Analysis of phosphorylation-dependent modulation of Kv1.1 potassium channels.

The voltage-gated potassium channel Kv1.1 contains phosphorylation sites for protein kinase A (PKA) and protein kinase C (PKC). To study Kv1.1 protein expression and cellular distribution in regard to its level of phosphorylation, the effects of PKA and PKC activation on Kv1.1 were investigated in HEK 293 cells stably transfected with Kv1.1 (HEK 293/1). Without kinase activation, HEK 293/1 cells carry unphosphorylated Kv1.1 protein in the plasma membranes, whereas large amounts of phosphorylated and unphosphorylated Kv1.1 protein were located intracellularly. Activation of PKA resulted in phosphorylation of intracellular Kv1.1 protein, followed by a rapid translocation of Kv1.1 into the plasma membrane. Patch-clamp analysis revealed an increase in current amplitude upon PKA activation and demonstrated differences in the voltage dependence of current activation between unphosphorylated and phosphorylated Kv1.1 channels. In contrast to PKA, even prolonged activation of PKC did not lead to direct phosphorylation of Kv1.1, but induced Kv1.1 protein synthesis. Thus, protein kinases have direct and indirect effects on the functional expression of voltage-gated potassium channels. Our data suggest that the synergistic action of protein kinases may play an important role in the fine-tuning of Kv channel function.

Differentiation-associated apoptosis of neural stem cells is effected by Bcl-2 overexpression: impact on cell lineage determination.

Apoptosis is an integral part of neural development. To elucidate the importance of programmed cell death on cell lineage determination we utilized murine PCC7-Mzl cells, a model system for neural differentiation. Treatment of pluripotent PCC7-Mzl stem cells with 0.1 microM all-trans retinoic acid (RA) causes a cease of proliferation and an initiation of differentiation into neurons, glial cells and fibroblasts. Simultaneously, a fraction of the cell culture (ca. 25%) dies within 24 h by apoptosis. We transfected PCC7-Mzl cells with the human bcl-2 cDNA and generated PCC7-Mz-Bcl-2 cell lines expressing two- to tenfold higher levels of Bcl-2 than parental cells. Overexpression of Bcl-2 resulted in hypophosphorylation of the retinoblastoma (Rb) protein and consequently prolonged the doubling time of the culture from 18 h to 23 h. RA-induced apoptosis was drastically reduced to 3 to 15% depending on the level of Bcl-2 expression. RA-induced caspase activation, cytochrome c release from the mitochondria to the cytosol and DNA fragmentation was completely blocked. Furthermore, treating Bcl-2 cultures with ceramide (10 microM), a second messenger mediating the RA-initiated death signal in parental cells, no longer caused DNA laddering. Bcl-2 overexpression did not interfere with the potential of PCC7-Mz cells to develop into neurons, glial cells and fibroblasts. However, the relative distribution of cell types in the culture was shifted such that the fraction of neurons was reduced to half (from 60 to 30%) with a concomitant increase in the number of glial and fibroblastoid cells. Furthermore, Bcl-2-overexpressing neurons, but not neurons of parental or mock-transfected PCC7-Mzl cultures, were able to grow as single cells.

Involvement of protein kinase Cdelta in contact-dependent inhibition of growth in human and murine fibroblasts.

There is evidence that protein kinase C delta (PKCdelta) is a tumor suppressor, although its physiological role has not been elucidated so far. Since important anti-proliferative signals are mediated by cell-cell contacts we studied whether PKCdelta is involved in contact-dependent inhibition of growth in human (FH109) and murine (NIH3T3) fibroblasts. Cell-cell contacts were imitated by the addition of glutardialdehyde-fixed cells to sparsely seeded fibroblasts. Downregulation of the PKC isoforms alpha, delta, epsilon, and mu after prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) resulted in a significant release from contact-inhibition in FH109 cells. Bryostatin 1 selectively prevented TPA-induced PKCdelta-downregulation and reversed TPA-induced release from contact-inhibition arguing for a role of PKCdelta in contact-inhibition. In accordance, the PKCdelta specific inhibitor Rottlerin (1 microM) totally abolished contact-inhibition. Interestingly, immunofluorescence revealed a rapid translocation of PKCdelta to the nucleus when cultures reached confluence with a peak in early-mid G1 phase. Nuclear translocation of PKCdelta in response to cell-cell contacts could also be demonstrated after subcellular fractionation by Western blotting and by measuring PKCdelta-activity after immunoprecipitation. Transient transfection of NIH3T3 cells with a dominant negative mutant of PKCdelta induced a transformed phenotype. We conclude that PKCdelta is involved in contact-dependent inhibition of growth.

