Biomedical subjects

J F Kuo

Publications and source records attributed to J F Kuo.

At least 73 records · Page 4Linked to original sources

PubMed1987

Strain-specific postnatal changes in the activity and tissue levels of protein kinase C.

The specific activity of protein kinase C from adult mouse lung and spleen was higher than in the corresponding tissues from neonatal mice. BALB/cBy mice had higher lung and spleen protein kinase C activities at both ages than did A/J mice, and the extent of this strain difference increased with age. These activity differences reflected the tissue levels of the 80 kD form of protein kinase C, as determined by quantitative immunoblotting. These genetic and ontogenetic differences provide an interesting model with which to study the regulation of protein kinase C gene expression.

Animals↗

PubMed1987

Immunocytochemical evidence for phorbol ester-induced directional translocations of protein kinase C in HL60, K562, CHO, and E7SKS cells: possible role in differentiation.

The effects of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the directions of protein kinase C (PKC) translocation in two leukemic cell lines (HL60 and K562) and two fibroblastic cell lines (CHO and E7SKS), related to their susceptibility to the differentiating effect of TPA, were examined. Immunocytochemical evidence indicated that TPA induced a redistribution (outward) of PKC to the plasma membrane in TPA-sensitive HL60 cells, whereas it caused a translocation (inward) of the enzyme to the nucleus or the perinuclear region in K562, CHO, and E7SKS cells, which are resistant to TPA in terms of cell growth and differentiation. Immunoblot analysis of the nuclear proteins from K562 cells revealed that TPA induced an increase in the amount of immunoreactive proteins. TPA, however, did not increase the amount of these immunoreactive species in nuclei isolated from CHO and E7SKS cells, indicating that the translocated PKC was associated only with perinuclear structures of the TPA-treated cells. It is suggested that directional redistribution of PKC to the plasma membrane, as opposed to the nuclear and perinuclear region, might represent an early event required for the TPA-induced differentiation and maturation of HL60 cells.

Animals↗

PubMed1987

Cyclic AMP-like effects of polyamines on phosphatidylcholine synthesis and protein phosphorylation in human promyelocytic leukemia HL60 cells. Comparison with the effects of phorbol ester.

Spermine or putrescine increased cAMP levels through a catalase-sensitive mechanism, resulting in, most notably, a dephosphorylation of protein A (Mr 45,000, pI 5.15) and protein B (Mr 45,000, pI 4.9) and slightly increased phosphatidylcholine (PC) synthesis in HL60 cells. Exogenous dibutyryl cAMP mimicked the polyamine effects. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) also promoted the protein dephosphorylation and PC synthesis, the effects augmented by R59022 and mimicked by exogenous 1-oleoyl-2-acetylglycerol. The effects of spermine (or dibutyryl cAMP) and TPA on PC synthesis were synergistic. It was suggested that cAMP-dependent protein kinase and protein kinase C might mediate, in an independent but inter-related manner, the effects of polyamines and TPA.

Bucladesine↗

PubMed1987

Differential effects of various protein kinase C activators on protein phosphorylation in human acute myeloblastic leukemia cell line KG-1 and its phorbol ester-resistant subline KG-1a.

Human myeloid leukemia KG-1 cells are induced to differentiate to macrophage-like cells by tumor-promoting phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Cells from the cloned subline, KG-1a, unlike the parental line, are resistant to the differentiating effect of TPA. In the present studies, we investigated in these cells protein phosphorylation stimulated by various protein kinase C activators, including 1-oleoyl-2-acetylglycerol in the presence of the diacylglycerol kinase inhibitor R59022, TPA, mezerein, and bryostatin. All the agents stimulated, to a greater extent and with a higher potency, phosphorylation of several proteins in KG-1 cells than in KG-1a cells. On the other hand, these agents markedly stimulated phosphorylation of other proteins in KG-1a cells compared to that in KG-1 cells. The findings indicated that the actions of the diacylglycerol, 1-oleoyl-2-acetylglycerol, and the non-metabolizable activators (TPA, mezerein, and bryostatin) were very similar but not fully equivalent; and that KG-1a cells exhibited altered (increased or decreased) phosphorylation patterns, perhaps related to the TPA resistance characteristic of this subline of cells.

