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S Jaken

Publications and source records attributed to S Jaken.

At least 73 records · Page 4Linked to original sources
PubMed1988

Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity.

Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.

Animals↗
PubMed1988

Early signals for keratinocyte differentiation: role of Ca2+-mediated inositol lipid metabolism in normal and neoplastic epidermal cells.

Differentiation of cultured keratinocytes is regulated by the Ca2+ concentration of the culture medium. Below 0.1 mM Ca2+, a monolayer of basal cells is formed which fully differentiates in response to a rise in medium Ca2+. A role for protein kinase C in this differentiation program has been suggested because phorbol esters induce epidermal differentiation in cells grown in reduced Ca2+ medium, and exogenously added phospholipase C (which increases cellular diacylglycerol) mimics phorbol ester action. These findings suggested that the external Ca2+ signal may lead to protein kinase C activation via stimulation of cellular phospholipase C activity. The effect of the external Ca2+ signal on phospholipase C was studied in cultures prelabeled with [3H]-inositol. Within 2 min after addition of Ca2+ to 1 mM, an increase in inositol phosphates was measured. This correlated with a decrease in radiolabeled phosphoinositides, suggesting that these were the source of the increased inositol phosphates. After 3 h in 1 mM Ca2+ medium, each of the inositol phosphates remained increased to 130-140% of control levels. Inositol phosphate metabolism in neoplastic epidermal cells was quantitatively similar to normal cells in response to the Ca2+ signal. Stimulation of phosphatidylinositol (PIP) metabolism appears to be mediated by a rise in intracellular free Ca2+ because Ca2+ ionophores A23187 and ionomycin also cause a similar rise in inositol phosphate levels. Phorbol esters did not increase PIP turnover but instead stimulated phosphatidylcholine metabolism. The induction of epidermal differentiation by phorbol esters was enhanced by ionomycin, suggesting that both protein kinase C activation, elevation of intracellular calcium and PIP turnover were important components of the signal for epidermal differentiation. These results demonstrate that the second messenger system for Ca2+-mediated keratinocyte differentiation may be through a direct effect on phospholipase C activity.

Animals↗
PubMed1988

Signal transduction for proliferation and differentiation in keratinocytes.

In mouse and human epidermis, the Ca2+ environment of the basal cell layer is substantially below serum Ca2+, while that of the granular cell layer is unusually high. Reduction of extracellular Ca2+ concentration (Cao) in the medium of keratinocyte cultures maintains a basal cell phenotype while serum Ca2+ concentrations induce terminal differentiation. Measurements of intracellular Ca2+ (Cai) by the use of Fura 2 and digital imaging technology reveal that Cai increases 10-20-fold in response to an increase in Cao and remains elevated. Concomitant with the rise in Cai is an increase in the metabolism of phosphatidylinositol (PI) to yield inositol phosphates and diacylglycerol. PI metabolism is also stimulated by calcium ionophores suggesting that a rise in Cai is directly responsible. The consequent increase in diacylglycerol and Cai would activate protein kinase C, an event known to trigger epidermal differentiation. Specific Cao and Cai determine the expression of individual markers of keratinocyte differentiation in vitro. These findings may account for the importance of the Ca2+ gradient for maintaining regulated growth and differentiation of the epidermis in vivo.

Animals↗
PubMed1987

Purification and characterization of three types of protein kinase C from rabbit brain cytosol.

Three types of protein kinase C were purified from rabbit brain cytosol. Each type has a molecular mass of approximately 80 kDa and serves as a receptor for phorbol esters. Polyclonal antibodies produced to two protein kinase C types were relatively type-specific, indicating that these proteins have unique antigenic determinants. We, therefore, characterized the enzymatic activities to determine if these proteins also had distinct biochemical properties. Type 1 protein kinase C was relatively less Ca2+-dependent than types 2 and 3. The addition of Ca2+ increased Vmax approximately 40% for type 1,600% for type 2, and 1400% for type 3 as compared to the Vmax measured at lower Ca2+ conditions. These results suggest that differences in primary structure can confer type-specific biochemical properties, and this in turn may provide the basis for protein kinase C type-specific stimulus-response coupling.

Animals↗
PubMed1987

Intracellular Ca2+-dependent protein kinase C activation mimics delayed effects of thyrotropin-releasing hormone on clonal pituitary cell excitability.

