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S Jaken

Publications and source records attributed to S Jaken.

At least 55 records · Page 3Linked to original sources
PubMed1992

Selective redistribution of protein kinase C isozymes by thapsigargin and staurosporine.

Protein kinase C (PKC) is the major cellular receptor for tumor promoting phorbol esters. Phorbol esters activate alpha-, beta-, delta- and epsilon-PKCs in GH4C1 rat pituitary cells and cause their redistribution from a soluble to a particulate fraction. We have now characterized the effect of several non-phorbol ester tumor promoters on PKC isozyme distribution in GH4C1 cells. The incomplete tumor promoter mezerein caused redistribution of alpha-, beta-, delta- and epsilon-PKCs. Thus, it did not display partial agonist activity. The phosphatase inhibitor okadaic acid did not cause redistribution of any isozyme. The calcium ATPase inhibitor thapsigargin and the ser/thr kinase inhibitor staurosporine caused redistribution of epsilon-PKC and, to a lesser extent, delta-PKC. Although the mechanism of the selective effect on delta- and epsilon-PKCs is not yet known, these data clearly demonstrate that their subcellular distribution can be regulated by a pathway that does not influence alpha- and beta-PKCs. Phorbol ester activation of epsilon-PKC was associated with appearance of a more slowly migrating immunoreactive band in the particulate fraction. Both epsilon-PKC forms accumulated phosphate during phorbol ester treatment. The phosphorylated forms of epsilon-PKC were preferentially recovered in the particulate fraction. Although staurosporine caused redistribution, it prevented the phorbol dibutyrate (PDBu)-mediated appearance of the upper band of the doublet and the increased phosphorylation of both bands. The PDBu-mediated redistribution of alpha- and beta-PKCs was not inhibited by staurosporine, even though staurosporine effectively inhibited PKC catalytic activity. Therefore, catalytic activity is not required for redistribution.

Alkaloids↗
PubMed1992

Hormone- and phorbol ester-activated protein kinase C isozymes mediate a reorganization of the actin cytoskeleton associated with prolactin secretion in GH4C1 cells.

TRH regulates PRL secretion and synthesis in GH4C1 rat pituitary cells. TRH responses are associated with activation of protein kinase C (PKC) isozymes and elevation of cytosolic calcium. To determine which PKC isozymes are involved in TRH-directed responses, we evaluated the effect of TRH on GH cell alpha-, beta-, delta-, and epsilon-PKC isozymes. Immunoblot analysis demonstrated that TRH caused rapid redistribution of all isozymes to a Triton X-100-insoluble (i.e. cytoskeletal) fraction. Corollary immunocytofluorescence studies demonstrated that redistributed PKCs accumulate in cell peripheries. Exocytosis involves reorganization of the cytoskeleton, therefore, each of the GH cell PKCs is appropriately located to phosphorylate proteins important for cytoskeleton organization. To determine the relative contributions of calcium and PKC signal transduction pathways in mediating TRH responses, the effects of potassium depolarization (which increases cytosolic calcium) and phorbol dibutyrate (which activates all PKC isozymes without increasing calcium) were compared. The data indicate that TRH-mediated reorganization of vinculin proceeds via a calcium-mediated pathway, whereas fragmentation of actin filaments proceeds via a PKC-dependent pathway. Selective down-modulation of epsilon-PKC with prolonged TRH-treatment was used to demonstrate that epsilon-PKC is not necessary for certain TRH-stimulated biological responses.

Actins↗
PubMed1992

Mechanism of the nephrogenic repair response. Studies on proliferation and vimentin expression after 35S-1,2-dichlorovinyl-L-cysteine nephrotoxicity in vivo and in cultured proximal tubule epithelial cells.