Bryostatins↗

PubMed1987

Protein kinase C in fibroblasts. Characteristics of its intracellular location during growth and after exposure to phorbol esters and other mitogens.

Using an N-bromosuccinimide cleavage fragment of histone H1 as a relatively specific substrate for protein kinase C, we evaluated the partitioning of this kinase activity between soluble and particulate cellular fractions in 3T3-L1 fibroblasts. In confluent, serum-deprived cells, protein kinase C activity was approximately equally divided between soluble and detergent-extractable particulate fractions; both rapidly growing and transformed cells appeared to contain higher levels of particulate enzyme activity. Soluble protein kinase C activity and immunoreactivity decreased to virtually undetectable levels after exposure of the cells to phorbol 12-myristate 13-acetate (PMA), associated with a commensurate increase in particulate kinase activity and immunoreactivity. In intact cells, PMA appeared to cause a shift of immunoreactive protein kinase C from the cytosol to the perinuclear region, as assessed by immunofluorescent microscopy; however; subcellular fractionation revealed that PMA caused increases in the protein kinase C activity associated primarily with non-nuclear membranes. Exposure of the cells to sn-1,2-dioctanoylglycerol resulted in a modest and transient membrane association of protein kinase C, whereas platelet-derived growth factor, fibroblast growth factor, and bombesin caused no detectable increases in the membrane association of the kinase. Activation of protein kinase C by growth factors in fibroblasts may occur without the gross disturbances in intracellular kinase location which occur in response to phorbol esters.

Animals↗

PubMed1987

Antileukemic agent alkyllysophospholipid regulates phosphorylation of distinct proteins in HL60 and K562 cells and differentiation of HL60 cells promoted by phorbol ester.

The tumor-promoting 12-0-tetradecanoylphorbol-13-acetate (TPA) stimulated phosphorylation of several proteins in block I (including protein Ia) and protein 3 in HL60 cells. The antileukemic agent alkyllysophospholipid (ALP) inhibited the TPA-stimulated phosphorylation of these proteins and the TPA-induced differentiation of the cells. In comparison, TPA only stimulated phosphorylation of protein 3 in K562 cells which, in contrast, were not induced to differentiate by TPA and lacked protein Ia and had a very high basal phosphorylation of protein B. ALP inhibited phosphorylation of protein 3 as well as protein B in K562 cells. The data suggest that the presence of distinct phosphoproteins and regulation of their phosphorylation may be related to the selective susceptibility of the two leukemia cell lines to the maturating effect of TPA and cytotoxicity of ALP.

Antineoplastic Agents↗

PubMed1987

Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C.

We have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the alpha subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to Gs, Gi, and Go, respectively. A single beta subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the alpha subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive Gs alpha relative to Gi alpha and Go alpha when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. alpha subunits of G-proteins were not detected in cilia detached from the nonchemosensory respiratory epithelium of the palate. Immunohistochemical studies using an antiserum against the beta subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the alpha subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. Prolonged incubation periods with these antisera tended to obliterate these differences in staining patterns, giving rise to staining by both antisera of the ciliary surface, the olfactory receptor cell membranes, the axon bundles, and the acinar cells of the glands. In addition to G-proteins, we have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Immunochemical characterization of protein kinase C in rat liver nuclei and subnuclear fractions.

A doublet of immunoreactive bands has been identified in rat liver nuclei, nuclear matrix and lamina by means of a polyclonal antibody against protein kinase C. The two polypeptides show an apparent molecular weight of 77 and 74 kDa on SDS-polyacrylamide gels, and appear to be tightly bound nuclear components, resistant to detergent and high salt extraction. Given the complexity of the genes encoding for protein kinase C, these two forms of the enzyme might be translational products specifically located in the nucleus, involved in the transduction to the genomic apparatus of regulatory signals generated by growth factors and tumor promoters.

Animals↗

PubMed1986

Immunocytochemical evidence for phorbol ester-induced protein kinase C translocation in HL60 cells.