Solid-phase synthesis and biological evaluation of a teleocidin library--discovery of a selective PKCdelta down regulator.

Protein kinaseC (PKC) is linked to the signal-induced modulation of a wide variety of cellular processes, such as growth, differentiation, secretion, apoptosis, and tumor development. The design and synthesis of small molecules that regulate these different cellular signaling systems is at the forefront of modern drug design. Herein we report a) an efficient method for the synthesis of indolactamV (6), a PKC activator, and its N13-des(methyl) analogues (19) using a regioselective organometallic transformation, a convenient aminomalonate derivative (10) to introduce the appropriate functionality and an enantiospecific enzymic hydrolysis as key steps; b) the use of this method in the first solid-phase synthesis of a teleocidin library modifying the N-13, C-12 and C-7 alkyl chains, and, therefore, producing a library of potential activators and/or inhibitors of PKC of the general structure (32); c) the activation of PKC by selected members of the library using a MARCKS translocation in vivo assay system; d) the observation that some of these analogues are nearly as effective as the natural PKC activators phorbol dibutyrate and (-)-indolactam V (6), and e) the observation that some of these analogues have different potential to induce down-regulation of members of the PKC gene family after chronic stimulation.

Production of ceramides causes apoptosis during early neural differentiation in vitro.

To investigate signal transduction pathways leading to apoptosis during the early phase of neurogenesis, we employed PCC7-Mz1 cells, which cease to proliferate and begin to differentiate into a stable pattern of neurons, astroglial cells, and fibroblasts upon incubation with retinoic acid (RA). As part of lineage determination, a sizable fraction of RA-treated cultures die by apoptosis. Applying natural long-chain C(16)-ceramides as well as membrane-permeable C(2)/C(6)-ceramide analogs caused apoptosis, whereas the biologically nonactive C(2)-dihydroceramide did not. Treating PCC7-Mz1 stem cells with a neutral sphingomyelinase or with the ceramidase inhibitor N-oleoylethanolamine elevated the endogenous ceramide levels and concomitantly induced apoptosis. Addition of RA caused an increase in ceramide levels within 3-5 h, which reached a maximum (up to 3.5-fold of control) between days 1 and 3 of differentiation. Differentiated PCC7-Mz1 cells did not respond with ceramide formation and apoptosis to RA treatment. The acidic sphingomyelinase contributed only weakly and the neutral Mg(2+)-dependent and Mg(2+)-independent sphingomyelinases not at all to the RA-mediated production of ceramides. However, ceramide increase was sensitive to the ceramide synthase inhibitor fumonisin B(1), suggesting a crucial role for the de novo synthesis pathway. Enzymatic assays revealed that ceramide synthase activity remained unaltered, whereas serine palmitoyltransferase (SPT), a key enzyme in ceramide synthesis, was activated approximately 2.5-fold by RA treatment. Activation of SPT seemed to be mediated via a post-translational mechanism because levels of the mRNAs coding for the two SPT subunits were unaffected. Expression of marker proteins shows that ceramide regulates apoptosis, rather than differentiation, during early neural differentiation.

Natural Product Synthesis on Polymeric Supports-Synthesis and Biological Evaluation of an Indolactam Library.

Potent activators of protein kinase C in fibroblasts: This property was determined for several indolactam V analogues (1) with a new cell-based assay system. This tumor-promoting indole alkaloid and analogues thereof can be synthesized efficiently on the solid phase. The key steps of the combinatorial approach are a regioselective amination of the indole ring and an enantioselective enzymatic reaction.

The protein kinase C (PKC) substrate GAP-43 is already expressed in neural precursor cells, colocalizes with PKCeta and binds calmodulin.

