known to contribute to the reorganization of cellular structure. The researchers determined that it directly interacts with molecular machinery that controls the storage and release of stockpiled calcium ions at the periphery of the cell where migration takes place. These ions play an important role in enabling cell movement and attachment, and the researchers showed that they could disrupt cellular calcium ion accumulation by switching off the gene encoding PAK1. They now aim to investigate how this mechanism contributes to cancer cell migration.

Clusterin, a protein associated with multiple functions, is expressed in a wide variety of mammalian tissues. Although clusterin is known to be involved in neurodegenerative diseases, ageing, and tumorigenesis, a detailed analysis of the consequences of gain- or loss-of-function approaches has yet to be performed to understand the underlying mechanisms of clusterin functions. Since clusterin levels change in neurological diseases, it is likely that clusterin contributes to cell death and degeneration in general. Zebrafish was investigated as a model system to study human diseases. During development, zebrafish clusterin was expressed in the notochord and nervous system. Embryonic overexpression of clusterin by mRNA microinjection did not affect axis formation, whereas its knock-down by anti-sense morpholino treatment resulted in neuronal cell death. To analyze the function of clusterin in neurodegeneration, a transgenic zebrafish was investigated, in which nitroreductase expression is regulated under the control of a neuron-specific huC promoter which is active between the stages of early neuronal precursors and mature neurons. Nitroreductase turns metronidazole into a cytotoxic agent that induces cell death within 12 h. After metronidazole treatment, transgenic zebrafish showed neuron-specific cell death. Interestingly, we also observed a dramatic induction of clusterin expression in the brain and spinal cord in these fish, suggesting a direct or indirect role of clusterin in neuronal cell death and thus, more generally, in neurodegeneration.

Osterix belongs to the SP gene family and is a core transcription factor responsible for osteoblast differentiation and bone formation. Activation of protein kinase A (PKA), a serine/threonine kinase, is essential for controlling bone formation and BMP-induced osteoblast differentiation. However, the relationship between Osterix and PKA is still unclear. In this report, we investigated the precise role of the PKA pathway in regulating Osterix during osteoblast differentiation. We found that PKA increased the protein level of Osterix; PKA phosphorylated Osterix, increased protein stability, and enhanced the transcriptional activity of Osterix. These results suggest that Osterix is a novel target of PKA, and PKA modulates osteoblast differentiation partially through the regulation of Osterix. J. Cell. Biochem. 115: 1808–1815, 2014. © 2014 Wiley Periodicals, Inc.

Store-operated calcium entry (SOCE) channels composed of Stim and Orai proteins play a critical role in diverse biological processes. Upon endoplasmic reticulum (ER)-mediated calcium (Ca(2+)) depletion, Stim proteins oligomerize with Orai to initiate Ca(2+) influx across the plasma membrane. The ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of ubiquilin 1 are involved in the degradation of presenilin and polyglutamine proteins. Through screening of Orai1 interaction partner(s) that might have an effect on SOCE, ubiquilin 1 was identified as a target of Orai1. However, the UBL and UBA domains of ubiquilin 1 were dispensable for this interaction. Additionally, ubiquilin 1 and Orai1 colocalized in the cytosolic compartment. Ubiquilin 1 increased the ubiquitination of Orai1, resulting in the formation of a high-molecular-weight form. MG132, a proteasome inhibitor, failed to block the degradation of Orai1, whereas bafilomycin A, a lysosome inhibitor, prevented Orai1 degradation. Confocal microscopy studies demonstrated that a fraction of Orai1 colocalized with ubiquilin 1 and the autophagosomal marker LC3. Because Orai1 is a constituent of SOCE, we determined the effect of ubiquilin 1 on Orai1-mediated Ca(2+) influx. As we expected, intracellular Ca(2+) mobilization, a process normally potentiated by Orai1, was downregulated by ubiquilin 1. Taken together, these findings suggest that ubiquilin 1 downregulates intracellular Ca(2+) mobilization and its downstream signaling by promoting the ubiquitination and lysosomal degradation of Orai1.

