Structure of anabolic ornithine carbamoyltransferase from Campylobacter jejuni at 2.7 Å resolution.

Anabolic ornithine transcarbamoylase (aOTC) catalyzes the reaction between carbamoyl phosphate (CP) and L-ornithine (ORN) to form L-citrulline and phosphate in the urea cycle and L-arginine biosynthesis. The crystal structure of unliganded aOTC from Campylobacter jejuni (Cje aOTC) was determined at 2.7 Å resolution and refined to an R(work) of 20.3% and an R(free) of 24.0%. Cje aOTC is a trimer that forms a head-to-head pseudohexamer in the asymmetric unit. Each monomer is composed of an N-terminal CP-binding domain and a C-terminal ORN-binding domain joined by two interdomain helices. The Cje aOTC structure presents an open conformation of the enzyme with a relatively flexible orientation of the ORN-binding domain respective to the CP-binding domain. The conformation of the B2-H3 loop (residues 68-78), which is involved in binding CP in an adjacent subunit of the trimer, differs from that seen in homologous proteins with CP bound. The loop containing the ORN-binding motif (DxxxSMG, residues 223-230) has a conformation that is different from those observed in unliganded OTC structures from other species, but is similar to those in structures with bound ORN analogs. The major differences in tertiary structure between Cje aOTC and human aOTC are described.

Developmentally spliced PKCbetaII provides a possible link between mTORC2 and Akt kinase to regulate 3T3-L1 adipocyte insulin-stimulated glucose transport.

Functional adipocyte glucose disposal is a key component of global glucose homeostasis. PKCbetaII is involved in rat skeletal muscle cell ISGT. Western blot analysis and real-time PCR revealed 3T3-L1 cells developmentally regulated PKCbeta splicing such that PKCbetaI was downregulated and PKCbetaII was upregulated during the course of differentiation. An initial glucose uptake screen using PKC inhibitor LY379196 pointed to a PKC isozyme other than PKCzeta mediating 3T3-L1 adipocyte ISGT. Subsequent use of PKCbetaII inhibitor CGP53353 pointed to a role for PKCbetaII in ISGT. Western blot analysis showed that CGP53353 specifically inhibited phosphorylation of PKCbetaII Serine 660. Subcellular fractionation and immunofluorescence demonstrated that PKCbetaII regulates GLUT4 translocation. Further Western blot, immunofluorescence and co-immunoprecipitation analysis reveal that PKCbetaII inhibition does not affect mTORC2 activity yet abrogates phosphorylation of Akt Serine 473. PKCbetaII regulates GLUT4 translocation by regulating Akt phosphorylation and thus activity.

Functional involvement of protein kinase C-betaII and its substrate, myristoylated alanine-rich C-kinase substrate (MARCKS), in insulin-stimulated glucose transport in L6 rat skeletal muscle cells.

AIMS/HYPOTHESIS: Insulin stimulates phosphorylation cascades, including phosphatidylinositol-3-kinase (PI3K), phosphatidylinositol-dependent kinase (PDK1), Akt, and protein kinase C (PKC). Myristoylated alanine-rich C-kinase substrate (MARCKS), a PKCbetaII substrate, could link the effects of insulin to insulin-stimulated glucose transport (ISGT) via phosphorylation of its effector domain since MARCKS has a role in cytoskeletal rearrangements. METHODS: We examined phosphoPKCbetaII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCbetaII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCbetaII and calmodulin inhibitors, respectively, before insulin treatment, using western blots. ISGT was examined after cells had been treated with inhibitors, small inhibitory RNA (siRNA) for MARCKS, or transfection with MARCKS mutated at a PKC site. MARCKS, PKCbetaII, GLUT4 and insulin receptor were immunoblotted in subcellular fractions with F-actin antibody immunoprecipitates to demonstrate changes following insulin treatment. GLUT4 membrane insertion was followed after insulin with or without CG53353. RESULTS: Insulin increased phosphoPKCbetaII(Ser660 and Thr641); LY294002 blocked this, indicating its activation by PI3K. Insulin treatment increased cytosolic phosphoMARCKS, decreased membrane MARCKS and increased membrane phospholipase D1 (PLD1), a protein regulating glucose transporter vesicle fusion resulted. PhosphoMARCKS was attenuated by CG53353 or MARCKS siRNA. MARCKS siRNA blocked ISGT. Association of PKCbetaII and GLUT4 with membrane F-actin was enhanced by insulin, as was that of cytosolic and membrane MARCKS. ISGT was attenuated in myocytes transfected with mutated MARCKS (Ser152Ala), whereas overproduction of wild-type MARCKS enhanced ISGT. CG53353 blocked insertion of GLUT4 into membranes of insulin treated cells. CONCLUSIONS/INTERPRETATION: The results suggest that PKCbetaII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.