Expression of the growth-associated protein of 43-kDa (GAP-43), which is described as a postmitotic, neuron-specific major protein kinase C (PKC) substrate, was investigated in the murine embryonic carcinoma cell line PCC7-Mz1 which develops into a brain-tissue-like pattern of neuronal, fibroblast-like and astroglial cells upon stimulation with all-trans retinoic acid (RA). GAP-43 expression was very low in stem cells, but increased on mRNA and protein level within the 12 h after differentiation was initiated. While the P1 promoter of the GAP-43 gene gave rise to a 1.6-kb mRNA and was already active at a very low level in PCC7-Mz1 stem cells, transcription of the P2 promoter, which resulted in a 1.4-kb mRNA, was completely blocked in stem cells but increased rapidly after RA treatment. Within the first 2 days of neural differentiation, GAP-43 was localized with the cytoplasmic membrane and the Golgi complex of proliferating neural precursor cells. Then, GAP-43 was translocated to the growth cones and neurites, and from day 6, when neurons began to acquire polarity, the protein was found in the axons. GAP-43 was never detected in the non-neuronal PCC7-Mz1 derivatives, i.e. in fibroblasts or glial cells. In the foetal rat brain (prenatal day F11), GAP-43 was expressed in the optic stalk, the lense plakode and in the postmitotic neurons of the marginal zone of the hindbrain. Moreover, in a layer between the ventricular and marginal zone of the hindbrain (F13) and forebrain (F15), GAP-43 was already expressed in mitotic neural precursor cells. In PCC7-Mz1 cultures, 2 days after addition of RA, GAP-43 became phosphorylated upon activation of PKC, and colocalized specifically with the novel PKC isoform eta. Phosphorylation of GAP-43 caused a disruption of its complex with calmodulin. These data demonstrate that GAP-43 is already a functional PKC substrate in prolific neuronal precursor cells, and may participate in neuronal cell lineage determination.

Activation of protein kinase C alpha and/or epsilon enhances transcription of the human endothelial nitric oxide synthase gene.

In primary human umbilical vein endothelial cells (HUVECs), incubation with phorbol-12-myristate-13-acetate (PMA) enhanced basal and bradykinin-stimulated nitric oxide production. In the HUVEC-derived cell line EA.hy 926, PMA and phorbol-12,13-dibutyrate stimulated endothelial nitric oxide synthase (NOS III) mRNA expression in a concentration- and time-dependent manner. Maximal mRNA expression (3.3-fold increase) was observed after 18 hr. NOS III protein and activity were increased to a similar extent. The specific protein kinase C (PKC) inhibitors bisindolylmaleimide I (1 microM), Gö 6976 [12-(2 cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo-[3, 4-c]carbazole] (1 microM), Ro-31-8220 [3-[1-[3(amidinothio)propyl-1H-inoyl-3-yl]3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate] (1 microM), and chelerythrine (3 microM) did not change NOS III expression when applied alone, but they all prevented the up-regulation of NOS III mRNA produced by PMA. Of the PKC isoforms expressed in EA.hy 926 cells (alpha, beta I, delta, epsilon, eta, zeta, lambda, and mu), only PKC alpha and PKC epsilon showed changes in protein expression after PMA treatment. Incubation of EA.hy 926 cells with PMA for 2-6 hr resulted in a translocation of PKC alpha and PKC epsilon from the cytosol to the cell membrane, indicating activation of these isoforms. After 24 hr of PMA incubation, both isoforms were down-regulated. The time course of activation and down-regulation of these two PKC isoforms correlated well with the PMA-stimulated increase in NOS III expression. When human endothelial cells (ECV 304 or EA.hy 926) were transiently or stably transfected with a 3.5-kb fragment of the human NOS III promoter driving a luciferase reporter gene, PMA stimulated promoter activity up to 2.5-fold. On the other hand, PMA did not change the stability of the NOS III mRNA. These data indicate that stimulation of PKC alpha, PKC epsilon, or both by active phorbol esters represents an efficacious pathway activating the human NOS III promoter in human endothelium.

Retinoic acid induces apoptosis-associated neural differentiation of a murine teratocarcinoma cell line.

