Protein Kinase CK2 Is Upregulated by Calorie Restriction and Induces Autophagy

Calorie restriction (CR) and the activation of autophagy extend healthspan by delaying the onset of age-associated diseases in most living organisms. Because protein kinase CK2 (CK2) downregulation induces cellular senescence and nematode aging, we investigated CK2’s role in CR and autophagy. This study indicated that CR upregulated CK2’s expression, thereby causing SIRT1 and AMP-activated protein kinase (AMPK) activation. CK2α overexpression, including antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760, stimulated autophagy initiation and nucleation markers (increase in ATG5, ATG7, LC3BII, beclin-1, and Ulk1, and decrease in SQSTM1/p62). The SIRT1 deacetylase, AKT, mammalian target of rapamycin (mTOR), AMPK, and forkhead homeobox type O (FoxO) 3a were involved in CK2-mediated autophagy. The treatment with the AKT inhibitor triciribine, the AMPK activator AICAR, or the SIRT1 activator resveratrol rescued a reduction in the expression of lgg-1 (the Caenorhabditis elegans ortholog of LC3B), bec-1 (the C. elegans ortholog of beclin-1), and unc-51 (the C. elegans ortholog of Ulk1), mediated by kin-10 (the C. elegans ortholog of CK2β) knockdown in nematodes. Thus, this study indicated that CK2 acted as a positive regulator in CR and autophagy, thereby suggesting that these four miRs’ antisense inhibitors can be used as CR mimetics or autophagy inducers.     

Regulation of protein kinase CKII by direct interaction with the C-terminal region of p47(phox)

Protein kinase CKII is a Ser/Thr kinase which is involved in many proliferation-related processes in the cell. p47(phox) is a component of the leukocyte NADPH oxidase, which is an important element of host defense against microbial infection. In this study, we demonstrate that a truncated form of the p47(phox) lacking its N-terminal region (p47(phox)/SH3-C), but not a truncated form of the p47(phox) lacking its C-terminal region (p47(phox)/N-SH3), undergoes better phosphorylation by CKII in the presence of arachidonic acid. The yeast two-hybrid test and the glutathione S-transferase (GST) pull-down assay showed that p47(phox) interacts specifically with the regulatory beta subunit (CKIIbeta), but not with the catalytic alpha subunit (CKIIalpha) of the tetrameric CKII holoenzyme. The binding of p47(phox) to CKIIbeta requires the C-terminal region of p47(phox) and is completely abolished by addition of spermine, indicating that a highly basic region in the C-terminal region of p47(phox) contributes to binding to CKIIbeta. In addition, p47(phox) stimulates the catalytic activity of CKII holoenzyme; this stimulation also requires the C-terminal region of p47(phox). Coimmunoprecipitation experiments showed that CKII holoenzyme interacts with p47(phox) in human neutrophils. Taken together, the present data indicate that the C-terminal region of p47(phox) plays a significant role in the arachidonic acid-dependent phosphorylation of p47(phox) by CKII and that the same region of p47(phox) associates directly with CKIIbeta and can modulate the catalytic activity of CKII holoenzyme.     

Localization of phospholipase C-gamma1 signaling in caveolae: importance in EGF-induced phosphoinositide hydrolysis but not in tyrosine phosphorylation

Upon epidermal growth factor treatment, phospholipase C-gamma1 (PLC-gamma1) translocates from cytosol to membrane where it is phosphorylated at tyrosine residues. Caveolae are small plasma membrane invaginations whose structural protein is caveolin. In this study, we show that the translocation of PLC-gamma1 and its tyrosine phosphorylation are localized in caveolae by caveolin-enriched low-density membrane (CM) preparation and immunostaining of cells. Pretreatment of cells with methyl-beta-cyclodextrin (MbetaCD), a chemical disrupting caveolae structure, inhibits the translocation of PLC-gamma1 to CM as well as phosphatidylinositol (PtdIns) turnover. However, MbetaCD shows no effect on tyrosine phosphorylation level of PLC-gamma1. Our findings suggest that, for proper signaling, PLC-gamma1 phosphorylation has to occur at PtdInsP(2)-enriched sites.     

