14-3-3 Protein mediates phosphorylation of microtubule-associated protein tau by serum- and glucocorticoid-induced protein kinase 1

The microtubule-associated protein, tau, is involved in numerous neuronal processes such as vesicle transport, microtubule-plasma membrane interaction and the intracellular localization of proteins. Tau is known to be phosphorylated by several kinases such as mitogen activated protein kinase, microtubule affinity regulating kinase, and protein kinase A. We found a putative serum- and glucocorticoid-induced protein kinase 1 (SGK1) phosphorylation site within the 207GSRSRTPSLP216 tau amino acid sequence. We report here that SGK1 phosphorylates Ser214 of Tau. Using a pull-down assay, we found that 14-3-3q interacts with SGK1 and tau to form a ternary protein complex that leads to phosphorylation of tau. 14-3-3 and phosphorylated tau were mainly co-localized in the nucleus of COS-1 cells. These results demonstrate that 14-3-3 scaffolds tau with SGK1 to facilitate the phosphorylation of tau at Ser214 and to regulate its subcellular localization.     

Human follicular dendritic cells express prostacyclin synthase: a novel mechanism to control T cell numbers in the germinal center

Stromal cells in the lymphoid organs provide a microenvironment where lymphocytes undergo various biological processes such as development, homing, clonal expansion, and differentiation. Follicular dendritic cells (FDCs) in the primary and secondary follicles of the peripheral lymphoid tissues interact with lymphocytes by contacting directly or producing diffusible molecules. To understand the biological role of human FDC at the molecular level, we developed a mAb, 3C8, that recognizes FDC but not bone marrow-derived cells. Through expression cloning and proteome analysis, we identified the protein that is recognized by 3C8 mAb, which revealed that FDC expresses prostacyclin synthase. The 3C8 protein purified from FDC-like cells indeed displayed the enzymatic activity of prostacyclin synthase and converted PGH2 into prostacyclin. In addition, prostacyclin significantly inhibited proliferation of T cells but delayed their spontaneous apoptosis. These findings may help explain why T cells constitute only a minor population compared with B cells in the germinal center.     

Trans-activation of mutant follicle-stimulating hormone receptors selectively generates only one of two hormone signals

Previously, we reported that a liganded LH receptor (LHR) is capable of activating itself (cis-activation) and other nonliganded LHRs to induce cAMP (trans-activation). Trans-activation of the LHR raises two crucial questions. Is trans-activation unique to LHR or common to other G protein-coupled receptors? Does trans-activation stimulate phospholipase Cbeta as it does adenylyl cyclase? To address these questions, two types of novel FSH receptors (FSHRs) were constructed, one defective in hormone binding and the other defective in signal generation. The FSHR, a G protein-coupled receptor, comprises two major domains, the N-terminal extracellular exodomain that binds the hormone and the membrane-associated endodomain that generates the hormone signals. For signal defective receptors, the exodomain was attached to glycosyl phosphatidylinositol (ExoGPI) or the transmembrane domain of CD8 immune receptor (ExoCD). ExoGPI and ExoCD can trans-activate another nonliganded FSH. Surprisingly, the trans-activation generates a signal to activate either adenylyl cyclase or phospholipase Cbeta, but not both. These results indicate that trans-activation in these mutant receptors is selective and limited in signal generation, thus providing new approaches to investigating the generation of different hormone signals and a novel means to selectively generate a particular hormone signal. Our data also suggest that the FSHR's exodomain could not trans-activate LHR.     

Mechanisms responsible for regulation of branched-chain amino acid catabolism

The branched-chain amino acids (BCAAs) are essential amino acids and therefore must be continuously available for protein synthesis. However, BCAAs are toxic at high concentrations as evidenced by maple syrup urine disease (MSUD), which explains why animals have such an efficient oxidative mechanism for their disposal. Nevertheless, it is clear that leucine is special among the BCAAs. Leucine promotes global protein synthesis by signaling an increase in translation, promotes insulin release, and inhibits autophagic protein degradation. However, leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway, thereby terminating its positive effects on body protein accretion. A strong case can therefore be made that the proper leucine concentration in the various compartments of the body is critically important for maintaining body protein levels beyond simply the need of this essential amino acid for protein synthesis. The goal of the work of this laboratory is to establish the importance of regulation of the branched chain alpha-ketoacid dehydrogenase complex (BCKDC) to growth and maintenance of body protein. We hypothesize that proper regulation of the activity state of BCKDC by way of its kinase (BDK) and its phosphatase (BDP) is critically important for body growth, tissue repair, and maintenance of body protein. We believe that growth and protection of body protein during illness and stress will be improved by therapeutic control of BCKDC activity. We also believe that it is possible that the negative effects of some drugs (PPAR alpha ligands) and dietary supplements (medium chain fatty acids) on growth and body protein maintenance can be countered by therapeutic control of BCDKC activity.     

The regulation of Clb5 kinase activity by mating factor

Mating factor was found to affect Clb5 kinase activity in Saccharomyces cerevisiae. Mating factor decreased Clb5 kinase activity in a time- and dose-dependent manner. The regulation of Clb5 kinase activity requires functional CLNs (G1 cyclins). Strains without functional CLNs still showed sensitivity to mating factor in the presence of moderately expressing Clb5. This type of mating factor sensitivity is thought to be induced by non-G1 arrest. It is apparent that mating factor treated cells contained inhibitor(s) of Clb5 kinase activity, suggesting that inhibition of Clb5 kinase activity is accompanied by a specific inhibitor. This notion is supported by mixing experiment. Nocodazole treatment showed that the effect of mating factor on Clb5 kinase activity occurred at G1 and connected to mitotic exit. Mating factor regulation of Clb5 kinase activity was found to be dependent on Sic1 protein.     

Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers

Early and reliable detection of plant transformation events is essential for establishing efficient transformation protocols. We have compared the effectiveness of using the gene encoding a green fluorescent protein (GFP) and a beta-glucuronidase (gus) as reporter genes for early detection of transgene expression in explants subjected to biolistic bombardment and Agrobacterium-mediated transformation. The results indicate that gfp gene is superior to gus gene in following transgene expression in transiently transformed materials in both methods of transformation. Using GFP as the screenable marker, we have optimized sorghum transformation with respect to the conditions for transformation, type of explants, promoters, and inbreds. These optimized conditions have been used to obtain stably transformed explants for subsequent regeneration.     

MAPK signaling is involved in camptothecin-induced cell death

Camptothecin, a topoisomerase I inhibitor, is a well-known anticancer drug. However, its mechanism has not been well studied in human gastric cancer cell lines. Camptothecin induced apoptotic cell death in human gastric cancer cell line AGS. Z-VAD-fmk, pan-caspase inhibitor, blocked apoptotic phenotypes induced by camptothecin suggesting that caspases are involved in camptothecin-induced cell death. An inhibitor of caspase-6 or -8 or -9 did not prevent cell death by camptothecin. Various protease inhibitors failed to prevent camptothecin-induced cell death. These results suggest that only few caspases are involved in camptothecin-induced cell death. Camptothecin induced phosphorylation of ERK1/2, JNK, and p38 MAPK, in a dose and time-dependent manner in AGS. Z-VAD-fmk did not affect MAPK signaling induced by camptothecin suggesting that caspase signaling occurs downstream of MAPK signaling. Blocking of p38 MAPK, but not ERK1/2, resulted in partial inhibition of cell death and PARP cleavage by camptothecin in AGS. Taken together, MAPK signaling is associated with apoptotic cell death by camptothecin.