Members of the TNF family can promote signals in myeloid cells and both positively and negatively regulate the production of pro-inflammatory cytokines depending on the target myeloid cell type. Using the yeast-two hybrid system, we identified transmembrane protein 126A (TMEM126A) as a binding partner for CD137L (4-1BB ligand). We found that TMEM126A associated and co-localized with CD137L in a mouse macrophage cell line and knockdown of TMEM126A with siRNA abolished the CD137L-induced tyrosine phosphorylation as well as the up-regulation of M-CSF, IL-1β and TN-C expressions. Knockdown of TMEM126A also blocked the down-regulation of IL-1β and IL-6 expressions induced by CD137L in thioglycollate-elicited primary peritoneal macrophages. Knockdown of TMEM126A by stable retroviral TMEM126A shRNA transduction also abolished CD137L-induced tyrosine phosphorylation and cell adherence. These findings identify a novel molecule that bridges TNF family cytokines and pro-inflammatory cytokine secretion in myeloid cells.

Naphthoquinone is found in the core structure of many natural compounds, most notably the K vitamins. Numerous molecules with the 1,4-naphthoquinone moiety are known to display distinct biological activities including anti-cancer, anti-inflammatory and anti-bacterial activities. Vitamin K2 and doxorubicin, which are used to treat bleeding and lymphoma respectively, belong to this class of chemicals. Although the exact mechanism of action of these molecules is still under investigation, it may include interactions with DNA, inhibition of topoisomerase II, and production of ROS, all of which contribute individually or in combination to DNA damage and cell death. Based on our previous study, 3 novel naphthoquinone derivatives were synthesized and their anti-proliferative activity against HCT116 colon cancer cells was assessed using FACS and Caspase assays. Using this analysis, we found that the production of ROS by naphthoquinones played a critical role in the induction of apoptosis, since pre-treatment of the cells with antioxidant NAC decreased the cytotoxicity level of these compounds.

Runt-related (RUNX) transcription factors play pivotal roles in neoplastic development and have tissue-specific developmental roles in hematopoiesis (RUNX1), osteogenesis (RUNX2), as well as neurogenesis and thymopoiesis (RUNX3). RUNX3 is a tumor suppressor in gastric carcinoma, and its expression is frequently inactivated by DNA methylation or its protein mislocalized in many cancer types, including gastric and breast cancer. Jun-activation domain-binding protein 1 (Jab1/CSN5), a component of the COP9 signalosome (CSN), is critical for nuclear export and the degradation of several tumor suppressor proteins, including p53, p27(Kip1), and Smad4. Here, we find that Jab1 facilitates nuclear export of RUNX3 that is controlled by CSN-associated kinases. RUNX3 sequestered in the cytoplasm is rapidly degraded through a proteasome-mediated pathway. Our results identify a novel mechanism of regulating nuclear export and protein stability of RUNX3 by the CSN complex.

Purmorphamine is a purine analog and it affects osteogenesis, in part, through the modulation of Sonic Hedgehog signaling pathway. However, the underlying mechanism has remained elusive. Previously, we identified two purmorphamine derivatives that modulate the expression of osteogenic markers includingRunx2, Dlx2 andHes1. However, the extents of osteogenesis varied depending on the assay employed, and this necessitates the identification of reliable markers. Here, we report that the expression of osteocalcin was most sensitive to the treatment of purmorphamine derivatives among genes we analyzed by RT-PCR. Purmorphamine derivatives differentially modulated the expression of early and late osteogenic markers; alkaline phosphatase and osteocalcin gene respectively. In addition, the expression of osteocalcin is also modulated differentially by purmorphamine derivatives over time. Although JNK activity is reported to be necessary for osteocalcin expression, its requirement varied among purmorphamine derivatives. Also the overall cytotoxic response to purmorphamine derivatives appeared slightly different.