Incorporation of beta-selenolo[3,2-b]pyrrolyl-alanine into proteins for phase determination in protein X-ray crystallography

beta-Selenolo[3,2-b]pyrrolyl-L-alanine that mimics tryptophan with the benzene ring of the indole moiety replaced by selenophene, was incorporated into human annexin V and barstar. This was achieved by fermentation and expression in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. The seleno- proteins were obtained in yields comparable to those of the wild-type proteins and exhibit full crystallographic isomorphism to the parent proteins, but expectedly show altered absorbance profiles and quenched tryptophan fluorescence. Since the occurrence of tryptophan residues in proteins is rare, incorporation of the electron-rich selenium-containing tryptophan surrogate into proteins represents a useful supplementation and even a promising novel alternative to selenomethionine for solving the phase problem in protein X-ray crystallography.     

Expression, purification, characterization and crystallization of flap endonuclease-1 from Methanococcus jannaschii

A gene coding for a protein homologous to the flap endonuclease-1 (FEN-1) was cloned from Methanococcus jannaschii, overexpressed, purified and characterized. The gene product from M. jannaschii shows 5' endo-/exonuclease and 5' pseudo-Y-endonuclease activities as observed in the FEN-1 in eukaryotes. In addition, Methanococcus jannaschii FEN-1 functions effectively at high concentrations of salt, unlike eukaryotic FEN-1. We have crystallized Methanococcus jannaschii FEN-1 and analyzed its preliminary character. The crystal belongs to the space group of P2(1) with unit cell dimensions of a = 58.93 A, b = 42.53 A, c = 62.62 A and beta = 92.250. A complete data set has been collected at 2.0 A resolution using a frozen crystal.     

The alpha-helical domain of Galphat determines specific interaction with regulator of G protein signaling 9

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.     

Trp-Lys-Tyr-Met-Val-Met activates mitogen-activated protein kinase via a PI-3 kinase-mediated pathway independent of PKC

Trp-Lys-Tyr-Met-Val-Met (WKYMVM) is a novel potent peptide which can stimulate phosphoinositide hydrolysis in U937 as well as U266 and HL-60 cells (Baek et al., J. Biol. Chem. 271, 8170 (1996)). The peptide also induces superoxide generation in human neutrophils (Seo et al., J. Immunol. 158, 1896 (1997)). However, the signaling pathway down-stream of PLC set in motion by the peptide is not yet completely understood. We studied the signaling pathway of the peptide with the goal of elucidating the mechanism of the peptide's action. WKYMVM induced a rapid and transient activation of the ERKs in human histiocytic lymphoma cells, U937. The ERK1 activation peaked at 5 min and returned to the basal level after 30 min. The ERK1 stimulation by the peptide was partially inhibited by pretreatment of the cells with pertussis toxin (PTX), implicating G-protein involvement in the peptide's action. Pretreatment of staurosporine, protein kinase C (PKC) inhibitor, or PKC down-regulating PMA had no impact on the ERK1 activation by the peptide, indicating that the signaling pathway is independent of PKC activation. Pretreatment of the cells with neomycin and intracellular Ca2+ mobilizing reagents had also no effect on the ERK1 activation by the peptide. However, pretreatment with wortmannin or LY294002, the inhibitor of phosphatidylinositol 3 kinase (PI-3K), strongly inhibited peptide-stimulated ERK1 activation. Our results suggest that PI-3K may be an important participant in the ERK cascade induced by the peptide. Furthermore, the treatment of U937 cells with the peptide activated p74Raf-1, an upstream kinase of ERK. Taken together, our results suggest that the peptide activate ERK via a G-protein/PI-3K/Ras/Raf-1 mediated signaling pathway in U937 cells.     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.