Biochemical and spectrophotometric studies of second messenger pathways transducing TRH signals in clonal pituitary (GH) cells have shown that TRH induces rapid turnover of phosphoinositides and changes in cytoplasmic Ca2+ as well as activation of protein kinase C (PKC) and secretion of PRL. Here we have used classical microelectrode and contemporary patch pipette recording techniques under current-clamp conditions to compare the effects of TRH receptor-coupled stimulation with direct activation of PKC on the excitability of GH3/B6 cells. With high resistance microelectrodes TRH induced a complex sequence of changes in membrane properties consisting of an initial 20- to 30-mV hyperpolarization associated with an increase in membrane conductance lasting less than a minute, followed by several minutes of low amplitude fluctuations and action potential activity superimposed on a modest increase in input resistance. Active phorbol ester induced a slowly developing hyperpolarization of about 5 mV and a modest increase in input resistance, followed by several minutes of low amplitude fluctuations and spontaneous action potential activity. Both the peptide- and phorbol ester-evoked changes in excitability were attenuated or completely lost during patch recordings in the whole cell mode. Dilute aqueous lysates of the clone restored various phases of the electrical response. The low amplitude fluctuations and action potential activity phase could be induced by either TRH or phorbol ester if the cells were dialyzed with intracellular electrolyte containing PKC and at least 50 nM Ca2+. These results demonstrate that the phosphoinositide/PKC circuit activated by TRH in clonal pituitary cells has electrically detectable effects on cell excitability, and these help to explain TRH's actions on electrical activity.

Action Potentials↗
PubMed1985

Initial events in phorbol ester binding to GH4C1 rat pituitary cells.

Phorbol ester receptors in GH4C1 rat pituitary cells have been studied using [3H]phorbol 12,13-dibutyrate (PDBu). Binding to intact cells was of higher affinity at 37 C than at 4 C (Kd, approximately 8 or approximately 34 nM, respectively). The dissociation rate (measured at 4 C) of PDBu from cells equilibrated at 37 C was slower than that from cells equilibrated at 4 C, indicating the temperature-dependent formation of a high affinity (more slowly dissociating) complex in intact cells. Binding to isolated membrane preparations was of low affinity (Kd, approximately 30 nM) at 4 C and 37 C; however, binding affinity was increased by including phosphatidylserine in the standard assay mixture (Kd, approximately 4 nM). Membrane binding did not account for all of the PDBu binding associated with intact cells. A cytosolic receptor dependent on phospholipids for binding was also studied. The sum of the receptors recovered in the cytosol plus membrane fractions of cell homogenates was very similar to the amount measured in intact cells. This indicates that the cytosolic receptor contributes significantly to the total receptors measured in the intact cell assay. Treatment of cultures with phorbol esters leads to a decrease in receptor concentration in the cytosol and a corresponding increase in receptor density in the membrane fraction. This redistribution indicates that phorbol esters stabilize the membrane association of the cytosolic receptor. The Kd for binding of PDBu to the cytosolic receptor (1.7 nM) and the ED50 for in vitro activation of protein kinase C (4.3 nM) were similar. This result is consistent with this enzyme being a mediator of certain phorbol ester-directed responses in GH4C1 cells.

Animals↗
PubMed1985

Increased diacylglycerol content with phospholipase C or hormone treatment: inhibition of phorbol ester binding and induction of phorbol ester-like biological responses.

The purpose of these studies was to determine whether increased cellular diacylglycerol could modulate phorbol ester receptor properties, in order to demonstrate that diacylglycerol can interact with and modulate the phorbol ester receptor in intact cells. Treatment of GH4C1 cells with bacterial phospholipase C caused an increase in cellular diacylglycerol. This was accompanied by increased PRL secretion and decreased epidermal growth factor (EGF) binding, two responses that also occur with phorbol ester treatment of GH4C1 cells. Phospholipase C treatment led to decreased apparent affinity for phorbol esters with no change in receptor number when measured in intact cells. This is consistent with increased concentrations of a competitive inhibitor of phorbol ester binding in treated cultures. Phospholipase C treatment caused a change in subcellular distribution of phorbol ester receptors, another response characteristic of phorbol ester treatment. TRH is known to activate endogenous phospholipase C activity in these cells, leading to a transient increase in diacylglycerol levels. TRH treatment also led to a transient change in subcellular distribution of phorbol ester receptors. In addition, a coordinate change in subcellular distribution of protein kinase C was observed. These data suggest that diacylglycerol is an endogenous ligand for the target for phorbol ester action in GH4C1 cells.

Animals↗
PubMed1984

Structural and chemical specificity of diacylglycerols for protein kinase C activation.