Studies were performed in vivo using 35S-(1,2-dichlorovinyl)-L-cysteine, a nephrotoxin that damages the S3 segment of the proximal tubule after metabolism to a reactive intermediate. Initiation of damage (35S covalent binding) was complete by 6 hour, and an early proliferative response was observed by 24 hour in the S2 or S3C segments. Necrosis in the S3M and increased blood urea nitrogen were maximal at 48 hours and were accompanied by an increase in proliferation of cells at the wound site. Regeneration was marked by the appearance of vimentin expressing cells that lacked brush border enzymes. The loss of differentiated character in the regenerative epithelium persisted after the proliferation (bromodeoxyuridine incorporation) had stopped; redifferentiation occurred between days 5 and 13. Much of the process was reproduced by culturing rat kidney proximal tubule epithelial cells in defined medium. As growth increased, the cells expressed vimentin and lost brush border marker enzymes. However, as the cells reached high density and stopped dividing there was an increase in brush border markers, as was seen in vivo. Vimentin expression did not decrease, however. The data support a mechanism for damage and nephrogenic repair composed of 1) interaction of the toxin with the target cells, 2) necrosis and exfoliation, 3) loss of differentiation and cell growth, 4) recovery of the damaged area and cessation of cell growth, and 5) differentiation of the quiescent cells. Nephrogenic repair may have similarities with the differentiation of the tubular epithelium during development.

Alkaline Phosphatase↗
PubMed1991

Differential regulation of protein kinase C isozymes by thyrotropin-releasing hormone in GH4C1 cells.

GH4C1 cells, which express Ca(2+)-dependent alpha- and beta- as well as Ca(2+)-independent gamma-, epsilon- and zeta-protein kinase C (PKC) isozymes, provide a cell culture model for studying isozyme-specific properties and functions. Hormonal activation of PKCs regulates the differentiated functions of these cells, namely secretion and synthesis of prolactin (PRL). We previously reported that thyrotropin-releasing hormone (TRH) selectively down-modulates epsilon-PKC with no effect on alpha- or beta-PKCs (Kiley, S.C., Schaap, D., Parker, P., Hsieh, L.-L., and Jaken, S. (1990) J. Biol. Chem. 265, 15704-15712). We now extend those studies to explore the relationship between TRH-stimulated diacylglycerol (DAG) levels and epsilon-PKC down-modulation. TRH stimulates three distinct DAG phases in GH cells. Phase 1 DAG peaks at 15 s, is accompanied by a 6-fold increase in intracellular Ca2+, and causes the redistribution of alpha-, beta-, delta, and epsilon-PKC isozymes from a soluble to a detergent-insoluble particulate compartment. Phase 2 DAG peaks at 10 min, is not associated with a Ca2+ signal, and does not activate PKC by any criteria tested. Phase 3 DAG peaks at 6 h and is sustained through 12 h. This novel DAG phase is not associated with increased intracellular Ca2+. The time course of phase 3 DAG formation corresponds to the time course of TRH-stimulated epsilon-PKC down-regulation; maximal effects are observed at 6-12 h for both events. Unlike alpha-, beta-, and delta-PKCs which are preferentially distributed in the soluble fraction of resting GH cells, epsilon-PKC is also distributed in the detergent-insoluble particulate fraction. The selective compartmentalization of epsilon-PKC in the particulate fraction may render this pool uniquely susceptible to proteolytic degradation. The time course of phase 3 DAG formation and epsilon-PKC down-modulation corresponds to the time course of decreasing PRL message synthesis in GH4 cells. The data suggests that loss of epsilon-PKC may be associated with the down-regulation of prolactin synthesis and that regulation of PRL gene transcription may be an epsilon-PKC-specific function in GH cells.

Animals↗
PubMed1991

Immunocytochemical localization of phospholipase C-gamma in rat embryo fibroblasts.