The loci of the Pim and Runx gene families have been identified as targets for viral insertions in CD2-myc mice. Synergistic cooperation between Pim and RUNX was also found in the CD2-Runx2 transgenic mouse lymphoma model. RUNX genes have come to prominence recently because of their roles as essential regulators of cell fate in development. Paradoxically, they appear to function either as tumor-suppressor genes or dominant oncogenes according to the cellular context. However, the molecular mechanism of the ambiguous roles played by this family of transcription factors in cancer has remained largely uninvestigated. Here we demonstrate that Pim-1 phosphorylates four Ser/Thr residues within the Runt domain and stabilizes RUNX3 protein. In addition, Pim-1 markedly altered the cellular localization of RUNX3 from the nucleus to the cytoplasm. Our results demonstrate that the subcellular localization of RUNX3 is altered by phosphorylation. We propose that RUNX family members may behave as oncogenes if mislocalized to a cellular micro-compartment.

The ubiquitin-mediated protein degradation pathway has been emphasized for the regulation of numerous cellular mechanisms and the significance of deubiquitination, mediated by deubiquitinating (DUB) enzymes, has been emerging as an essential regulatory step to control these cellular mechanisms. Previously, we demonstrated a human DUB enzyme, HeLa DUB-1, expressed in human ovarian cancer cells. Here, we report human USP36, which has the extension of the C-terminal region of HeLa DUB-1 and has conserved amino acid domains as previously shown in other DUBs. Human USP36, encoding a DUB enzyme, was isolated from ovarian cancer cells using RT-PCR and characterized. We identified DUB enzyme activity of USP36 by analyzing its capability to cleave the ubiquitin. Interestingly, structural and immunoprecipitation analyses revealed for the first time that USP36 contains the PEST motif and is polyubiquitinated.

The Runt domain transcription factors (RUNXs) play essential roles in normal development and neoplasias. Genetic analyses of animals and humans have revealed the involvement of RUNX1 in hematopoiesis and leukemia, RUNX2 in osteogenesis and cleidocranial dysplasia, and RUNX3 in the development of T-cells and dorsal root ganglion neurons and in the genesis of gastric cancer. Here we report that RUNX3 is a target of the acetyltransferase activity of p300. The p300-dependent acetylation of three lysine residues protects RUNX3 from ubiquitin ligase Smurf-mediated degradation. The extent of the acetylation is up-regulated by the transforming growth factor-beta signaling pathway and down-regulated by histone deacetylase activities. Our findings demonstrate that the level of RUNX3 protein is controlled by the competitive acetylation and deacetylation of the three lysine residues, revealing a new mechanism for the posttranslational regulation of RUNX3 expression.

