The Experts below are selected from a list of 1410 Experts worldwide ranked by ideXlab platform
Robert H. Lenox - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional down-regulation of MARCKS gene expression in immortalized hippocampal cells by lithium.
Journal of neurochemistry, 2008Co-Authors: Le Wang, Xingge Liu, Robert H. LenoxAbstract:The gene (Macs) for the mouse myristoylated alanine-rich C kinase substrate (MARCKS) encodes a prominent substrate for Protein kinase C that has been implicated in processes requiring signal dependent changes in actin-membrane plasticity and cytoskeletal restructuring. We have previously demonstrated that MARCKS Protein is significantly down-regulated in rat hippocampus and in an immortalized hippocampal cell line (HN33.dw) following long-term exposure to lithium at clinically relevant concentrations (1 mm). Our current studies have examined transcriptional and post-transcriptional events that may underlie the lithium-induced down-regulation of MARCKS Protein in the cultured hippocampal cell model system. MARCKS mRNA and Protein expression were found to be concomitantly down-regulated following exposure of the HN33.dw cells to chronic lithium. Whereas the stability of MARCKS mRNA remained unchanged in the presence of lithium, nuclear run-off assay indicated that the transcription of nascent MARCKS mRNA was significantly reduced (≈50%) in the cells that had been treated with lithium for 7 days. Transient transfection of HN33.dw cells with a mouse cloned Macs promoter (993-bp) showed that the Macs promoter activity was attenuated to the same extent after chronic (7–10 days), but not subacute (24 h), lithium exposure. The inhibition of the Macs promoter was found to be dependent upon the presence of a 280-bp promoter region between −993-bp and −713-bp relative to the translation start site, suggesting that this region is a potential lithium-responsive region of Macs promoter (LRR). Mutant promoter lacking the LRR not only did not respond to chronic lithium exposure but also had significantly reduced promoter activity, suggesting that chronic lithium exposure represses the transcriptional activity of activator(s) bound to the promoter. Taken together, our data indicate that transcriptional inhibition of the Macs gene underlies the lithium-induced down-regulation of MARCKS expression in the immortalized hippocampal cells.
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Chronic lithium-induced down-regulation of MARCKS in immortalized hippocampal cells: potentiation by muscarinic receptor activation.
Journal of neurochemistry, 2002Co-Authors: David G. Watson, Robert H. LenoxAbstract:: Previous studies in our laboratory have demonstrated that exposure of rats to chronic lithium results in a significant reduction in the hippocampus of levels of the Protein kinase C (PKC) phosphoProtein substrate MARCKS (myristoylated alanine-rich C kinase substrate), which persists after withdrawal and is not observed following acute administration. In an immortalized hippocampal cell line (HN33), we have determined that phorbol esters rapidly down-regulate PKC activity and lead to a subsequent PKC-dependent reduction in content of MARCKS Protein. We now report that chronic exposure of HN33 cells to LiCl (1–10 mM) produces a dose- and time-dependent down-regulation of MARCKS Protein. The lithium-induced reduction in MARCKS is dependent on the concentration of inositol present in the medium and is reversed and prevented in the presence of elevated inositol concentrations. When HN33 cells were exposed to lithium at clinically relevant concentrations (1 mM) under limiting inositol conditions, activation of muscarinic receptor-coupled phosphoinositide signaling significantly potentiated the lithium-induced down-regulation of MARCKS Protein. It has been suggested that a major action of lithium in the brain is linked to its inositol monophosphatase inhibitory activity in receptor-mediated signaling through the inositol trisphosphate/diacylglycerol pathway, resulting in a relative inositol depletion. Our data provide evidence that this initial action of lithium may translate into a PKC-dependent long-term down-regulation of MARCKS Protein expression in the hippocampus.
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Phorbol Ester‐ and Retinoic Acid‐Induced Regulation of the Protein Kinase C Substrate MARCKS in Immortalized Hippocampal Cells
Journal of neurochemistry, 2002Co-Authors: D. G. Watson, Bruce H. Wainer, Robert H. LenoxAbstract:: The expression of MARCKS, a major Protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS Protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 µM phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to 90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS Protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS Protein in PMA-treated cultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.
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phorbol ester and retinoic acid induced regulation of the Protein kinase c substrate MARCKS in immortalized hippocampal cells
Journal of Neurochemistry, 2002Co-Authors: D. G. Watson, Bruce H. Wainer, Robert H. LenoxAbstract:: The expression of MARCKS, a major Protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS Protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 µM phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to 90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS Protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS Protein in PMA-treated cultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.
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Myristoylation alters retinoic acid-induced down-regulation of MARCKS in immortalized hippocampal cells.