Incubation with all-trans retinoic acid (RA) induces PCC7-Mz1 embryonic carcinoma cells to cease proliferation and to develop into a tissue-like pattern of neuronal, astroglial, and fibroblast-like derivatives over a period of several days. Concomitant with the induction of differentiation by RA, a sizable fraction of the Mz1 stem cells detaches and dies, with the maximal level of cell death achieved after 10 h of RA treatment. This RA-induced cell death fulfills all criteria of apoptosis, including nuclear condensation, intranucleosomal DNA degradation, expression of cysteine aspases (caspases), and the formation of apoptotic bodies. Apoptosis could be suppressed by the pan-caspase inhibitor zVAD-fmk (benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone). Induction of apoptosis required at least 2 h of incubation with RA and followed the same RA concentration (EC50 = 10(-7) M RA) and time dependence as the induction of differentiation as delineated by the expression of the neuron-specific protein kinase C substrate GAP-43. RA-induced apoptosis increased with the plating density of PCC7-Mz1 cells. This effect was not due to deprivation of an essential nutrient or factor from the medium because apoptosis was not significantly affected by an increase of the concentration of fetal calf serum. In addition to RA, apoptosis could be induced by DNA-damaging treatment (UV light, cisplatin, methanesulfonic acid methyl ester) and cell cycle-arresting agents (hydroxyurea) as well as by serum depletion. Because inhibition of transcription and translation caused cell death efficiently even in the presence of serum, the synthesis of apoptosis-inhibiting factors by the cultured cells is indicated. Neither ApoI/Fas antibody nor glutamate induced apoptosis. Mz1 cells that have entered a differentiation pathway in response to RA treatment become increasingly less sensitive to apoptosis. This may be due in part to the expression of the bcl-2 proto-oncogene, which was detectable on the mRNA and protein level beginning 4 days after the addition of RA. The intracellular signaling pathway leading to apoptosis does not involve conventional or novel members of the protein kinase C gene family. Neither activation of protein kinase C by phorbol esters (phorbol 12,13-dibutyrate) nor inhibition by specific inhibitors (GF109203X, Gö 6976) and long-term treatment with phorbol 12,13-dibutyrate, in the presence or absence of RA, significantly influenced the amount or rate of apoptosis of PCC7-Mz1 cells. We conclude that the apoptotic activity following RA treatment of cultured PCC7-Mz1 cells probably is controlled by the same cascade of gene regulatory events that govern the early cell lineage determinations in this in vitro model system of neural development.

Expression of protein kinase C gene family members is temporally and spatially regulated during neural development in vitro.

We used primary cultures of rat hippocampal neurons and PCC7-Mz1 cells to correlate the expression of the protein kinase C (PKC) gene family with specific events during neural differentiation. Multipotent PCC7-Mz1 embryonic carcinoma stem cells develop into a tissue-like pattern of neuronal, fibroblast-like and astroglial cells by all-trans retinoic acid (RA) treatment. Western blot analyses demonstrate that PKCalpha, betaI, gamma, theta, mu, lambda, and zeta were constitutively expressed but the expression of PKCbetaII, delta, epsilon, and eta was up-regulated three days after addition of RA when cells mature morphologically. While the protein levels of the PKC isoforms betaII, delta and eta decreased after d6, when the major phenotypical alterations of the developing neurons were completed, PKCepsilon expression remained at a high level. Immunofluorescence studies demonstrated that PKCalpha, lambda and zeta were constantly expressed in stem cells and the arising cell types. PKCdelta was detected in all differentiated cell types, whereby PKCbetaII, gamma, epsilon, and zeta were solely found in the neuronal derivatives with PKCgamma predominantly located in the nuclei. PKCeta was weakly expressed at the Golgi complex of stem cells but expanded throughout the entire somata of all developing neurons. In contrast, PKCbetaII was abundant only in the somata of a minor fraction of all neurons (approximately 2.5%). Also, PKCepsilon was exclusively synthesized by a subpopulation of neurons (40+/-5%), where it was localized in the somata and in the axons. PKCzeta was persistently expressed in two forms, the full-length PKCzeta and the constitutively active, proteolytic product PKMzeta, reasoning that permanent PKCzeta activity is important for PCC7-Mz1 physiology. Fractionation of extracts from undifferentiated and differentiating PCC7-Mz1 cells revealed that the conventional cPKCalpha was partly and the cPKCbetaI and the novel nPKCs delta and epsilon were mainly membrane bound, implying that they were also in an active state. However, when using the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate) to monitor cellular PKC activity, we observed that activation of PKC by phorbol ester was required for complete MARCKS phosphorylation and its translocation from the membrane to the cytoplasm. Our data show that the cell type-specific expression, subcellular localization and activation of PKCs are regulated in an isoform-specific manner during neurogenesis suggesting that they are involved in the control of neural development and in particular in neuronal differentiation.