Protein kinase A (PKA), a serine/threonine kinase, regulates bone formation, and enhances Bone morphogenetic protein (BMP)-induced osteoblast differentiation. However, the mechanisms of how PKA controls the cellular response to BMP are not well known. We investigated the effects of modulating PKA activity during BMP2-induced osteoblast differentiation, and found that PKA regulates the function of Dlx3. Dlx3 plays crucial roles in osteoblast differentiation and it is expressed in most skeletal elements during development. We found that PKA activation increases BMP2-induced expression of Dlx3 protein, and enhances the protein stability, DNA binding, and transcriptional activity of Dlx3. In addition, PKA activation induces the phosphorylation of Dlx3 at consensus PKA phosphorylation target site(s). Lastly, substitution of serine 10 in Dlx3 to alanine significantly reduces, if not completely abolishes, the phosphorylation of Dlx3 and the regulation of Dlx3 function by PKA. These results suggest that Dlx3 is a novel target of PKA, and that PKA mediates BMP signaling during osteoblast differentiation, at least in part, by phosphorylating Dlx3 and modulating the protein stability and function of Dlx3.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a distinct open reading frame called K15 at a position equivalent to the gene encoding LMP2A of Epstein-Barr virus (EBV). K15 isolates from body cavity-based lymphoma (BCBL) cells exhibited a dramatic sequence variation and a complex splicing pattern. However, all K15 alleles are organized similarly with the potential SH2 and SH3 binding motifs in their cytoplasmic regions. Northern blot analysis showed that K15 was weakly expressed in latently infected BCBL-1 cells, and the level of its expression was significantly induced by tetradecanoyl phorbol acetate stimulation. K15 encoded 40- to 55-kDa proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was localized at the cytoplasm and plasma membrane. To demonstrate the signal-transducing activity of the K15 protein, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8α polypeptide was replaced with that of KSHV K15. While the CD8-K15 chimera was not capable of eliciting cellular signal transduction upon stimulation with an anti-CD8 antibody, it significantly inhibited B-cell receptor signaling, as evidenced by a suppression of tyrosine phosphorylation and intracellular calcium mobilization. This inhibition required the putative SH2 or SH3 binding motif in the cytoplasmic region of K15. Biochemical study of CD8-K15 chimeras showed that the cytoplasmic region of K15 was constitutively tyrosine phosphorylated and that the tyrosine residue within the putative SH2 binding motif of K15 was a primary site of phosphorylation. These results demonstrate that KSHV K15 resembles LMP2A in genomic location, splicing pattern, and protein structure and by the presence of functional signal-transducing motifs in the cytoplasmic region. Thus, KSHV K15 is likely a distant evolutionary relative of EBV LMP2A.

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus that is most closely related to the human Kaposi’s sarcoma-associated herpesvirus (KSHV). We have identified a distinct open reading frame at the left end of RRV and designated it R1. The position of the R1 gene is equivalent to that of the saimiri transforming protein (STP) of herpesvirus saimiri (HVS) and of K1 of KSHV, other members of the gamma-2 or rhadinovirus subgroup of herpesviruses. The R1 sequence revealed an open reading frame encoding a product of 423 amino acids that was predicted to contain an extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic tail reflective of a type I membrane-bound protein. The predicted structural motifs of R1, including the presence of immunoreceptor tyrosine-based activation motifs, resembled those in K1 of KSHV but were distinct from those of STP. R1 sequences from four independent isolates from three different macaque species revealed 0.95 to 7.3% divergence over the 423 amino acids. Variation was located predominantly within the predicted extracellular domain. The R1 protein migrated at 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was extensively glycosylated. Tagged R1 protein was localized to the cytoplasmic and plasma membranes of transfected cells. Expression of the R1 gene in Rat-1 fibroblasts induced morphologic changes and focus formation, and injection of R1-expressing cells into nude mice induced the formation of multifocal tumors. A recombinant herpesvirus in which the STP oncogene of HVS was replaced by R1 immortalized T lymphocytes to interleukin-2-independent growth. These results indicate that R1 is an oncogene of RRV.

We report the molecular cloning and sequence of the cDNA coding for the biotin-containing subunit of the chloroplastic acetylcoenzyme A (CoA) carboxylase (ACCase) of Arabidopsis thaliana (CAC1). The 3[prime] end of the CAC1 sequence, coding for a peptide of 94 amino acids, which includes a putative biotinylation motif, was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The resulting GST-CAC1 fusion protein was biotinylated in vivo, indicating that CAC1 codes for a biotin-containing protein. Antibodies generated to the GST-CAC1 protein reacted solely with the 38-kD biotin-containing polypeptide of Arabidopsis. Furthermore, these antibodies inhibited ACCase activity in extracts from Arabidopsis leaves. The deduced amino acid sequence of CAC1 has an apparent N-terminal chloroplast-targeting transit peptide. The CAC1 protein is coded by a single Arabidopsis gene, and its mRNA accumulates to the highest levels in organs that are undergoing rapid growth. The amino acid sequence of the CAC1 protein is most similar to the biotin carboxyl-carrier protein component of eubacterial ACCases. These characterizations identify CAC1 as the biotin-containing subunit of the plastidic, heteromeric ACCase of Arabidopsis. The results support the ancient origin of the two structurally distinct ACCases of plants.

