Phorbol Ester-induced Translocation Research Articles

β3-Chimaerin, a novel member of the chimaerin Rac-GAP family

Chimaerins are a family of diacylglycerol- and phorbol ester-regulated GTPase activating proteins (GAPs) for the small G-protein Rac. Extensive evidence indicates that these proteins play important roles in development, axon guidance, metabolism, cell motility, and T cell activation. Four isoforms have been reported to-date, which are products of CHN1 (α1- and α2-chimaerins) and CHN2 (β1- and β2-chimaerins) genes. Although these gene products are assumed to be generated by alternative splicing, bioinformatics analysis of the CHN2 gene revealed that β1- and β2-chimaerins are the products of alternative transcription start sites (TSSs) in different promoter regions. Furthermore, we found an additional TSS in CHN2 gene that leads to a novel product, which we named β3-chimaerin. Expression profile analysis revealed predominantly low levels for the β3-chimaerin transcript, with higher expression levels in epididymis, plasma blood leucocytes, spleen, thymus, as well as various areas of the brain. In addition to the prototypical SH2, C1, and Rac-GAP domains, β3-chimaerin has a unique N-terminal domain. Studies in cells established that β3-chimaerin has Rac-GAP activity and is responsive to phorbol esters. The enhanced responsiveness of β3-chimaerin for phorbol ester-induced translocation relative to β2-chimaerin suggests differential ligand accessibility to the C1 domain.

ARF1-regulated coatomer directs the steady-state localization of protein kinase C epsilon at the Golgi apparatus

Protein kinase C epsilon (PKCε) contributes to multiple signaling pathways affecting human disease. The function of PKCε requires it to undergo changes in subcellular distribution in response to signaling events. While the mechanisms underlying this translocation are incompletely understood, it involves the receptor for activated C kinase protein (RACK2/β′-COP). This receptor also functions as a vesicle coat protein in the secretory pathway where it is regulated by the small GTP-binding protein ADP-ribosylation factor, ARF1. We inhibited ARF1 activation to test the requirement for RACK2/β′-COP in PKCε localization in NIH3T3 fibroblasts. We found that steady-state localization of PKCε at the Golgi complex requires ARF1-regulated RACK2/β′-COP function. By contrast, we did not observe any defects in phorbol ester-induced translocation when ARF1 was inhibited. We also found that PKCε bound to isolated membranes through two distinct mechanisms. One mechanism was dependent upon RACK2/β′-COP while a second was RACK2/β′-COP-independent and stimulated by phorbol esters. Finally, we show that RACK2/β′-COP affects the subcellular distribution of a constitutively active form of PKCε, in a manner similar to what we observed for wild-type PKCε. Together, our data support a role for RACK2/β′-COP in the steady-state localization of PKCε at the Golgi apparatus, which may be independent of its role during PKCε translocation to the cell surface.

Synthesis and Antiapoptotic Activity of a Novel Analogue of the Neutral Sphingomyelinase Inhibitor Scyphostatin

The enantioselective synthesis of an analogue of scyphostatin, a potent inhibitor of the neutral sphingomyelinase, is described. The synthesis starts with cyclohexanone and a protected D-serine derivative. The key step is an asymmetric hydroxylation to access a hydroxycyclohexanone, which is transformed into a substituted hydroxycyclohexenone. This is converted into the scyphostatin analogue 14, a chemically and metabolically stabilised compound lacking the epoxy function of the natural congener and carrying a palmitic acid group instead of the native trienoyl residue. An evaluation of the biological activity of 14 revealed neutral sphingomyelinase inhibition in several in vivo test systems (monocytes, macrophages, hepatocytes) monitoring antiapoptotic effects and the inversion of phorbolester-induced translocation of green fluorescent protein labelled kinase (protein kinase C-alpha).

Phorbol ester-induced translocation of PKC epsilon to the nucleus in fibroblasts: identification of nuclear PKC epsilon-associating proteins

We show that phorbol ester treatment of NIH 3T3 fibroblasts induces rapid translocation of PKCε from a perinuclear site to the nucleus, extending findings in PC12 and NG108-15 cells and in myocytes. We have immunoprecipitated the PKCε from nuclei isolated from phorbol ester-treated fibroblasts and identified six proteins which associate with nuclear PKCε. These have been characterised as matrin 3, transferrin, Rac GTPase activating protein 1, vimentin, β-actin and annexin II by MALDI–TOF–MS. We have confirmed that these proteins associate with PKCε by gel overlay and/or dot blotting assays. The role of these PKCε-associating proteins in the nucleus and their interaction with PKCε are considered.

Protein kinase C in rat brain is altered by developmental lead exposure.