Effect of energy and lysine intake in gestation on sow performance.

Nutrient intake during gestation has an impact on gestation parameters and subsequent lactation performance. The objectives of this experiment were to determine the impact of feeding two levels of amino acids in gestation on sow BW changes in gestation and lactation, and litter size, and to evaluate a factorial method for determining daily energy requirements. At mating, 419 sows (Camborough 15; Pig Improvement Canada, Acme, AB) were assigned randomly within Parities 1, 2 or 3+ to a gestation diet containing either 0.44% (low lysine) or 0.55% (high lysine) total lysine and 3,100 kcal DE/kg; other indispensable amino acids were adjusted to lysine based on ideal protein ratios. Feed allowance in gestation was determined factorially using estimated DE requirements for maintenance, maternal gain, and conceptus growth. Sows were allowed free access to the lactation diet. Gestation BW gain from d 0 to 110 was affected by parity (61.2, 60.0, and 42.3 kg for Parity 1, 2, and 3+, respectively; P < 0.05) but not (P > 0.10) by gestation lysine level. Sow BW changes from d 0 of lactation to weaning were affected by parity (0.5, 6.8, and 5.8 kg for Parity 1, 2, and 3+, respectively; P < 0.01) and gestation BW gain (P < 0.01), but not by gestation lysine level (5.0 vs 3.8 kg for low and high lysine, respectively; P > 0.10). Total piglets born was affected by parity (11.5, 12.1, and 12.5, for Parity 1, 2, and 3+, respectively; P < 0.01) and increased with increasing sow BW gain (P < 0.05). Total piglets born alive (mean = 11.2) was increased with increasing sow BW gain (P < 0.05). Total litter weight born alive was affected by parity (15.9, 18.6, and 19.4 kg for Parities 1, 2, and 3+, respectively; P < 0.01) and gestation BW gain (P < 0.05). The model used to determine daily energy intake requirements resulted in an average BW gain of 10.6 kg above the targets set by the model. Total lysine intakes greater than 10.6 g/d in gestation did not improve sow productivity. Setting target weight gains in gestation and feeding to meet these targets may not always provide predictable results due to a number of factors that affect the energy requirement in the sow.

Effect of nutrient intake in lactation on sow performance: determining the threonine requirement of the high-producing lactating sow.

Reproductive performance is steadily increasing within the pork industry; logically, amino acid requirements need to be redefined for sows producing larger litters. The objective of this study was to determine the threonine requirement of the high-producing lactating sow and to determine the effect of lysine on this requirement. A total of 419 PIC C-15 sows were assigned randomly to treatment within parity groups (1, 2, and 3+) and gestation treatment at d 110 of gestation. Lactation diets were formulated to contain 0.80% total lysine (tLYS) with 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, or 0.65% total threonine (tTHR) or 1.06% tLYS with 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, or 0.70% tTHR. Litters were standardized to a minimum of 11 piglets within 48 h after farrowing, and sows had free access to feed throughout lactation (lactation length = 20.1 +/- 0.1 d). Sow ADFI exceeded expectation, averaging 6.90, 7.40, and 7.20 kg/d for Parities 1, 2, and 3+, respectively. Daily tLYS intake was 58 g/d (47 g of apparent ileal digestible lysine [dLYS] per day) and 74 g/d (59 g dLYS/d) for the low- and high-lysine group, respectively. Lysine intake did not affect sow or litter performance (P > 0.10). Sows gained an average of 4.8 kg in lactation. Using regression analysis, BW gain was maximized at 0.54% tTHR for all parity groups (quadratic; P < 0.05). Litter weaning weight (67.1, 67.9, and 66.2 kg for Parities 1, 2, and 3+, respectively) and litter weight gain (2.49, 2.53, and 2.44 kg/d for Parities 1, 2, and 3+, respectively) were maximized at 0.53% tTHR using regression analysis, for all parity groups (quadratic; P < 0.05). Based on regression analysis, plasma urea nitrogen on d 10 and 18 was minimized at 0.54% tTHR (P < 0.05). Lysine levels in excess of 58 g of tLYS/d did not benefit sow or litter performance. The requirement for threonine to minimize sow tissue mobilization was 37, 40, and 38 g tTHR/d (28, 30, and 30 g of apparent ileal digestible threonine [dTHR] per day) for Parities 1, 2, and 3+ sows, respectively. The threonine required to maximize litter performance was 36, 39, and 38 g of tTHR/d (28, 30, and 29 g of dTHR/d) for Parities 1, 2, and 3+ sows, respectively. Alternatively, the requirement can be expressed as 14.3 g tTHR (11.8 g dTHR) per kilogram of litter gain.