FGF-2 is involved in cell survival, proliferation, apoptosis, andangiogenesis in a wide variety of cells. FRGRs, PI3K and MAPkinases are well known mediators of FGF signaling. Despite itsknown roles during many developmental processes, includingosteogenesis, there are few known targets of FGF-2. In thepresent study, we identified Bcl2-A1 and Bcl-xL as two prominenttargets involved in promoting cell survival. Pretreatmentof ATDC5 cells with FGF-2 increased cell survival, whilesiRNAs specific for Bcl2-A1 and Bcl-xL compromised the anti-apoptotic effect of FGF-2, sensitized the cells to apoptosistriggered by TNF-α. Chemical inhibition of FGFR, NFkB, andPI3K activity by PD173074, pyrrolidine dithiocarbamate, andLY294002 respectively abrogated the FGF-2-mediated inductionof Bcl2-A1 and Bcl-xL expression. Taken together, our datademonstrate that a subset of Bcl2 family proteins are the targetsof FGF-2 signaling that promotes the survival of ATDC5cells. [BMB reports 2012; 45(5): 287-292]

Herpesvirus saimiri (HVS), a T-lymphotropic monkey herpesvirus, induces fulminant T-cell lymphoma in non-natural primate hosts. In addition, it can immortalize human T-cells in vitro. HVS tyrosine kinase-interacting protein (Tip) is an essential viral gene required for T-cell transformation both in vitro and in vivo. In this study, we found that Tip interacts with the STAT6 transcription factor and induces phosphorylation of STAT6 in T-cells. The interaction with STAT6 requires the Tyr127 residue and Lck-binding domain of Tip, which are indispensable for interleukin (IL)-2-independent T-cell transformation by HVS. It was also demonstrated that Tip induces nuclear translocation of STAT6, as well as activation of STAT6-dependent transcription in Jurkat T-cells. Interestingly, the phosphorylated STAT6 mainly colocalized with vesicles containing Tip within T-cells, but was barely detectable in the nucleus. However, nuclear translocation of phospho-STAT6 and transcriptional activation of STAT6 by IL-4 stimulation were not affected significantly in T-cells expressing Tip. Collectively, these findings suggest that the constitutive activation of STAT6 by Tip in T-cells may contribute to IL-2-independent T-cell transformation by HVS.

Dlx5 transcription factor plays important roles in osteoblast differentiation and its transcription is regulated by many osteogenic signals including BMP-2. Recent studies suggest that the function of Dlx5 is also regulated post-translationally by protein kinases such as p38 and CaMKII. Protein kinase A (PKA) is involved in several steps of osteoblast differentiation and its activity has been shown necessary, yet not sufficient, for BMP-induced osteoblast differentiation. PKA is a ubiquitous cellular kinase that phosphorylates serine and threonine residues(s) of target proteins. In this study, we investigated the potential regulation of Dlx5 function by PKA in osteoblast differentiation. We found that PKA phosphorylates Dlx5 and that PKA activation increases the protein stability, osteogenic activity and transcriptional activity of Dlx5. We also found that BMP-2 increases the protein level of Dlx5 in a PKA activity-dependent manner. These results suggest that PKA activity enhances the osteogenic function of Dlx5, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Dlx5, at least in part, through PKA.