The structural and chemical specificity of diacylglycerols, lipid components of the quaternary complex for protein kinase C activation, have been evaluated. The ether-linked analogs of the diacyl lipids, either dialkyl or alkyl acyl, were not effective activators of protein kinase C and thus had little influence on reducing the Ca++ requirement of the enzyme. Ester-linked compounds such as 1-palmitoyl-sn-2-butyrylglycerol were as effective as dioleoylglycerol in stimulating protein phosphorylation. Increasing the carbon number at the sn-2 position from two to four resulted in enhanced enzymatic activity, suggesting that the chain length at the secondary hydroxyl is also of importance. These data clearly establish the necessity of the sn-1 carbonyl group of ester-linked glycerolipids in the protein kinase C activation complex.

Animals↗
PubMed1984

Mechanism of action of the phorbol ester tumor promoters: specific receptors for lipophilic ligands.

Cells and tissue preparations specifically bind the phorbol ester tumor promoters. The agreement in structure-activity relationships between binding and biological response strongly argues that these binding sites function as phorbol ester receptors. Upon subcellular fractionation, the phorbol ester binding activity is particulate. In addition, a phorbol ester apo-receptor can be detected in cytosol which requires phospholipids for reconstitution. This apo-receptor appears to correspond to protein kinase C. Diacylglycerols, the probable natural activators of protein kinase C, competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogs. In certain systems, heterogeneity of phorbol ester binding is found. An outstanding issue therefore is whether protein kinase C is the phorbol ester receptor or whether it is only the most abundant class of receptor. Although this question remains unresolved, we can demonstrate heterogeneity of phorbol ester binding by reconstitution of apo-receptor into a heterogeneous lipid environment.

Animals↗
PubMed1984

Analysis of membrane and cytosolic phorbol ester receptors.

Specific phorbol ester receptors are found in the particulate fraction of cells. In addition, cytosol contains a phorbol ester apo-receptor, which requires phospholipids for reconstitution. The apo-receptor corresponds to protein kinase C, and the quantitatively major membrane receptor appears to be a protein kinase C-phospholipid complex. The ability to reconstitute the phorbol ester apo-receptor into different lipid domains now makes it possible to begin elucidation of the role of the lipid domain in phorbol ester action. Studies reviewed here indicate that diglycerides competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogues. Highly lipophilic phorbol esters inhibit effectively only if they are incorporated into the lipid phase, indicating that it is the membrane-dissolved form of the ligand which is recognized. The binding affinity of 3H-phorbol 12,13-dibutyrate for holo-receptor depends markedly (greater than 20-fold range) on the phospholipid environment, and heterogeneous phorbol ester binding (i.e., curved Scatchard plots) can be generated by use of heterogeneous lipid environments in the reconstitution. The possible existence of other phorbol ester receptors in addition to protein kinase C-phospholipid complexes remains to be resolved.

Animals↗
PubMed1983

Phorbol ester receptors and the in vitro effects of tumor promoters.

The evidence for the multistage nature of tumor promotion in vivo and for multiple subclasses of phorbol ester receptors in vitro argues that multiple mechanisms of tumor promotion exist. The existence of multiple mechanisms suggests that brute force assay for tumor promoters in vivo may be inadequate and that understanding of mechanisms may be essential. The interest in the phorbol esters is not primarily that they are environmental hazards for man, but rather that they provide a probe for phorbol ester receptors. These receptors are found in people, and modulation of their activity may play a role in tumor promotion in man.

Animals↗
PubMed1983

Phorbol ester receptors-insights into the initial events in the mechanism of action of the phorbol esters.

Specific phorbol ester receptors are found in the particulate fraction of cells. In addition, cytosol contains a phorbol ester apo-receptor, which requires phospholipids for reconstitution. The apo-receptor corresponds to protein kinase C, and the quantitatively major membrane receptor appears to be a protein kinase C-phospholipid complex. The ability to reconstitute the phorbol ester apo-receptor into different lipid domains permits analysis of the role of the lipid domain in phorbol ester receptor function. Studies reviewed here indicate that diacylglycerols competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogs. Highly lipophilic phorbol esters only inhibit effectively if incorporated into the lipid phase, indicating that the membrane dissolved form of the ligand can be recognized. The binding affinity of [3H]phorbol 12,13-dibutyrate for holo-receptor depends markedly (greater than 20-fold range) on the phospholipid environment, and heterogeneous phorbol ester binding (i.e., curved Scatchard plots) can be generated by use of heterogeneous lipid environments in the reconstitution. The possible existence of other phorbol ester receptors in addition to protein kinase C-phospholipid complexes remains to be resolved.

Animals↗