Insulin regulates alternative splicing of protein kinase C beta II through a phosphatidylinositol 3-kinase-dependent pathway involving the nuclear serine/arginine-rich splicing factor, SRp40, in skeletal muscle cells.

Insulin regulates the inclusion of the exon encoding protein kinase C (PKC) betaII mRNA. In this report, we show that insulin regulates this exon inclusion (alternative splicing) via the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway through the phosphorylation state of SRp40, a factor required for insulin-regulated splice site selection for PKCbetaII mRNA. By taking advantage of a well known inhibitor of PI 3-kinase, LY294002, we demonstrated that pretreatment of L6 myotubes with LY294002 blocked insulin-induced PKCbetaII exon inclusion as well as phosphorylation of SRp40. In the absence of LY294002, overexpression of SRp40 in L6 cells mimicked insulin-induced exon inclusion. When antisense oligonucleotides targeted to a putative SRp40-binding sequence in the betaII-betaI intron were transfected into L6 cells, insulin effects on splicing and glucose uptake were blocked. Taken together, these results demonstrate a role for SRp40 in insulin-mediated alternative splicing independent of changes in SRp40 concentration but dependent on serine phosphorylation of SRp40 via a PI 3-kinase signaling pathway. This switch in PKC isozyme expression is important for increases in the glucose transport effect of insulin. Significantly, insulin regulation of PKCbetaII exon inclusion occurred in the absence of cell growth and differentiation demonstrating that insulin-induced alternative splicing of PKCbetaII mRNA in L6 cells occurs in response to a metabolic change.

Urkinase: structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases.

Acetate kinase, an enzyme widely distributed in the Bacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the alpha-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.

Adult bone marrow stromal cells differentiate into neural cells in vitro.

Bone marrow stromal cells (BMSC) normally give rise to bone, cartilage, and mesenchymal cells. Recently, bone marrow cells have been shown to have the capacity to differentiate into myocytes, hepatocytes, and glial cells. We now demonstrate that human and mouse BMSC can be induced to differentiate into neural cells under experimental cell culture conditions. BMSC cultured in the presence of EGF or BDNF expressed the protein and mRNA for nestin, a marker of neural precursors. These cultures also expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN). When labeled human or mouse BMSC were cultured with rat fetal mesencephalic or striatal cells, a small proportion of BMSC-derived cells differentiated into neuron-like cells expressing NeuN and glial cells expressing GFAP.

Protein kinase Ctheta expression is increased upon differentiation of human skeletal muscle cells: dysregulation in type 2 diabetic patients and a possible role for protein kinase Ctheta in insulin-stimulated glycogen synthase activity.