Rat embryo fibroblasts (REF52) exhibit a distinctive, transformation-sensitive distribution of alpha-protein kinase C (alpha-PKC). Receptor-mediated activation of phospholipase C (PLC)-gamma generates diacylglycerol, the major cellular activator of PKC. Immunofluorescence techniques were used to investigate the subcellular localization of two PLC isozymes (PLC-gamma and PLC-delta) in normal and simian virus 40-transformed REF52 cells to determine (i) if PLC colocalizes with alpha-PKC and (ii) if PLC isozyme distribution is sensitive to transformation. PLC-delta was not detected in either cell type. In REF52 cells, PLC-gamma was associated with the actin cytoskeleton and was evenly distributed along the length of the actin microfilaments. PLC-gamma was coincident with alpha-PKC at the points where the filaments are anchored to the membrane (i.e., the focal contacts). Cytoskeletal association of PLC-gamma was not transformation sensitive, although the actin cytoskeleton was more disordered in simian virus 40-transformed cells. In REF52 cells, platelet-derived growth factor induced tyrosine phosphorylation of both soluble and cytoskeletal PLC-gamma. Tyrosine phosphorylation of PLC-gamma did not seem to be a determinant of its subcellular localization, but there was a detectable increase in cytoskeleton-associated PLC-gamma in response to platelet-derived growth factor treatment.

Animals↗
PubMed1991

Regulation of motility in bovine brain endothelial cells.

Scatter factor (SF) is a fibroblast-derived cytokine which stimulates motility of epithelial and vascular endothelial cells. We used a quantitative assay based on migration of cells from microcarrier beads to flat surfaces to study the regulation of motility in bovine brain endothelial cells (BBEC). Peptide growth factors (EGF, ECGF, basic FGF) did not stimulate migration. Tumor promoting phorbol esters (PMA, PDD) markedly stimulated migration, while inactive phorbol esters (4a-PDD, phorbol-13,20-diacetate) did not affect migration. Both SF- and PMA-stimulated migration were inhibited by 1) TGF-beta; 2) protein kinase inhibitors (e.g., staurosporine, K-252a); 3) activators of the adenylate cyclase signaling pathway (e.g., dibutyryl cyclic AMP, theophylline); 4) cycloheximide; and 5) anti-cytoskeleton agents (e.g., cytochalasin B, colcemid). However, PMA and SF pathways were distinguishable: 1) PMA induced additional migration at saturating SF concentrations; 2) the onset of migration-stimulation was immediate for PMA and delayed for SF; and 3) down-modulation of protein kinase C (PKC) ablated PMA but not SF responsiveness. Assessment of PKC by (3H)-phorbol ester (PDBu) binding and by immunoblot showed 1) scatter factor does not cause significant redistribution or down-modulation of PDBu binding or alpha-PKC; and 2) PDBu mediates redistribution and down-modulation of both binding and alpha-PKC. These findings suggest two pathways for BBEC motility: a PKC-dependent pathway and an SF-stimulated/PKC-independent pathway.

Animals↗
PubMed1991

Scatter factor stimulates migration of vascular endothelium and capillary-like tube formation.

Scatter factors (SFs) are heat- and trypsin-sensitive cytokines secreted by fibroblastic and vascular smooth muscle cell lines which stimulate motility of normal epithelium, carcinoma cells, and vascular endothelium. Human and mouse SFs have been purified and identified as 90 kD heterodimeric proteins consisting of heavy (58 kD) and light (31 kD) disulfide-bonded subunits. Partial amino acid sequence data from SF-derived tryptic peptides indicate marked sequence homology with hepatocyte growth factors, suggesting a common multigene family. In this chapter we describe the regulation by SF of vascular endothelial cell chemotaxis and chemokinesis; migration from microcarrier beads to flat surfaces; invasion through porous filters coated with reconstituted basement membrane; secretion of plasminogen activator; and in vitro capillary-like tube formation on a basement membrane surface.

Animals↗
PubMed1991

Overexpression of the alpha-type protein kinase (PK) C in LLC-PK1 cells does not lead to a proportional increase in the induction of two 12-O-tetradecanoylphorbol-13-acetate-inducible genes.