Activation of gp130 by IL-6/soluble IL-6 receptor induces neuronal differentiation.

Interleukin-6 (IL-6) on target cells binds to the specific IL-6 receptor (IL-6R) and subsequently induces homodimerization of the signal-transducing protein gp130. Cells which express gp130 but no IL-6R and which therefore do not respond to IL-6 can be stimulated by the complex of IL-6 and soluble IL-6R (slL-6R). Here we show that on rat pheochromocytoma cells (PC12), the combination of IL-6 and slL-6R but not IL-6 alone induces expression of c-fos, GAP-43 and neuron-specific enolase followed by neuron-specific differentiation and formation of a neuronal network. The differentiation was dose-and time-dependent and followed the same kinetics as nerve-growth factor (NGF)-induced differentiation. The responses of PC12 cells to IL-6/sIL-6R and NGF were additive, suggesting independent signaling pathways. We demonstrate that activation of gp130 generates a neuronal differentiation signal that is equivalent to and independent of trk/NGF receptor tyrosine kinase. Interestingly, the failure of IL-6 to induce differentiation of PC12 cells is not due to lack of surface expression of IL-6R as IL-6 alone triggered expression of GAP-43 mRNA and protein. We hypothesize that PC12 cells express more gp130 than IL-6R and that the extent of activated gp130 molecules determines the quality of the response.

p42 MAPK phosphorylates 80 kDa MARCKS at Ser-113.

It is demonstrated here that p42 MAPKinase (p42 MAPK) phosphorylates the Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) at Ser-113. In permeabilised Swiss 3T3 cells activation of protein kinase C (PKC) leads to p42 MAPK activation, but only the protein kinase C sites in MARCKS become phosphorylated and not Ser-113. The mitogen platelet-derived growth factor (PDGF) elicits the same response. These results demonstrate that while Ser-113 is a substrate for p42 MAPK in vitro and can be phosphorylated in vivo as shown by Taniguchi et al. [(1994) J. Biol. Chem. 269, 18299-18302], its phosphorylation is not subject to acute regulation by p42 MAPK in Swiss 3T3 cells.

Phosphorylation of GAP-43 (growth-associated protein of 43 kDa) by conventional, novel and atypical isotypes of the protein kinase C gene family: differences between oligopeptide and polypeptide phosphorylation.

GAP-43 (growth-associated protein of 43 kDa; also known as neuromodulin, P-57, B-50 and F-1) is a neuronal calmodulin binding protein and a major protein kinase C (PKC) substrate in mammalian brain. Here we describe the phosphorylation by and the site specificity of different PKC isotypes. The conventional PKC beta 1 and the novel PKCs delta and epsilon effectively phosphorylated recombinant GAP-43 in vitro; atypical PKC zeta did not. The K(m) values (between 0.6 and 2.3 microM) were very low, demonstrating a high-affinity interaction between kinase and substrate. All PKC isotypes were shown to phosphorylate serine-41 in GAP-43. When using a 19-amino-acid oligopeptide based on the GAP-43 phosphorylation site as substrate, there was a significant difference compared with polypeptide phosphorylation. The V(max) values of PKC beta 1 and PKC epsilon were much higher for this oligopeptide than for the complete protein (up to 10-fold); in contrast, their apparent affinities for the peptide were much lower (up to 100-fold) than for the intact GAP-43 polypeptide. Furthermore, phosphorylation of the GAP-43 oligopeptide by PKC beta 1 was more sensitive to a catalytic-site inhibitor than was phosphorylation of intact GAP-43. These results suggest that there are multiple sites of interaction between GAP-43 and PKC.

In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase C mu.