Protein kinase C (PKCtheta) is a key enzyme in regulating a variety of cellular functions, including growth and differentiation. PKCtheta is the most abundant PKC isoform expressed in skeletal muscle; however, its role in differentiation and metabolism is not clear. We examined the effect of muscle cell differentiation on PKCtheta expression in human skeletal muscle cells from normal and type 2 diabetic subjects. Low levels of PKCtheta messenger RNA (mRNA) and protein were detected in human myoblasts from both types of subjects. Upon differentiation into myotubes, PKCtheta mRNA and protein were increased 12-fold in myotubes from normal subjects. In human skeletal muscle cells obtained from type 2 diabetic subjects, increases in PKCtheta mRNA and protein were not observed upon differentiation into myotubes although expression of other markers of differentiation and fusion increased. Cells from type 2 diabetic subjects also exhibited decreased insulin-stimulated glycogen synthase activity. To determine whether the up-regulation of PKCtheta was important for the metabolic actions of insulin, PKCtheta was overexpressed in L6 rat skeletal muscle cells. Increased expression of PKCtheta occurred with differentiation of skeletal muscle myoblasts to myotubes. Glycogen synthase activity was further increased in L6 myotubes stably transfected with the complementary DNA for PKCtheta. The decreased expression of PKCtheta found in cells from type 2 diabetic subjects may be linked to insulin resistance and decreased glycogen synthase activity.

Acute glucose-induced downregulation of PKC-betaII accelerates cultured VSMC proliferation.

Accelerated vascular smooth muscle cell (VSMC) proliferation contributes to the formation of atherosclerotic lesions. To investigate protein kinase C (PKC)-betaII functions with regard to glucose-induced VSMC proliferation, human VSMC from aorta (AoSMC), a clonal VSMC line of rat aorta (A10), and A10 cells overexpressing PKC-betaI (betaI-A10) and PKC-betaII (betaII-A10) were studied with the use of three techniques to evaluate glucose effects on aspects affecting proliferation. High glucose (25 mM) increased DNA synthesis and accelerated cell proliferation compared with normal glucose (5.5 mM) in AoSMC and A10 cells, but not in betaI-A10 and betaII-A10 cells. The PKC-betaII specific inhibitor CGP-53353 inhibited glucose-induced cell proliferation and DNA synthesis in AoSMC and A10 cells. In flow cytometry analysis, high glucose increased the percentage of A10 cells at 12 h after cell cycle initiation but did not increase the percentage of betaI-A10 or betaII-A10 cells entering S phase. PKC-betaII protein levels decreased before the peak of DNA synthesis, and high glucose further decreased PKC-betaII mRNA and protein levels in AoSMC and A10 cells. These results suggest that high glucose downregulates endogenous PKC-betaII, which then alters the normal inhibitory role of PKC-betaII in cell cycle progression, resulting in the stimulation of VSMC proliferation through acceleration of the cell cycle.

Protein kinases as therapeutic targets.

Protein kinases and phosphatases are likely targets for the development of therapeutic drugs since they are involved in specific signaling pathways which regulate cell functions such as metabolism, cell cycle progression, cell adhesion, vascular function and angiogenesis. Protein phosphorylation and dephosphorylation serve as molecular switches for modulating these processes and the level and duration of each is a highly regulated process in normal cells. Several compounds that inhibit the activity of tyrosine kinases are being evaluated as cancer therapeutic agents in clinical trials. Diabetes and complications of diabetes also involve deregulated levels of protein kinases. New approaches for regulating kinase gene expression include specific antisense oligonucleotides for inhibiting post-transcriptional processing of the messenger RNA, naturally occurring products and their chemical derivatives to inhibit kinase activity, and monoclonal antibodies to inhibit receptor linked kinases. Inhibition of phosphatases also serves to alter the duration of phosphorylation by kinases. Considerations for development of effective inhibitors include non-specific actions of compounds, cellular uptake, multiple intracellular targets that can dilute the effective cellular concentration of an agent, and tissue specificity. Kinase inhibitors may allow other therapeutic agents additional time to become effective and they may act synergistically with current treatments.

Ectopic expression of protein kinase CbetaII, -delta, and -epsilon, but not -betaI or -zeta, provide for insulin stimulation of glucose uptake in NIH-3T3 cells.