Phorbol esters, by activating protein kinase C (PKC), induce the expression of the urokinase-type plasminogen activator (uPA) gene and the proto-oncogene c-fos in LLC-PK1 (PK1) porcine kidney epithelial cells. To investigate the role of PKC in the regulation of these two 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible genes, the alpha-type PKC, the predominant subtype present in the PK1 cells, was overexpressed in this cell line. Two clonal PK1 derivatives overexpressing the alpha PKC 15- and 20-fold, respectively, were established. Compared with the parental and control cells, only a modest but substantially sustained (2- to 3-fold) increase in the accumulation of uPA as well as c-fos mRNAs were observed by TPA in these cells. These results indicate that the extent of induction of these genes mediated by TPA was not proportional to the amounts of alpha-type PKC stably overexpressed in these cells, suggesting that factor(s) downstream of the activation of the alpha PKC appear to be rate limiting for the induction of both TPA-inducible genes in PK1 cells.

Animals↗
PubMed1991

Biochemical and immunological characterization of renal protein kinase C.

Protein kinase C (PKC) plays an important role in regulation of renal transport and metabolic function. To understand the role of a specific PKC isozyme in renal homeostasis, alpha-PKC content, regulation, and localization have been characterized. Immunoadsorption assays were used to determine that 34% of the total rat kidney PKC (measured as phorbol ester receptors) was alpha-PKC. Immunohistochemical staining with alpha-PKC-specific monoclonal antibodies determined that alpha-PKC was present throughout the nephron and was especially concentrated in proximal tubules and papillary collecting ducts. In general, the S3 segment of the proximal tubule stained more intensely than the S1-S2 segments. Cortical collecting ducts stained poorly for alpha-PKC. Interstitial cells of the papilla also stained for alpha-PKC. Subcellular distribution of alpha-PKC could not be determined in tissue sections; however, in cultured proximal tubule epithelial cells, alpha-PKC was localized not only in cytoplasm but also in cell-cell borders and focal contacts. Chromatography of rat kidney soluble fraction revealed two endogenous kinase inhibitors, one which is PKC specific and one which is a more general kinase inhibitor. The presence of negative regulators of PKC activity suggests that both activation and inactivation of PKC are important for normal renal function.

Animals↗
PubMed1990

Protein kinase C heterogeneity in GH4C1 rat pituitary cells. Characterization of a Ca2(+)-independent phorbol ester receptor.

Clonal GH4C1 rat pituitary cells are heterogeneous with respect to phorbol dibutyrate receptors (PDBu-R) and protein kinase C (PKC) content. GH cell PDBu-Rs can be separated into two categories based on Ca2(+)-modulation of receptor affinity. Approximately 70% of the cytosolic PDBu-Rs demonstrate Ca2(+)-sensitive receptor affinity and redistribute from the soluble to the particulate fraction in the presence of excess Ca2+. The other 30% of the receptors remain in the cytosol in the presence of excess Ca2+. Their receptor affinity is Ca2(+)-independent. Northern blot hybridization and immunoblot analysis showed that GH4C1 cells express Ca2(+)-independent epsilon-PKC as well as Ca2(+)-dependent alpha- and beta-PKCs. Cell lysis in Ca2+ caused the redistribution of greater than 95% of alpha- and beta-PKC to the particulate fraction, whereas approximately 90% of the epsilon-PKC remained in the cytosol. In contrast, brief treatment of GH cell cultures with PDBu or thyrotropin-releasing hormone caused redistribution of all three isozymes. Prolonged treatment with PDBu down-modulated all three isozymes but at different rates and to different extents. In contrast, prolonged thyrotropin-releasing hormone treatment selectively down-modulated epsilon-PKC. These results demonstrate that GH cells have both Ca2(+)-sensitive and -insensitive PKCs and PDBu-Rs and that both populations are regulated by agonists that control prolactin synthesis and secretion by these cells.

Animals↗
PubMed1990

Protein kinase C is localized in focal contacts of normal but not transformed fibroblasts.