To study enzymatic activity and activation conditions of the recently identified novel protein kinase C mu (PKC mu) subtype, epitope tagged PKC mu was propagated in the baculovirus expression system and was purified to homogeneity. PKC mu displays high affinity phorbol ester binding (Kd=7 nM) resulting in enhanced phosphatidylserine-dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns-4,5-P2, were found to promote PKC mu kinase activity. Two peptides derived from the glycogen synthase, GS-peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine-rich C-kinase substrate) served as an in vitro substrate for PKC mu too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKC mu.

PRK1 phosphorylates MARCKS at the PKC sites: serine 152, serine 156 and serine 163.

The 80kDa Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) is a major in vivo substrate of protein kinase C (PKC). Here we report that MARCKS is a major substrate for the lipid-activated PKC-related kinase (PRK1) in cell extracts. Furthermore, PRK1 is shown to phosphorylate MARCKS on the same sites as PKC in vitro. Thus, control of MARCKS phosphorylation on these previously identified 'PKC' sites may be regulated under certain circumstances by PRK as well as PKC mediated signalling pathways. The implications for MARCKS as a marker of PKC activation and as a point of signal convergence are discussed.

The myristoylated alanine-rich C-kinase substrate (MARCKS) is sequentially phosphorylated by conventional, novel and atypical isotypes of protein kinase C.

The myristoylated alanine-rich C-kinase substrate (MARCKS) is the major protein kinase C (PKC) substrate in many cell types including fibroblasts and brain cells. Here we describe the phosphorylation of MARCKS and the site specificity for different PKC isotypes. Conventional (c)PKC beta 1, novel (n)PKC delta and nPKC epsilon efficiently phosphorylated the MARCKS protein in vitro. The Km values were extremely low, reflecting a high affinity between kinases and substrate. The apparent affinity of nPKC delta (Km = 0.06 microM) was higher than that of nPKC epsilon and cPKC beta 1 (Km = 0.32 microM). The rate of substrate phosphorylation was inversely correlated with affinity and decreased in the order nPKC epsilon > cPKC beta 1 > nPKC delta. Atypical (a)PKC zeta did not phosphorylate the intact MARCKS protein. However, a 25-amino-acid peptide deduced from the MARCKS phosphorylation domain, was efficiently phosphorylated by aPKC zeta as well as by the other three PKC. Site analysis revealed that only serine residues S152, S156 and S163 were phosphorylated, with S163 phosphorylated highest, followed by S156 and S152; in contrast, S160 and S167 were not phosphorylated. No further PKC phosphorylation sites could be detected in MARCKS. The phosphorylation pattern was independent of the type of PKC isotype used. Kinetic analysis showed, that MARCKS is sequentially phosphorylated in the order S156 > S163 > S152 by cPKC, nPKC and aPKC. There was no dramatic difference in the sequential phosphorylation of MARCKS detectable when comparing the four PKC isotypes. The results are discussed in the context of the functional significance of MARCKS phosphorylation.

The human CCKB/gastrin receptor transfected into rat1 fibroblasts mediates activation of MAP kinase, p74raf-1 kinase, and mitogenesis.

The human cholecystokinin (CCK)B/gastrin receptor was stably transfected into Rat1 fibroblasts to examine the signaling pathways mediated by this seven-transmembrane, G protein-linked receptor. We report here that binding of CCK-8 or gastrin to the CCKB/gastrin receptor induced phosphoinositide breakdown and led to a rapid, transient, and concentration-dependent increase in intracellular Ca2+, which was completely blocked by a specific CCKB receptor antagonist. The peptides also stimulated tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin. Both CCK-8 and gastrin induced a dose- and time-dependent activation of MAP kinase and p74raf-1 kinase in the transfected Rat1 cells. These effects could be dissociated from protein kinase C activation and were not dependent on a functional Gi protein. Finally, both CCK-8 and gastrin induced DNA synthesis in Rat1 cells transfected with the human CCKB/gastrin receptor through a pertussis toxin-insensitive pathway. These results indicate that the neuropeptides gastrin and CCK can activate multiple signal transduction pathways and act as sole mitogens by binding to the CCKB/gastrin receptor transfected into Rat1 fibroblasts.

Expression of a reporter gene is reduced by a ribozyme in transgenic plants.