Insulin regulates a diverse array of signaling pathways involved in the control of growth, differentiation, proliferation, and metabolism. Insulin increases in glucose uptake via a protein kinase C-dependent pathway in target tissues such as fat and muscle are well documented. Insulin-regulated events, however, occur in all cells. The utilization of glucose as a preferred energy source is a ubiquitous event in eukaryotic cells. In NIH-3T3 fibroblasts, insulin treatment increased levels of the cPKC and nPKC activator, diacylglycerol. Insulin-responsive 2-[(3)H]deoxyglucose uptake was stimulated in a dose-dependent manner. The overexpression of protein kinase C (PKC)betaI, -betaII, -delta, -epsilon, and -zeta was used to investigate the specificity of PKC isozymes for insulin-sensitive glucose uptake. The stable overexpression of PKCbetaII, -delta, and -epsilon resulted in increases in insulin-stimulated 2-[(3)H]deoxyglucose uptake compared to vector control cells, while basal 2-deoxyglucose uptake levels were not elevated. Overexpression of PKCbetaI and PKCzeta isozymes had no further effect on basal or insulin-stimulated 2-deoxyglucose uptake. The PKC-specific inhibitor, CGP41251, blocked insulin effects on 2-deoxyglucose uptake but not its effects on tyrosine phosphorylation of cellular substrates. Insulin-stimulated 3-O-methylglucose uptake was also greater in cells overexpressing PKCbetaII, -delta, and -epsilon, compared to control cells. The increased responsiveness was not accompanied by conversion of 3T3 cells to the adipocyte phenotype or the increased expression of insulin receptors or glucose transporters (GLUT1-type). Insulin-stimulated recruitment of GLUT1 to plasma membranes of cells overexpressing PKCbetaII, -delta, and -epsilon, was greater than that in control cells. The data suggest that more than one PKC isozyme is involved in insulin signaling pathways in fibroblasts, resulting in increased GLUT1 transporter recruitment to cell membranes.

A shift from normal to high glucose levels stimulates cell proliferation in drug sensitive MCF-7 human breast cancer cells but not in multidrug resistant MCF-7/ADR cells which overproduce PKC-betaII.

Glucose concentration may be an important factor in breast cancer cell proliferation because the prevalence of breast cancer is high in diabetic patients. To determine the role of protein kinase C (PKC)-betaII in regulating MCF-7 cell proliferation at different glucose concentrations, the effects of glucose and a PKC-betaII-specific inhibitor (CGP53353) were examined in cultures of MCF-7 human breast cancer cell line and its multidrug resistant variant (MCF-7/ADR). Cell proliferation and DNA synthesis of MCF-7 were increased when glucose concentration in the culture medium was increased from normal (5.5 mM) to high (25 mM) levels. However, MCF-7/ADR cell proliferation and DNA synthesis were unaffected by the increase in glucose. PKC-betaII protein and the corresponding mRNA levels were 4- to 5-fold higher in MCF-7/ADR than in MCF-7 cells. High glucose-induced decreases of PKC-betaII protein and mRNA levels were observed during the DNA synthesis phase in MCF-7 but not in MCF-7/ADR cells. Decreases in PKC-betaII mRNA and protein levels below a critical threshold in response to high glucose levels may account for glucose-stimulated proliferation of MCF-7 cells. Cultures of multidrug resistant MCF-7/ADR cells reach maximal cell density in medium containing normal (5.5 mM) glucose levels and are not induced to grow further in response to high (25 mM) glucose. Our results demonstrate a link between high glucose-induced PKC-betaII isozyme down-regulation with concomitant acceleration of cell cycle progression in MCF-7 cells.

Hypoxia-associated induction of early growth response-1 gene expression.

The paradigm for the response to hypoxia is erythropoietin gene expression; activation of hypoxia-inducible factor-1 (HIF-1) results in erythropoietin production. Previously, we found that oxygen deprivation induced tissue factor, especially in mononuclear phagocytes, by an early growth response (Egr-1)-dependent pathway without involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C., Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8298-8303). Now, we show that cultured monocytes subjected to hypoxia (pO2 approximately 12 torr) displayed increased Egr-1 expression because of de novo biosynthesis, with a approximately 10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase constructs localized elements responsible for hypoxia-enhanced expression to -424/-65, a region including EBS (ets binding site)-SRE (serum response element)-EBS and SRE-EBS-SRE sites. Further studies with each of these regions ligated to the basal thymidine kinase promoter and luciferase demonstrated that EBS sites in the element spanning -424/-375 were critical for hypoxia-enhanceable gene expression. These data suggested that an activated ets factor, such as Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced activation of Elk-1, and suppression of Elk-1 blocked up-regulation of Egr-1 transcription. The signaling cascade preceding Elk-1 activation in response to oxygen deprivation was traced to activation of protein kinase C-betaII, Raf, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase and mitogen-activated protein kinases. Comparable hypoxia-mediated Egr-1 induction and activation were observed in cultured hepatoma-derived cells deficient in HIF-1beta and wild-type hepatoma cells, indicating that the HIF-1 and Egr-1 pathways are initiated independently in response to oxygen deprivation. We propose that activation of Egr-1 in response to hypoxia induces a different facet of the adaptive response than HIF-1, one component of which causes expression of tissue factor, resulting in fibrin deposition.