The absence of learning-related redistribution of hippocampal protein kinase C (PKC) has been correlated with impairment of learning performance induced by developmental lead (Pb) exposure. This study was designed to examine whether the properties of brain PKC are altered by chronic Pb exposure during development. Two-tenth percent Pb acetate was administered to pregnant and lactating dams and then administered to weanlings in drinking water until postnatal day (PN) 56. Effects of Pb on translocation of PKC were studied in brain slices prepared from hippocampus. When the slices were treated with 0.33 microM phorbol-12, 13-dibutyrate (PDBu) for 15 min, a significant increase in PKC activity was observed in the membrane fraction of hippocampal slices from Pb-exposed rats, suggesting that chronic Pb exposure potentiates PDBu-activated PKC translocation. Data obtained from saturation binding assays in the frontal cortices of Pb-exposed rats showed a decrease in the dissociation constant (KD) in both membrane and cytosolic PKC. A decrease in the total binding sites (Bmax) of [3H]PDBu binding was only observed in membrane PKC. Furthermore, developmental Pb exposure decreased PKC-gamma, but not PKC-alpha, -betaII, and -epsilon in the membrane fraction of the hippocampus and the frontal cortex. These results indicate that chronic Pb exposure during development increases phorbol ester binding affinity, enhances phorbol ester-induced translocation of PKC, and down-regulates membrane PKC, mainly PKC-gamma.

The Catalytic Domain of Protein Kinase C Chimeras Modulates the Affinity and Targeting of Phorbol Ester-induced Translocation

Emerging evidence suggests important differences among protein kinase C (PKC) isozymes in terms of their regulation and biological functions. PKC is regulated by multiple interdependent mechanisms, including enzymatic activation, translocation of the enzyme in response to activation, phosphorylation, and proteolysis. As part of our ongoing studies to define the factors contributing to the specificity of PKC isozymes, we prepared chimeras between the catalytic and regulatory domains of PKCalpha, -delta, and -epsilon. These chimeras, which preserve the overall structure of the native PKC enzymes, were stably expressed in NIH 3T3 fibroblasts. Their intracellular distribution was similar to that of the endogenous enzymes, and they responded with translocation upon treatment with phorbol 12-myristate 13-acetate (PMA). We found that the potency of PMA for translocation of the PKCalpha/x chimeras from the soluble fraction was influenced by the catalytic domain. The ED50 for translocation of PKCalpha/alpha was 26 nM, in marked contrast to the ED50 of 0.9 nM in the case of the PKCalpha/epsilon chimera. In addition to this increase in potency, the site of translocation was also changed; the PKCalpha/epsilon chimera translocated mainly into the cytoskeletal fraction. PKCx/epsilon chimeras displayed twin isoforms with different mobilities on Western blots. PMA treatment increased the proportion of the higher mobility isoform. The two PKCx/epsilon isoforms differed in their localization; moreover, their localization pattern depended on the regulatory domain. Our results emphasize the complex contributions of the regulatory and catalytic domains to the overall behavior of PKC.

Inhibitors of actin polymerization and calmodulin binding enhance protein kinase C-induced translocation of MARCKS in C6 glioma cells

MARCKS ( myristoylated alanine- rich C- kinase substrate) is known to interact with calmodulin, actin filaments, and anionic phospholipids at a central basic domain which is also the site of phosphorylation by protein kinase C (PKC). In the present study, cytochalasin D (CD) and calmodulin antagonists were used to examine the influence of F-actin and calmodulin on membrane interaction of MARCKS in C6 glioma cells. CD treatment for 1 h disrupted F-actin filaments, increased membrane bound immunoreactive MARCKS (from 51% to 62% of total), yet markedly enhanced the amount of MARCKS translocated to the cytosolic fraction in response to the phorbol ester 4 β-12- O-tetradecanoylphorbol 13-acetate. In contrast, CD treatment had no effect on phorbol ester-stimulated phosphorylation of MARCKS or on translocation of PKC α to the membrane fraction. Staurosporine also increased membrane association of MARCKS in a PKC-independent manner, as no change in MARCKS phosphorylation was noted and bis-indolylmaleimide (a more specific PKC inhibitor) did not alter MARCKS distribution. Staurosporine inhibited the phorbol ester-induced translocation of MARCKS but not of PKC α in both CD pretreated and untreated cells. Calmodulin antagonists (trifluoperazine, calmidazolium) had little effect on the cellular distribution or phosphorylation of MARCKS, but were synergistic with phorbol ester in translocating MARCKS from the membrane without a further increase in its phosphorylation. We conclude that cytoskeletal integrity is not required for phosphorylation and translocation of MARCKS in response to activated PKC, but that interaction with both F-actin and calmodulin might serve to independently modulate PKC-regulated localization and function of MARCKS at cellular membranes.