A chimeric gene encoding a ribozyme under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into transgenic tobacco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression assays in plant protoplasts. The ribozyme gene was transferred into transgenic tobacco plants expressing an rbcS-npt chimeric gene as an indicator. Five double transformants out of sixteen exhibited a reduction in the amount of active NPT enzyme. To measure the amount of ribozyme produced, in the absence of its target, the ribozyme and target genes were separated by genetic segregation. The steady-state concentrations of ribozyme and target RNA were shown to be similar in the resulting single transformants. Direct evidence for a correlation between reduced npt gene expression and ribozyme expression was provided by crossing a plant containing only the ribozyme gene with a transgenic plant expressing the npt gene under control of the 35S promoter, i.e. the same promoter used to direct ribozyme expression. The expression of npt was reduced in all progeny containing both transgenes. Both steady-state levels of npt mRNA and amounts of active NPT enzyme are decreased. In addition, our data indicate that, at least in stable transformants, a large excess of ribozyme over target is not a prerequisite for achieving a significant reduction in target gene expression.

Bombesin, endothelin and platelet-derived growth factor induce rapid translocation of the myristoylated alanine-rich C-kinase substrate in Swiss 3T3 cells.

We analyzed the effect of growth factors on the localization of the 80-kDa acidic myristoylated alanine-rich C-kinase substrate (80-kDa MARCKS), the major protein kinase C (PKC) substrate, in Swiss 3T3 fibroblasts. Virtually all 80-kDa MARCKS of quiescent cultures of these cells was membrane bound. However, within 40 min after addition of bombesin (10 nM) to these cells, the content of 80-kDa MARCKS in the cytoplasmic fraction increased 25-fold. Phosphorylated 80-kDa MARCKS was detectable in the cytoplasmic fraction as early as 30 s after addition of bombesin and the translocation was sustained for 6 h i.e. until 80-kDa MARCKS became down-regulated. The ability of bombesin to stimulate translocation of 80-kDa MARCKS was dose-dependent (concentration required to produce 50% of the effect was 0.6 nM bombesin) and was abolished by the specific antagonist [Leu14,13 psi 14CH2NH]bombesin. Furthermore, platelet-derived growth factor (PDGF) stimulated a dose-dependent (concentration required to produce 50% of the effect was 3 ng/ml) translocation which was comparable to that induced by bombesin in terms of kinetics and magnitude. Translocation was independent of continuous protein synthesis, but dependent on active PKC. Depletion or inhibition of PKC activity abolished the 80-kDa MARCKS translocation induced by either bombesin or PDGF. Furthermore, the neuropeptides beta-endothelin, bradykinin, and vasopressin, which are known to stimulate PKC activity, also promoted translocation. In contrast, epidermal growth factor, insulin and forskolin, which do not activate PKC, failed to cause such an effect. Translocation of 80-kDa MARCKS was also observed in Rat1 cells treated with phorbol ester, PDGF and beta-endothelin. We conclude that the translocation of 80-kDa MARCKS from the membrane to the cytosol is an early response to a variety of growth-promoting factors that stimulate PKC through different signal-transduction pathways.

Overexpression of the myristoylated alanine-rich C-kinase substrate in Rat1 cells increases sensitivity to calmodulin antagonists.

The acidic 80-kDa myristoylated alanine-rich C-kinase substrate protein (80-kDa MARCKS) is the major protein-kinase-C substrate in rodent fibroblasts. To elucidate its function, we transfected the cDNA coding for the 80-kDa MARCKS protein into Rat1 fibroblasts. One clone, called Rat1-80K, expressed 4.5 +/- 0.8-fold and 9.5 +/- 1.5-fold higher levels of 80-kDa MARCKS protein under quiescent and growing conditions, respectively, compared to mock or untransfected control cells. Southern-blot and Northern-blot analyses of Rat1-80K showed intact integration and correct transcription of the introduced 80-kDa MARCKS gene. The overexpressed 80-kDa MARCKS protein was phosphorylated and translocated from the membrane to the cytoplasmic fraction. Since 80-kDa MARCKS has been described as a calmodulin-binding protein in in vitro studies, we investigated the effects of the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and triflouperazine on the entry into the S-phase of the cell cycle in intact cells. DNA synthesis by Rat1-80K cells was more sensitive to either N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or triflouperazine than that of control cells. Our results suggest that overexpression of the 80-kDa MARCKS protein reduces the free concentration of calmodulin in the cell.