The ability of tumor promoting 12-O-tetradecanoylphorbol-13-acetate (TPA) to redistribute protein kinase C in human promyelocytic leukemic HL60 cells was investigated. It was found that TPA caused a rapid translocation (within 10 min) of protein kinase C from the cytosolic (soluble) fraction to the particulate (membrane) fraction, as determined indirectly by assaying for the enzyme activity or by immunoblotting of the enzyme protein in the isolated subcellular fractions. Immunocytochemical localization of the enzyme demonstrated directly that the TPA caused an enzyme translocation t the plasma membrane. These findings suggest that translocation to the plasma membrane of the enzyme may represent initial events related to the TPA effect on terminal differentiation of HL60 cells to monocytes/macrophages.

Cell Compartmentation↗

PubMed1986

Synthetic myelin basic protein peptide analogs are specific inhibitors of phospholipid/calcium-dependent protein kinase (protein kinase C).

Synthetic peptide analogs of the bovine myelin basic protein (MBP) corresponding to residues 104-118 were found to specifically inhibit phospholipid/ Ca2+-dependent protein kinase (protein kinase C). The peptides [Ala107]MBP (104-118) and [Ala113]MBP (104-118) inhibited protein phosphorylation of intact MBP, histone H1 and peptide phosphorylation with MBP(104-123), MBP(104-118) or [Ala105]MBP (104-118) as substrates. The inhibitor peptides [Ala107]MBP(104-118) and [Ala113]MBP (104-118), containing alanine in place of the arginine recognition sites, apparently inhibited the enzyme noncompetitively with respect to substrates, with IC50 values ranging from 46-145 and 28-62 microM, respectively. These peptide analogs did not inhibit cyclic AMP-dependent protein kinase or myosin light chain kinase but inhibited phospholipid/Ca2+-dependent phosphorylation of endogenous proteins in the total, solubilized fraction of rat brain.

Amino Acid Sequence↗

PubMed1986

Immunological quantitation of phospholipid/Ca2+-dependent protein kinase and its fragments. Tissue levels, subcellular distribution, and ontogenetic changes in brain and heart.

Levels of phospholipid/Ca2+-dependent protein kinase (protein kinase C, 80 kDa) and its presumed proteolytic fragments were quantified in a variety of animal tissues and cultured human leukemic cell lines (HL60 and K562) using an immunoblot analysis technique. Of many tissues examined, the rat brain and HL60 cells were by far the richest sources of the 80-kDa native enzyme, with its concentration estimated to be about 2-3 microM in both tissues. The major enzyme species detected in most tissues, however, was the 67-kDa fragment; the 80-kDa native enzyme was present in a smaller amount except in spleen which contained nearly equal amounts of both enzyme species. It was also found that HL60 and K562 leukemic cells contained the 50-kDa species instead of the 67-kDa species. A study of the subcellular distribution of the 80- and 67-kDa species showed the enzyme to be localized predominantly in the soluble fraction for some tissues (e.g. heart) and nearly equally distributed between soluble and particulate fractions in others (e.g. spleen). In the brain, however, the majority of the enzyme was present in the particulate fraction, in agreement with the findings made with immunocytochemical localization of the enzyme. The total enzyme content in developing rat brain and heart increased during the first 2 to 4 weeks following birth and decreased to 60% of peak levels in the adult. The present immunological method, showing for the first time that the tissue levels of phospholipid/Ca2+-dependent protein kinase and its fragments can be quantitated, would be useful for studies on the regulation of cellular events and pathophysiology of tissues thought to be involved in this multi-functional protein phosphorylation system.

Animals↗

PubMed1986

Phospholipid/calcium-dependent protein kinase (protein kinase C) system: a major site of bioregulation.

The substrate specificity determinants of protein kinase C are probed using synthetic peptides encompassing the major phosphorylation site serine 115 in bovine MBP. The results indicate that basic residues arginine 107 and 113 N-terminal to the phosphorylation site are essential for the substrate activity of the peptides. Substitutions of these basic residues by alanine cause a marked decrease in their substrate activity and the resulting peptide analogs become specific and rather potent inhibitors of protein kinase C. Leukemic cells are particularly abundant in protein kinase C and its substrate proteins, and the enzyme system has been shown to play a key role in cell growth. The agents that stimulate protein kinase C include tumor promoting phorbol esters (such as TPA) and mezerein, and the putative second messenger diacylglycerol. Many antineoplastic agents, on the other hand, inhibit the enzyme which include adriamycin, tamoxifen, alkyl-lysophospholipid, selenium, retinal and lipoidal amine CP-46, 665-1. Immunocytochemical studies of protein kinase C in leukemic cells indicate that it is localized in the plasma membrane, cytoplasm, nucleus and Golgi apparatus, and the subcellular distribution of the enzyme might be related to the phases of the cell cycle. TPA induces translocation of the enzyme to plasma membrane, suggesting an additional mode of action for the tumor promotor.

