Protein Ser/Thr kinase CK2 (casein kinase II) is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-GlcNAc on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344) on the same protein. We use protein semisynthesis to show that Thr344 phosphorylation increases CK2α cellular stability via Pin1 interaction whereas Ser347 glycosylation appears to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with the site-specifically modified phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that CK2 kinase substrate selectivity is modulated by these specific posttranslational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5-triphosphorylated, 2,5-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2,5-oligoadenylate synthetases (2,5-OASs), inactivated by 5-phosphatase and completely degraded by 2-phosphodiesterase (2-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2,5-OASs and RNase L have been molecularly cloned and studied well, the identification of 2-PDE has remained elusive. Here, we describe the first identification of 2-PDE, the third key enzyme of the 2-5A system. We found a putative 2-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2-PDE by mass spectrometry. Based on the full-length sequence of the human 2-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2-PDE expressed in mammalian cells certainly cleaved the 2,5-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2-PDE were found, the human 2-PDE was considered to be a unique enzyme without isoform. Suppression of 2-PDE by a small interfering RNA and a 2-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments.

Bone is maintained by two cell types, bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts express two factors, osteoprotegerin and receptor activator of NF-kappaB ligand (RANKL), inhibiting and promoting osteoclast differentiation, respectively. In contrast, modulators of bone resorption expressed by osteoclasts have not been so well studied enough. In the present study, we demonstrate proteome analysis of secreted proteins during osteoclast differentiation to elucidate the molecular mechanism of bone resorption and bone remodeling. To achieve this objective, we chose RAW264.7 cells with RANKL as a homogeneous osteoclast differentiation model and used two methods, two-dimensional gel electrophoresis (2-DE) and isotope-coded affinity tags (ICAT) analysis with two-dimensional liquid chromatography. We found 23 spots in 2-DE and 19 proteins in ICAT analysis which were expressed differently during osteoclast differentiation. These two methods gave us closely related but different information about proteins, suggesting they are complementary or at least supplementary methods at present. Cathepsins, osteopontin, legumain, macrophage inflammatory protein-1alpha, and other proteins were observed as up- or down-regulated proteins and are discussed in the context of osteoclast differentiation and bone resorption. In addition to confirming previous observations, this study indicates novel proteins related to osteoclast differentiation which are potential therapeutic targets for the treatment of bone diseases, such as osteoporosis.

In iodine-bridged binuclear Pt compounds, R(4)[Pt(2)(pop)(4)I]nH(2)O and R(')(2)[Pt(2)(pop)(4)I]nH(2)O (pop=P(2)O5H2-2), electronic structures on the PtPtI chains have been controlled between a diamagnetic charge-density-wave (CDW) state and a paramagnetic charge-polarization (CP) state by modification of the counterions (R, R') located between chains. In the R=(C2H5)2NH+2 compound, a pressure-induced CP to CDW transition with a drastic color change is identified. This transition is accompanied by a large hysteresis loop within which photoinduced transition between CDW and CP can be driven by selecting the excitation photon energy.

Pravastatin sodium (CS-514) is a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in cholesterol biosynthesis. This compound is obtained by microbial hydroxylation of sodium ML-236B (compactin) carboxylate. The soluble cytochrome P-450 was induced by sodium ML-236B carboxylate in Streptomyces carbophilus of Actinomycetes as detected in its cell-free extract. This cytochrome P-450 was designated as cytochrome P-450,,, after its origin. Cytochrome P-450,,, was purified by successive chromatography on anion-exchange, gel filtration and hydroxyapatite columns. On hydroxyapatite cytochrome P-450,,, was further separated into minor and major peaks, designated cytochrome P-450,,,_1 and cytochrome P-450,,,-2, respectively. Each peak yielded a single band on sodium dodecyl sulfate/polyacrylamide gels with molecular masses of 46 1 kDa. The activity hydroxylating sodium ML-236B carboxylate to pravastatin sodium was reconstituted in the presence of an electron transport system, an NADPH-generating system and oxygen. The Ks values of the cytochromes P-450,,,-1 and P-450,,,.2 for sodium ML-236B carboxylate were 179 pM and 229 pM, respectively. The CO versus reduced difference spectra of both cytochromes P-450 showed an absorption maximum at 448.5 nm. Their substrate difference spectra with sodium ML-236B carboxylate showed an absorption maximum at 386 nm. Amino acid analysis indicated that cytochrome P-450,,,-1 and P-450,,,-2 contained 46% and 47% hydrophobic residues, respectively. On Western blotting, cytochromes P-45OS,,-and P-450,,,-2 were immunologically identical.Pravastatin sodium (the generic name for CS-514, hereafter referred to as pravastatin) [l -41 is obtained by hydroxylation of sodium ML-236B carboxylate [ 5 , 6 ] at the 68 position (Fig.

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We report the observation of nuclear quadrupole resonance (NQR) of Cu from the sites near the doping center in the spin-Peierls system CuGe 1-xSixO3. The signal appears as the satellites in the Cu NQR spectrum, and has a suppressed nuclear spin-lattice relaxation rate indicative of a singlet correlation rather than an enhanced magnetic correlation near the doping center. Signal loss of Cu nuclei with no neighboring Si is also observed. We conclude from these observations that the doping-induced moments are not in the vicinity of the doping center but rather away from it.