Transformed cells differ from normal cells in that they fail to respond to normal signals for regulation of growth and differentiation. This disordered signal transduction probably contributes to maintenance of the transformed phenotype. Several lines of evidence suggest that changes in the Ca2(+)- and phospholipid-dependent protein kinase, protein kinase C (PKC), may be important for transformation. To determine the role of PKC in transformation, we compared the levels and subcellular distribution of total phorbol ester receptors and PKC in normal and SV40-transformed rat embryo fibroblasts (REF52 cells). We also used our alpha-PKC (Type 3)-specific monoclonal antibodies to compare alpha-PKC content and regulation. We found no differences in quantity or subcellular distribution of PKC in 100,000 x g soluble and pelletable fractions. Downmodulation, which represents a feedback loop for limiting PKC activity, occurs to the same extent in both cell types. A major difference between the normal and transformed cells was revealed by immunofluorescence of alpha-PKC. In normal cels, alpha-PKC is tightly associated with the cytoskeleton and appears to be organized into focal contacts because it colocalizes with talin. In contrast, in SV40-REF52 cells, alpha-PKC is not tightly associated with the cytoskeleton and does not colocalize with talin. The difference in subcellular localizations correlates with a loss of two alpha-PKC-binding proteins in the transformed cells. These results indicate that inappropriate subcellular location of alpha-PKC may contribute to maintenance of the transformed phenotype.

Animals↗
PubMed1990

Protein kinase C and tumor promoters.

The complexity of PKC has made it difficult to define the precise biochemical processes associated with the various PKC-related cellular responses observed. In the past year, we have seen progress in complementary approaches that are helping to solve the puzzles. These include purification and characterization of individual isozymes, expression of normal and mutant PKCs, immunolocalization, and identification of specific activators and inhibitors. All of these will be useful in identifying the primary targets of PKC phosphorylation and their function.

Animals↗
PubMed1990

Activation of alpha-protein kinase C leads to association with detergent-insoluble components of GH4C1 cells.

TRH and phorbol dibutyrate (PDBu) stimulate PRL secretion and synthesis from GH4C1 rat pituitary cells through activation of protein kinase C (PKC). TRH responses are mediated by increases in cellular levels of two PKC activators, Ca2+ and diacylglycerol (DAG), whereas PDBu acts as a DAG analog. We conducted experiments to compare the effects of Ca2+ and PDBu/DAG on alpha-PKC redistribution and to determine to what components of the particulate fraction activated alpha-PKC associates. Subcellular fractionation experiments demonstrated that TRH and PDBu both caused chelator-stable association of alpha-PKC with the particulate fraction. In contrast, Ca2+-mediated association with the particulate fraction was not chelator stable. Immunocytofluorescence experiments also demonstrated that TRH, PDBu, and increased cytosolic Ca2+ (due to ionomycin or K+ depolarization) caused redistribution. The effect of TRH was rapid and transient, similar to TRH stimulation of phospholipase C. The translocated alpha-PKC in the particulate fraction from TRH- or PDBu-treated cultures was not solubilized with Triton X-100. In comparable studies using an immunofluorescence assay, alpha-PKC immunofluorescence remained in detergent-insoluble preparations from TRH- and PDBu-stimulated, but not resting cells. The association of activated alpha-PKC with chelator- and detergent-insoluble material suggested that activated alpha-PKC may be associated with membrane and cytoskeletal components.

Animals↗
PubMed1989

Phorbol ester and interferon-gamma modulation of epidermal growth factor receptors on human amniotic (WISH) cells.

In this study we report that pretreatment of human amniotic (WISH) cells with interferon gamma (IFN-gamma) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the down-modulation of epidermal growth factor (EGF) receptors with respect to both receptor number and affinity. Scatchard analysis of EGF binding in the absence of both IFN-gamma and TPA indicated biphasic binding whereas addition of TPA resulted in the loss of the higher affinity class of sites. Pretreatment with IFN-gamma for 24 h enhanced the TPA-induced inhibition of EGF binding whereas IFN-gamma alone had no effect on binding. Protein kinase C (Ca2+/phospholipid-dependent enzyme) was examined as a possible mediator of IFN-induced EGF-receptor modulation; pretreatment of cells with IFN-gamma affected neither the binding of [3H]phorbol 12,13-dibutyrate to membrane or cytosolic fractions nor the protein kinase C activity, suggesting that IFN-gamma pretreatment did not result in translocation or activation of protein kinase C.

Cell Compartmentation↗
PubMed1989

Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells.