Anti cancer agent 5-FU (Fluoro Uracil) is a prodrug that can be metabolized and then activated to interfere with RNA and DNA homeostasis. However, the majority of administered 5-FU is known to be catabolized in vivo in the liver where Dihydropyrimidine dehydrogenase (DPD) is abundantly expressed to degrade 5-FU. The biological factors that correlate with the response to 5-FU-based chemotherapy have been proposed to include uridine phosphorylase (UPP), thymidine phosphorylase (TPP), p53 and microsatellite instability. Among these, the expression of UPP is known to be controlled by cytokines such as TNF-alpha, IL1 and IFN-gamma. Our preliminary study using a DNA microarray technique showed that basic fibroblast growth factor (bFGF) markedly induced the expression of UPP1 at the transcription level. In the present study, we investigated whether bFGF could modulate the expression of UPP1 in osteo-lineage cells and examined the sensitivity of these cells to 5-FU mediated apoptosis.

Viral proteins of gamma-2 herpesviruses, such as LMP2A of Epstein Barr virus (EBV) and Tip of herpesvirus saimiri (HVS) dysregulate lymphocyte signaling by interacting with Src family kinases. K15 open reading frame of Kaposi's sarcoma associated herpesvirus (KSHV), located at the right end of the viral genome, encodes several splicing variants differing in numbers of transmembrane domains. Previously, we demonstrated that the cytoplasmic tail of the K15 protein interfered with B cell receptor signal transduction to cellular tyrosine phosphorylation and calcium mobilization. However, the detailed mechanism underlying this phenomenon was not understood. In the C-terminal cytoplasmic region of K15, putative binding domains for Src-SH2 and -SH3 were identified. In this study, we attempted to characterize these modular elements and cellular binding protein(s) by GST pull down and co-immunoprecipitation assays. These studies revealed that K15 interacted with the major B cell tyrosine kinase Lyn. In vitro kinase and transient co-expression assays showed that the expression of K15 protein resulted in activation of Lyn kinase activity. In addition, GST pull down assay suggested that the SH2 domain of Lyn alone was necessary for interaction with the C-terminal SH2B (YEEV) of K15, but the addition of Lyn SH3 to the SH2 domain increases the binding affinity to K15 protein. The data from luciferase assays indicate that K15 expression in BJAB cells induced NFAT and AP1 activities. The tyrosine residue in the C-terminal end of K15 required for the Lyn interaction appeared to be essential for NFAT/AP1 activation, highlighting the significance of the C-terminal SH2B of K15 as a modular element in interfering with B lymphocyte signaling through interaction with Lyn kinase.

Herpesvirus saimiri (HVS) is divided into three subgroups, A, B, and C, based on sequence divergence at the left end of genomic DNA in which the saimiri transforming protein (STP) resides. Subgroup A and C strains transform primary common marmoset lymphocytes to interleukin-2-independent growth, whereas subgroup B strains do not. To investigate the nononcogenic phenotype of the subgroup B viruses, STP genes from seven subgroup B virus isolates were cloned and sequenced. Consistent with the lack of oncogenic activity of HVS subgroup B viruses, STP-B was deficient for transforming activity in rodent fibroblast cells. Sequence comparison reveals that STP-B lacks the signal-transducing modules found in STP proteins of the other subgroups, collagen repeats and an authentic SH2 binding motif. Substitution mutations demonstrated that the lack of collagen repeats but not an SH2 binding motif contributed to the nontransforming phenotype of STP-B. Introduction of the collagen repeat sequence induced oligomerization of STP-B, resulting in activation of NF-κB activity and deregulation of cell growth control. These results demonstrate that the collagen repeat sequence is a determinant of the degree of HVS STP transforming activity.