Animals↗

PubMed1986

Effects of selenium compounds on phospholipid/Ca2+-dependent protein kinase (protein kinase C) system from human leukemic cells.

Selenium compounds (selenium dioxide, selenious acid, and selenic acid) were found to inhibit phospholipid/Ca2+-dependent protein kinase (protein kinase C) and the phorbol ester-stimulated phosphorylation of endogenous substrate proteins from HL60 cells. Kinetic analysis indicated that selenium dioxide (SeO2) inhibited the enzyme noncompetitively with respect to phosphatidylserine (apparent Ki, 60 microM) and Ca2+ (apparent Ki, 68 microM). The inhibitory effect of SeO2 on protein kinase C was additive to that of another inhibitor of the enzyme (alkyl-lysophospholipid) when present together. SeO2 was also equally inhibitory to myosin light chain kinase, a calmodulin/Ca2+-dependent class of protein kinase. It, however, affected only very slightly cyclic adenosine 3':5'-monophosphate-dependent protein kinase. It is suggested that inhibition of Ca2+-dependent reactions might be related to the anticarcinogenic property of selenium.

Animals↗

PubMed1986

Immunocytochemical localization of protein kinase C in identified neuronal compartments of rat brain.

Polyclonal antisera to the phospholipid/Ca2+-dependent protein kinase have been used to study the distribution of the enzyme in identified neurons of several brain regions. The results indicated that the enzyme was concentrated in synaptic terminals of mossy fibers, Golgi II neurons and Purkinje neurons in the cerebellum, and in granule cell terminals in the hippocampus. These synapses have different physiological properties and utilize different neurotransmitters. Electron microscopic results indicated that the enzyme was concentrated in presynaptic terminals. Thus, the protein kinase may play a broad role in Ca2+-related events of the presynaptic terminal during neurotransmission. Light- and electron-microscopic immunocytochemical analysis also indicated that the enzyme was inside the nucleus concentrated in a region adjacent to the inner nuclear membrane, where it may play a role in the regulation of neuronal function.

Animals↗

PubMed1985

Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways.

Exposure of serum-deprived 3T3-L1 fibroblasts to phorbol 12-myristate 13-acetate (PMA), synthetic diacylglycerols, platelet-derived growth factor (PDGF), or pituitary fibroblast growth factor (FGF) resulted in stimulated phosphorylation of an acidic, multicomponent, soluble protein of Mr 80,000. Phosphorylation of this protein was promoted to a lesser extent by epidermal growth factor; however, neither insulin nor dibutyryl cAMP was effective. Phosphoamino acid analysis and peptide mapping of the Mr 80,000 32P-protein after exposure of fibroblasts to PDGF revealed identical patterns to those obtained with PMA or diacylglycerols. In contrast to the Mr 80,000 protein, proteins of Mr 22,000 (and pI 4.4) and Mr 31,000 were also phosphorylated in response to insulin as well as to PMA, diacylglycerols, epidermal growth factor, PDGF, and FGF in these cells. Similar findings were noted in fully differentiated 3T3-L1 adipocytes. Preincubation of the cells with high concentrations of active phorbol esters abolished specific [3H]phorbol 12,13-dibutyrate binding, protein kinase C activity, and immunoreactivity and also prevented stimulated phosphorylation of the Mr 80,000 protein by PMA, diacylglycerols, PDGF, or FGF, supporting the contention that this effect was mediated through protein kinase C. The stimulated phosphorylation of the Mr 22,000 and 31,000 proteins in response to PMA was also abolished by such pretreatment. In contrast, the ability of insulin, PDGF, and FGF to promote phosphorylation of the Mr 22,000 and 31,000 proteins was unaffected in the protein kinase C-deficient cells. We conclude that PDGF and FGF may exert some of their effects on these cells through at least two distinct pathways of protein phosphorylation, phorbol ester-like (P) activation of protein kinase C, and an insulin-like (I) pathway exemplified by phosphorylation of the Mr 22,000 and 31,000 proteins.