Aquaporin-1 expression in proximal tubule epithelial cells of human kidney is regulated by hyperosmolarity and contrast agents.

Primary cells of renal proximal tubule epithelium (S1 segment) of human kidney (HRPTE cells) up-regulate aquaporin-1 (AQP-1) expression in response to hyperosmolarity. NaCl and D(+)-raffinose increased (2-2.5 fold) AQP-1 expression when medium osmolarity was 400 and 500 mOsm/kg.H2O. Urea did not have this effect. Unlike our previous findings with mIMCD-3 cells, vasopressin (10(-8)M) did not affect AQP-1 expression in HRPTE cells in isosmolar or NaCl-enriched hyperosmolar conditions. Furthermore, HRPTE cells increased (3-4 fold) AQP-1 expression when exposed to hyperosmolar Reno-60 and Hypaque-76 (diatrizoates, ionic) contrast agents at 400 and 500 mOsm/kg.H2O. Isosmolar (290 mOsm/kg H2O) Visipaque (iodixanol, non-ionic) at 10% (v/v) concentrations also increased AQP-1 expression, and 25% v/v of Visipaque rendered morphological alterations of HRPTE cells and a 3-fold increase in AQP-1 expression after 24h exposure. Finally, semi-quantitative RT-PCR of HRPTE cells subjected to various isosmolar or hyperosmolar conditions demonstrated up-regulation of AQP-1 mRNA and protein levels. Our results suggest AQP-1 up-regulation in HRPTE cells exposed to environmental stresses such as hyperosmolarity and high doses of isosmolar contrast agents.

Acute hyperglycemia regulates transcription and posttranscriptional stability of PKCbetaII mRNA in vascular smooth muscle cells.

Acute hyperglycemia may contribute to the progression of atherosclerosis by regulating protein kinase C (PKC) isozymes and by accelerating vascular smooth muscle cell (VSMC) proliferation. We investigated acute glucose regulation of PKCbeta gene expression in A10 cells, a rat aortic smooth muscle cell line. Western blot analysis showed that PKCbetaII protein levels decreased with high glucose (25 mM) compared to normal glucose (5.5 mM), whereas PKCbetaI levels were unaltered. PKCbeta mRNA levels were depleted by 60-75% in hyperglycemic conditions. To elucidate whether high glucose regulated PKCbeta expression via the common promoter for PKCbetaI and PKCbetaII, deletion constructs of the PKCbeta promoter ligated to CAT as reporter gene were transfected into A10 cells. Construct D (-411 to +179CAT) showed quenching in high glucose (25 mM) and suggested the involvement of a carbohydrate response element in the 5' promoter region of the PKCbeta gene. In actinomycin D-treated A10 cells, a 60% decrease in PKCbeta mRNA with high glucose treatment indicated that posttranscriptional destabilization by glucose was also occurring. We have demonstrated that glucose-induced posttranscriptional destabilization of PKCbetaII message is mediated via a nuclease activity present in the cytosol. The specificity of glucose to posttranscriptionally destabilize PKCbetaII mRNA, but not the PKCbetaI mRNA, was confirmed in both A10 cells and primary cultures from human aorta.

TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis.

The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity-purified protein was identical to that of the RNA-binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCbetaII-dependent FUS phosphorylation. Moreover, suppression of PKCbetaII activity in BCR/ABL-expressing cells by treatment with the PKCbetaII inhibitor CGP53353, or by expression of a dominant-negative PKCbetaII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) beta-chain expression, and accelerated G-CSF-stimulated differentiation. Downregulation of FUS expression in BCR/ABL-expressing 32Dcl3 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression.