The STP oncoproteins of the herpesvirus saimiri (HVS) subgroup A strain 11 and subgroup C strain 488 are now found to be stably associated with tumor necrosis factor receptor-associated factor (TRAF) 1, 2, or 3. Mutational analyses identified residues of PXQXT/S in STP-A11 as critical for TRAF association. In addition, a somewhat divergent region of STP-C488 is critical for TRAF association. Mutational analysis also revealed that STP-C488 induced NF-κB activation that was correlated with its ability to associate with TRAFs. The HVS STP-C488 P 10 →R mutant was deficient in human T-lymphocyte transformation to interleukin-2-independent growth but showed wild-type phenotype for marmoset T-lymphocyte transformation in vitro and in vivo. The STP-C488 P 10 →R mutant was also defective in Rat-1 fibroblast transformation, and fibroblast cell transformation was blocked by a TRAF2 dominant-negative mutant. These data implicate TRAFs in STP-C488-mediated transformation of human lymphocytes and rodent fibroblasts. Other factors are implicated in immortalization of common marmoset T lymphocytes and may also be critical in the transformation of human lymphocytes and rodent fibroblasts.

Kaposi's sarcoma-associated herpesvirus (KSHV) is consistently identified in Kaposi's sarcoma and body cavity-based lymphoma. KSHV encodes a transforming protein called K1 which is structurally similar to lymphocyte receptors. We have found that a highly conserved region of the cytoplasmic domain of K1 resembles the sequence of immunoreceptor tyrosine-based activation motifs (ITAMs). To demonstrate the signal-transducing activity of K1, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8alpha polypeptide was replaced with that of KSHV K1. Expression of the CD8-K1 chimera in B cells induced cellular tyrosine phosphorylation and intracellular calcium mobilization upon stimulation with an anti-CD8 antibody. Mutational analyses showed that the putative ITAM of K1 was required for its signal-transducing activity. Furthermore, tyrosine residues of the putative ITAM of K1 were phosphorylated upon stimulation, and this allowed subsequent binding of SH2-containing proteins. These results demonstrate that the KSHV transforming protein K1 contains a functional ITAM in its cytoplasmic domain and that it can transduce signals to induce cellular activation.

Recognition of phosphorylated serine/threonine-containing motifs by 14-3-3 depends on the dimerization of 14-3-3. However, the molecular cues that control 14-3-3 dimerization are not well understood. In order to identify proteins that control 14-3-3 dimerization, we analyzed proteins that have effects on 14-3-3 dimerization and report that protein kinase A (PKA) phosphorylates 14-3-3zeta at a specific residue (Ser58). Phosphorylation by PKA leads to modulation of 14-3-3zeta dimerization and affect its interaction with partner proteins. Substitution of Ser58 to Ala completely abolished phosphorylation of 14-3-3zeta by PKA. A phospho-mimic mutant of 14-3-3zeta, Ser58 to Glu substitution, failed to form homodimers, showed reduced interaction with 14-3-3epsilon and p53, and could not enhance transcriptional activity of p53. Moreover, activation of PKA decreases and inhibition of PKA increases the dimerization of 14-3-3zeta and the functional interaction of 14-3-3zeta with p53. Therefore, our results suggest that PKA is a new member of protein kinases that can phosphorylate and impair the function of 14-3-3.

Protein kinase C (PKC) signaling regulates osteoblast differentiation, but little is known about its downstream effectors. We examined the effect of modulating PKC activity on osteogenic transcription factors and found that the protein level of Msx2 is affected. Msx2 is induced by osteogenic signals such as BMPs and it plays critical roles in bone formation and osteoblast differentiation. Here, we examined the role of PKC signaling in regulating the function of Msx2. We found that the inhibition of PKC signaling enhances osteogenic differentiation in BMP2-stimulated C2C12 cells. Treatment with inhibitors of PKC activity or overexpression of kinase-defective (KD), dominant-negative mutant PKC isoforms strongly reduced the level of Msx2 protein. Several PKC isoforms (α, β, δ, and ζ) interacted with Msx2, and PKCβ phosphorylated Msx2 at Thr135 and Thr141. Msx2 repressed the transcriptional activity of the osteogenic transcription factor Runx2, and this repression was relieved by inhibition of PKC activity or overexpression of the KD mutant PKC isoforms. In addition, PKC prolonged the half-life of Msx2 protein. These results suggest that PKC signaling modulates osteoblast differentiation, at least in part, through the regulation of Msx2.