Animals↗

PubMed1985

Effect of tamoxifen, a nonsteroidal antiestrogen, on phospholipid/calcium-dependent protein kinase and phosphorylation of its endogenous substrate proteins from the rat brain and ovary.

Antiestrogens (tamoxifen, clomiphene and nafoxidine) were found to inhibit phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C), whereas estrogens (estradiol and diethylstilbesterol) and the weakly estrogenic chlorotrianisene were inactive. Kinetic analysis indicated that the antiestrogens inhibited PL/Ca-PK competitively with respect to phosphatidylserine (Ki = 16-27 microM), but non-competitively with Ca2+ (Ki = 14-30 microM). Tamoxifen, but not diethylstilbesterol, also inhibited the phospholipid/Ca2+-dependent phosphorylation of various endogenous proteins from the total, solubilized fraction of the rat brain and ovary. Myosin light chain kinase, a calmodulin/Ca2+-dependent class of protein kinase, was similarly inhibited by tamoxifen; the drug, however, was without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases. It is suggested that PL/Ca-PK, by virtue of the hydrophobic interactions required for the enzyme activation, may represent a potential site of action for the lipophilic antiestrogens, in addition to the commonly recognized intracellular estrogen receptors.

Animals↗

PubMed1985

Substrate specificity of phospholipid/Ca2+-dependent protein kinase as probed with synthetic peptide fragments of the bovine myelin basic protein.

The substrate specificity of phospholipid/Ca2+-dependent protein kinase (protein kinase C) was studied using synthetic peptides, in particular those corresponding to the amino acid sequence around serine 115 in bovine myelin basic protein (MBP). It was found that MBP (104-118) and MBP (104-123) were substrates for the enzyme, with apparent Km values of 14 and 10 microM, respectively. Neither MBP (111-118) nor MBP (111-123) were phosphorylated, indicating that an additional segment of sequence extending toward the N terminus, but not toward the C terminus, was essential for the substrate activity of the peptides. Of the alanine-substituted analogs examined, [Ala 105] MBP (104-118) was comparable to the parent peptide, whereas [Ala 107] MBP (104-118) and [Ala 113] MBP-(104-118) were much poorer substrates. These findings indicated that lysine 105 was not essential, but both arginine 107 and arginine 113 were important specificity determinants. Initial studies revealed that [Ala 113] MBP (104-118) inhibited phosphorylation by the enzyme of the parent peptide and, to a lesser extent, the intact MBP(1-170). Serine 115 was the only site phosphorylated in the analog peptides [Ala 105] MBP (104-118) and [Ala 107]MBP (104-118). In the parent peptide, serine 115 was the initial site of phosphorylation but after prolonged phosphorylation other sites became phosphorylated (serine 110 and/or serine 112), further supporting the concept that arginine residues act as essential substrate specificity determinants for phospholipid/Ca2+-dependent protein kinase.

Animals↗

PubMed1985

Inhibition of phospholipid/Ca2+-dependent protein kinase and phosphorylation of leukemic cell proteins by CP-46,665-1, a novel antineoplastic lipoidal amine.

CP-46,665-1, an antineoplastic lipoidal amine, was found to inhibit phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C), with an IC50 (concentration causing a 50% inhibition) of 10 microM. Its inhibition of the enzyme was reversed by phosphatidylserine, but not by Ca2+. The agent also inhibited the enzyme activity which was further augmented by 12-0-tetradecanoylphorbol-13-acetate (TPA), mezerein or diolein. Phosphorylation of endogenous proteins from HL-60 cells by the enzyme, with or without being further augmented by TPA, was inhibited by CP-46,665-1 as well as by alkyllysophospholipid (an antineoplastic agent). CP-46,665-1, while without effect on cyclic AMP-dependent protein kinase, also inhibited myosin light chain kinase (a calmodulin/Ca2+-dependent protein kinase). The present findings suggest that inhibition of the Ca2+-effector enzymes may be related in part to the antimetastatic activity of the lipoidal amine.

Animals↗