The roles of protein kinase C beta I and beta II in vascular smooth muscle cell proliferation.

The role of protein kinase C (PKC) on proliferation of A10 vascular smooth muscle cells (VSMC) was studied by overexpressing specific PKC-beta I and -beta II isozymes. PKC-beta I and -beta II are derived from alternative splicing of the exon encoding the carboxy-terminal (C-terminal) 50 or 52 amino acids, respectively. The differential functions of the two isozymes with regard to cell proliferation, DNA synthesis, and the cell cycle were investigated in A10 cells, a clonal cell line of VSMC from rat aorta, and in A10 cells overexpressing PKC-beta I and PKC-beta II (beta I-A10 and beta II-A10). PKC levels were increased three- to fourfold in heterogeneous cultures of stably transfected cells. Although doubling time of A10 cells was 36 h, the cell doubling time in beta I-A10 cells decreased by 12 h, and, in contrast, the doubling time of beta II-A10 cells increased by 12 h compared to A10 cells. The increase of [3H]thymidine (TdR) incorporation was accelerated and increased in beta I-A10 cells, but slowed and diminished in beta II-A10 cells compared to A10 and control cells transfected with empty vector. Cell cycle analysis of beta I-A10 cells showed an acceleration of S phase entry while beta II-A10 cells slowed S phase entry. These results suggest that PKC-beta I and PKC-beta II regulate cell proliferation bidirectionally and that PKC-beta I and PKC-beta II may have distinct and opposing functions as cell cycle check point mediators during late G1 phase and may regulate S phase entry in A10 VSMC.

Insulin regulates protein kinase CbetaII expression through enhanced exon inclusion in L6 skeletal muscle cells. A novel mechanism of insulin- and insulin-like growth factor-i-induced 5' splice site selection.

The protein kinase Cbeta (PKCbeta) gene encodes two isoforms, PKCbetaI and PKCbetaII, as a result of alternative splicing. The unique mechanism that underlies insulin-induced alternative splicing of PKCbeta pre-mRNA was examined in L6 myotubes. Mature PKCbetaII mRNA and protein rapidly increased >3-fold following acute insulin treatment, while PKCbetaI mRNA and protein levels remained unchanged. Mature PKCbetaII mRNA resulted from inclusion of the PKCbetaII-specific exon rather than from selection of an alternative polyadenylation site. Increased PKCbetaII expression was also not likely accounted for by transcriptional activation of the gene or increased stabilization of the PKCbetaII mRNA, and suggest that PKCbetaII expression is regulated primarily at the level of alternative splicing. Insulin effects on exon inclusion were observed as early as 15 min after insulin treatment; by 20 min, a new 5'-splice site variant of PKCbetaII was also observed. After 30 min, the longer 5'-splice site variant became the predominate species through activation of a downstream 5' splice site. Similar results were obtained using IGF-I. Although the role of this new PKCbetaII mRNA species is presently unknown, inclusion of either PKCbetaII-specific exon results in the same PKCbetaII protein.

Effects of interferon and PKC modulators on human glioma protein kinase C, cell proliferation, and cell cycle.

The in-vitro effects of human interferon alpha-2b (HuIFN alpha-2b), protein kinase C (PKC) agonist [TPA (12-0-tetra-decanoyl-phorbol-13-acetate)] and PKC inhibitor (calphostin C) on human glioma (U-373 MG) PKC activity, cell proliferation and cell cycle were compared. HuIFN alpha-2b and TPA increased PKC activity, elevated the number of cells in DNA synthesis (S) phase and decreased cell proliferation by similar magnitudes. Calphostin C inhibited PKC activity, increased the number of cells in S phase and produced strong cytotoxic effects (IC50 150 nM). Higher concentrations of calphostin C with or without serum induced an additional block in gap2 and mitosis. We conclude that HuIFN alpha-2b's mode of action may be directly or indirectly affecting PKC. The response produced by HuIFN alpha-2b is similar to TPA (potent PKC activation and S phase arrest).