Biomedical subjects

W K Berlin

Publications and source records attributed to W K Berlin.

5 recordsLinked to original sources

PubMed1999

Phosphorylation and glycosylation of nucleoporins.

The nuclear pore complex mediates macromolecular transport between the nucleus and cytoplasm. Many nuclear pore components (nucleoporins) are modified by both phosphate and O-linked N-acetylglucosamine (O-GlcNAc). Among its many functions, protein phosphorylation plays essential roles in cell cycle progression. The role of O-GlcNAc addition is unknown. Here, levels of nucleoporin phosphorylation and glycosylation during cell cycle progression are examined. Whereas nuclear pore glycoproteins are phosphorylated in a cell-cycle-dependent manner, levels of O-GlcNAc remain constant. The major nucleoporin p62 can be phosphorylated in vitro by protein kinase A and glycogen synthase kinase (GSK)-3alpha but not by cyclin B/cdc2 or GSK-3beta. The consensus sites of these kinases resemble sites which can be glycosylated by O-GlcNAc transferase. These data are consistent with a model that O-GlcNAc limits nucleoporin hyperphosphorylation during M-phase and hastens the resumption of regulated nuclear transport at the completion of cell division.

Acetylglucosamine↗

PubMed1998

Nuclear glycogen and glycogen synthase kinase 3.

Glycogen is the principal storage form of glucose in animal cells. It accumulates in electron-dense cytoplasmic granules and is synthesized by glycogen synthase (GS), the rate-limiting enzyme of glycogen deposition. Glycogen synthase kinase-3 (GSK-3) is a protein kinase that phosphorylates GS. Two nearly identical forms of GSK-3 exist: GSK-3 alpha and GSK-3 beta. Both are constitutively active in resting cells and their activity can be modulated by hormones and growth factors. GSK-3 is implicated in the regulation of many physiological responses in mammalian cells by phosphorylating substrates including neuronal cell adhesion molecule, neurofilaments, synapsin I, and tau. Recent observations point to functions for glycogen and glycogen metabolism in the nucleus. GSK-3 phosphorylates several transcription factors, and we have recently shown that it modifies the major nuclear pore protein p62. It also regulates PK1, a protein kinase required for maintaining the interphase state and for DNA replication in cycling Xenopus egg extracts. Recently, glycogen was shown to be required for nuclear reformation in vitro using ovulated Xenopus laevis egg lysates. Because neither glycogen nor GSK-3 has been localized to the nuclear envelope or intranuclear sites, glycogen and GSK-3 activites were measured in rat liver nuclei and nuclear reformation extracts. Significant quantities of glycogen-like material co-purified with the rat-liver nuclear envelope. GSK-3 is also highly enriched in the glycogen pellet of egg extracts of Xenopus that is required for nuclear assembly in vitro. Based on the finding that enzymes of glycogen metabolism copurify with glycogen, we propose that glycogen may serve a structural role as a scaffold for nuclear assembly and sequestration of critical kinases and phosphatases in the nucleus.

Animals↗

PubMed1995

Characterization of nuclear pore protein p62 produced using baculovirus.

Nuclear pore glycoproteins are essential components of the nuclear import apparatus in eukaryotes. In vertebrates, the most abundant of these glycoproteins is a molecule called p62. Like other O-linked N-acetylglucosamine glycoproteins, p62 is normally modified in the cytoplasm and cannot be overexpressed and conveniently collected in a secreted form. We devised an efficient scheme for expression and purification of recombinant p62 from Sf9 cells that may have general applicability for this class of glycoproteins. The purified rat p62 bound to wheat germ agglutinin, consistent with modification by O-linked N-acetylglucosamine. Carbohydrate analysis, in conjunction with amino acid analysis, revealed that baculovirus-expressed rat p62 contains 5-6 mol of N-acetylglucosamine/mol of p62. As observed by circular dichroism, purified p62 expressed in the baculovirus system or in Escherichia coli share essentially the same secondary structure. Purified glycosylated rat p62 will be critical in determining the role of N-acetylglucosamine in both nuclear transport and assembly of the nuclear pore complex.

Acetylglucosamine↗

PubMed1995

Inhibition of iodide transport in rat thyroid cells using N-substituted anthranilic acid derivatives.

The purpose of this study was to test the effects of chloride channel blockers on iodide uptake in thyroid cells, in the hope of eventually using these blockers to identify and isolate a putative iodide transporter. The chloride channel blockers used in this report are derivatives of N-substituted anthranilic acid and were synthesized using published procedures. For these studies FRTL-5 cells, a line of continuous-growing rat thyroid cells, were used as a model system to study effects on iodide transport. In these cells, there are at least two ways for transmembrane iodide movements, a sodium-dependent influx step and a proposed channel that normally mediates iodide efflux. Two derivatives studied decreased iodide accumulation in FRTL-5 cells, but were found also to lower intracellular pH and ATP levels. To simplify interpretation of the effect of the drugs on iodide transport, we extended the studies using plasma membrane vesicles made from pig thyroid. Iodide entry in these vesicles depended on a sodium gradient and was independent of ATP levels. Iodide transport in plasma membrane vesicles and FRTL-5 cells was measured at 30 sec when the uptake was nearly linear and therefore likely to reflect iodide entry. The uptake was measured using three concentrations of iodide and three of drug. Kinetic analysis of the data described a competitive inhibition by the drugs with a Ki of approximately 250 microM. In summary, N-substituted anthranilic acid derivatives reversibly inhibit iodide entry in FRTL-5 cells and pig plasma membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1994

alpha 2-8 Sialic acid polymers: size, structure, and compositional analysis.

A series of three variable assay procedures is described to provide overlapping information on the size, structure, and composition of the alpha 2-8 linked polysialic acid chains present on a wide variety of critical cell surface glycoproteins. Technical advances in instrumentation have permitted the development of new applications for a methodology involving the sequential use of periodate and borohydride to modify terminal sialic acid residues. The procedures described here provide a rapid and facile assay for (a) the determination of polysialic acid chain length, (b) the simultaneous identification of N-acetylneuraminic acid, N-glycolyneuraminic acid, and KDN (deaminated sialic acid) when present in a single preparation, (c) the ability to distinguish qualitatively between reducing and nonreducing polymers, and (d) the ability to determine the number of chains bound to a glycoprotein of known molecular weight.

Carbohydrate Conformation↗