Possible involvement of protein kinase C-ϵ in phorbol ester-induced growth inhibition of human lymphoblastic cells

Sustained activation of members of the protein kinase C (PKC) family is known to influence the growth and differentiation of various cell types, however, the specific roles for individual isoforms mediating these cellular events have yet to be elucidated. Activation of PKC by phorbol esters leads to growth inhibition in certain cell lines. The HT58 human B lymphoblastic cell may serve as a cellular model system to investigate the participation of individual isoforms in the initial events of growth arrest induced by phorbol ester. Determination of cell cycle and investigation of apoptosis were performed by flow cytometric measurements. Phorbol ester-induced translocation and down-regulation of the conventional α, β and the novel ϵ isoforms of PKC were demonstrated by Western blot analysis. At lower concentrations (0.5 ng/ml) phorbol myristate acetate (PMA) stimulated a G1 arrest with retention of viability in the human HT58 B lymphoblastic cell. The protein kinase inhibitor staurosporine at a concentration of 25 nM did not significantly alter HT58 cell viability. However, staurosporine (25 nM) induced apoptosis in cells preincubated for 4 hr with 0.5–1.0 ng/ml PMA. The translocation of PKC-ϵ was observed within 30 min exposure to 0.5 ng/ml PMA. After a 4 hr treatment, evidence for down-regulation and an altered phosphorylation state of PKC-ϵ was seen. In contrast, the conventional α and β isoforms were practically uneffected by this PMA treatment. At higher PMA concentrations (50 ng/ml) the α and β isoforms showed a significant down-regulation. The preferential alterations in PKC-ϵ observed under the conditions required for PMA to influence the growth and survival of HT58 cells suggest a role for the Ca 2+-independent ϵ isoform in mediating the initial events of the phorbol ester stimulated cellular responses.

C2 Region-derived Peptides Inhibit Translocation and Function of β Protein Kinase C in Vivo

RACK1 is a protein kinase C (PKC)-binding protein that fulfills the criteria previously established for a receptor for activated C-kinase (RACK). If binding of PKC to RACK anchors the activated enzyme near its protein substrates, then inhibition of this binding should inhibit translocation and function of the enzyme in vivo. Here, we have identified such inhibitors that mimic the RACK1-binding site on beta PKC. We first found that a C2-containing fragment, but not a C1-containing fragment of beta PKC, bound to RACK1 and inhibited subsequent beta PKC binding. The RACK1-binding site was further mapped; peptides beta C2-1 (beta PKC(209-216), beta C2-2 (beta PKC(186-198)), and beta C2-4 (beta PKC(218-226), but not a number of control peptides, bound to RACK1 and inhibited the C2 fragment binding to RACK1. Peptides beta C2-1, beta C2-2, and beta C2-4 specifically inhibited phorbol ester-induced translocation of the C2-containing isozymes in cardiac myocytes and insulin-induced beta PKC translocation and function in Xenopus oocytes. Therefore, peptides corresponding to amino acids 186-198, and 209-226 within the C2 region of the beta PKC are specific inhibitors for functions mediated by beta PKC.

Effects of sodium on PKC translocation; relationship to neurotransmitter release.

Our previous work using Na+ channel activators such tityustoxin (TsTX), indicated that local increases in Na+ modulate glutamate release from synaptosomes. We have now investigated the role of the Ca2+/phospholipid-dependent protein kinase (PKC) in mediating this effect. TsTX and KCl stimulate 'fast' glutamate release to the same extent but TsTX is more effective than KCl in enhancing the 'slow' phase of release. KCl greatly stimulates PKC translocation. However, TsTX inhibits basal and phorbol ester-induced translocation while the Na(+)-ionophore, gramicidin D, has no effect. Taken together, these data suggest TsTX mediated localized Na+ entry inhibits PKC translocation and that this effect may be associated with recruitment of vesicles to the readily releasable pool.

Alterations in protein kinase C type III-α during heat shock of rat embryo fibroblasts

Heat shock treatment of rat embryo fibroblasts resulted in a 60% increase in cytosolic protein kinase C activity, in contrast to phorbol ester-induced translocation to the membrane. During reversal of the cells back to the normal temperature a decrease in cytosolic PKC activity was observed and paralleled by an increase in protamine kinase activity. Cell lysates prepared from heat shock-treated cells show a marked calcium/phospholipid-dependent phosphorylation of several endogenous PKC substrate proteins, while the 28-kDa stress protein was shown to be a PKC substrate. These cells express the TYPE III-α isoform of PKC and, thus, the alterations induced within cells exposed to hyperthermic treatment may reflect a functional significance with regard to the regulation of this specific isoform.

Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.

Concanavalin A- or phorbol ester-induced translocation of protein kinase C in thymoma cells from a patient with myasthenia gravis

We have examined the localization of phospholipid/calcium-dependent protein kinase (protein kinase C) in thymoma cells, which were resected from a patient with myasthenia gravis. Upon stimulation with 4 μg/ml of Concanavalin A (Con A) for 30 min, protein kinase C activity in the cytosolic fraction was increased from 7.44 pmol/min per 10 7 cells to 11.42 pmol/min per 10 7 cells. On the other hand, membrane-associated protein kinase C activity was decreased from 1.69 to 0.71 pmol/min per 10 7 cells. On the contrary, 10 −7 mol/l tetradecanoyl phorbol acetate (TPA) decreased cytosolic protein kinase C activity from 9.31 to 8.04 pmol/min per 10 7 cells, while membrane-associated protein kinase C activity was increased from 1.69 to 2.94 pmol/min per 10 7 cells. Several endogenous phosphorylated proteins (mol wt 20000, 23000) were observed as inferred by SDS-polyacrylamide gel electrophoresis. These findings indicated that Con A and TPA produce differential translocation of protein kinase C.