We have examined the immunocytochemical localization of protein kinase C (PKC) in NIH 3T3 cells using mAbs that recognize Type 3 PKC. In control cells, the immunofluorescent staining was similar with mAbs directed to either the catalytic or the regulatory domain of PKC. Type 3 PKC localized in a diffuse cytoplasmic pattern, while the nuclei were apparently unstained. Cytoskeletal components also were Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a redistribution of PKC with a specific increase in nuclear PKC. Compared to control cells, the staining with the anticatalytic domain mAbs changed markedly, covering the entire cell surface. In contrast, the staining by the antiregulatory domain mAb did not cover the cell surface and the nuclei remained unstained; these results suggest that PKC activation leads to a conformational change of the regulatory domain such that the epitope recognized by the antiregulatory domain mAb is not readily accessible. We have demonstrated by three criteria that PMA treatment specifically increased PKC in the nucleus: (a) immunofluorescent staining in isolated nuclei increased; (b) Western blots showed that our mAbs detected only one protein, the 82-kD PKC, whose level increased in nuclear lysates from PMA-treated cells; and (c) PKC activity increased in nuclear lysates. In fractionation studies we demonstrated that PKC specifically localized to the nuclear envelope fraction. These results demonstrate that PMA activation leads to a rapid redistribution of Type 3 PKC to the nuclear envelope, and suggests that this isozyme may play a role in mediating PKC-induced changes in gene expression.

Animals↗
PubMed1989

Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts.

We have used immunocytofluorescence techniques to determine the subcellular distribution of the Ca2+, phospholipid-dependent protein kinase, protein kinase C (PKC). Using monoclonal antibodies that are specific for Type 3 (alpha) PKC, we have determined that there are least two pools of PKC in normal rat embryo fibroblasts (REF52 cells): diffuse cytoplasmic and fiber-associated. Extraction with chelators and detergent before fixing and staining removes the cytoplasmic PKC. The fiber-associated staining remains in these cytoskeleton preparations. The cytoskeleton Type 3 PKC staining closely resembles that of the focal contact protein vinculin and colocalizes with another focal contact protein, talin. Cytochalasin, but not colchicine, coordinately disrupts the staining pattern of vinculin and PKC. Activation of PKC by treatment with phorbol esters causes depolymerization of microfilaments and reorganization of vinculin staining. We propose that Type 3 PKC is a modulatory component of the focal contact and has a primary role in regulation of the association of microfilament bundles with the plasma membrane.

Actin Cytoskeleton↗
PubMed1989

Human multidrug resistant KB cells overexpress protein kinase C: involvement in drug resistance.

Among the many phenotypic characteristics of multidrug resistance (MDR), the presence of P-glycoprotein is nearly always observed, and it appears that the plasma membrane of the multidrug resistant cell is integrally involved in controlling drug resistance. Another membrane-associated protein kinase, protein kinase C (PKC), has been shown to regulate the flow of information to the cell interior and to control the efflux of a number of different compounds. We therefore initiated a study of PKC and MDR. We found that multidrug resistant sublines from both mouse sarcoma 180 and human KB lines exhibited 80-90% increases in basal PKC activity. The mechanism of the increase appears to be quite different in the two cell lines. The human KB cells overexpress the alpha isozyme of PKC, commensurate with the increase in alpha-PKC protein, whereas the mouse cells do not overexpress alpha-mRNA but increase alpha-PKC protein. Furthermore, it appears that PKC activity plays a functional role in drug resistance, since inhibition of endogenous PKC activity by staurosporine resulted in decreased resistance to Adriamycin. We also found that phosphorylation of MDR cell membrane vesicles by purified PKC, followed by immunoprecipitation of P-glycoprotein with monoclonal antibody C219, resulted in a level of phosphorylation of P-glycoprotein that was greater than the endogenous phosphorylation level. The data presented indicate that MDR cells of diverse species exhibited enhanced PKC activity but that the mechanisms were different. The increased kinase activity may have biological relevance to MDR since PKC appears to be coupled to P-glycoprotein function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