Biochemical and biophysical research communications, 2000Co-Authors: Le Wang, David G. Watson, Robert H. LenoxAbstract:Abstract The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent PKC-substrate in the brain, which has been implicated in brain development, cytoskeletal remodeling, calcium/calmodulin signaling, and neuroplasticity. The sequence of the Macs gene codes for a Protein that has three highly conserved domains including a 5′ myristoylation region and a 25-amino-acid phosphorylation site domain (PSD), which are involved in anchoring MARCKS to the cellular membrane. In this study, we examined the role of the myristoylation signal in the regulation of MARCKS in transfected rat hippocampal cells (H19-7) following retinoic acid (RA) treatment. A mutant MARCKS lacking the myristoylation signal was engineered by substitution of alanine for glycine at position 2 of the Macs gene and was found to be exclusively expressed in the cytosol fraction of transfected cells. Exposure of the wild-type MARCKS-transfected cells to RA resulted in an apparent shift of MARCKS from the membrane to the cytosol, while the total Protein of wild-type MARCKS was not significantly changed. In contrast, RA-exposed cells transfected with the mutant MARCKS revealed a dramatic reduction of expression of MARCKS Protein in both cytosol and total Protein fractions. These data suggest that the absence of the myristoyl moiety may not only alter the anchoring of the Protein to the membrane but also play a novel role in modulating cellular levels of MARCKS Protein in response to RA.
Kenneth B Adler - One of the best experts on this subject based on the ideXlab platform.
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Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein
2016Co-Authors: Laura E. Ott, Kenneth B Adler, Mary K. Sheats, Eui Jae Sung, Adam T. Melvin, Jason M. Haugh, Samuel L. JonesAbstract:Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of Protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino
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An Inhaled Inhibitor of Myristoylated Alanine-Rich C Kinase Substrate Reverses LPS-Induced Acute Lung Injury in Mice.
American journal of respiratory cell and molecular biology, 2016Co-Authors: Qi Yin, Anne L. Crews, Shijing Fang, Joungjoa Park, Indu Parikh, Kenneth B AdlerAbstract:Intratracheal instillation of bacterial LPS is a well-established model of acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). Because the myristoylated alanine-rich C kinase substrate (MARCKS) Protein is involved in neutrophil migration and proinflammatory cytokine production, we examined whether an aerosolized peptide that inhibits MARCKS function could attenuate LPS-induced lung injury in mice. The peptide, BIO-11006, was delivered at 50 μM via inhalation either just before intratracheal instillation of 5 μg of LPS into Balb/C mice, or 4, 12, 24, or 36 hours after LPS instillation. Effects of BIO-11006 were evaluated via analysis of mouse disease-related behavior, lung histology, bronchoalveolar lavage fluid total Protein, neutrophil counts and percentages, cytokine (KC [CXCl1, mouse IL-8 equivalent] and TNF-α) expression, and activation of NF-κB in lung tissue. Treatment with aerosolized BIO-11006 at 0, 4, 12, 24, and even 36 hours after LPS instillation reversed the disease pro...
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abstract 4042 MARCKS Protein inhibitors attenuate cancer cell migration metastasis
Cancer Research, 2014Co-Authors: Walker Long, Anne L. Crews, Shijing Fang, Indu Parikh, Qi Yen, Kenneth B AdlerAbstract:Background: Metastasis causes most deaths from cancer. In a number of studies, high expressions of the MARCKS Protein (Myristoylated Alanine-Rich C Kinase Substrate, a 332-amino acid Protein known to play a key role in normal cell motion) in cancer cells have been associated with higher rates of metastasis in breast, lung, prostate, brain, skin and other neoplasias. Recently, Chen et al. showed that the 24-amino acid N-terminal sequence of MARCKS, called the MANS peptide, blocked metastasis of lung cancer cells orthotopically injected into mouse lungs (http://www.ncbi.nlm.nih.gov/pubmed/23955080). Unfortunately, for various reasons, the MANS peptide is not practical as an anti-cancer drug candidate. We have identified shorter and far more soluble N-terminal peptide inhibitors of MARCKS that appear to block migration of cancer cells in vitro and metastasis in mice in-vivo at lower concentrations than the MANS peptide, and when administered either IP or as an inhaled aerosol. Methods: For the in vitro cancer cell migration assays, 1 X 105 of three separate human lung cancer cell lines (A549, PC-9, and CL1-5) were cultured in Transwell ® plates (24-well, 8-um pore size). Either PBS (control), MANS, or 3 different N-terminal peptide inhibitor analogs of MANS; BIO-11006, BIO-11002, and BIO-10901 were incubated with the cells for 12 hrs, and cells that migrated from the upper to lower surface of the chambers were stained with hematoxylin and counted. For the in vivo metastasis evaluations, PC-9 cells were orthotopically-injected into the lungs of NOD-SCID mice with or without preincubation with either MANS or BIO-11006, and animals were given MANS or BIO-11006 IP after 7 days and every 3 days up until day 25, when they were harvested and examined. Another group of orthotopically injected animals received treatment with BIO-11006 as an inhaled aerosol starting at day 4 and every day thereafter until day 25. Animals were euthanized at day 26; locations and magnitudes of systemic metastases were assessed macroscopically and microscopically. Results: All of the N-terminal peptide inhibitors showed concentration - dependent attenuation of in vitro migration of each of the cancer cell lines, with an inhibition of close to 75% at the highest concentration (100μM). The smaller MANS analogs appeared to be the most effective at the lower concentrations. IP injection of MANS or BIO-11006 dramatically inhibited metastasis of the primary tumor to other sites in the lung, heart and diaphragm, with BIO-11006 showing potent inhibition of metastasis even at a concentration an order of magnitude lower than MANS. Administration of 50 uM BIO-11006daily via inhaled aerosol had the same inhibitory effect on metastasis as IP injection of 50 uM every three days. Conclusion: N-terminal peptide inhibitors of MARCKS suitable for development as potential new anti-cancer drugs show substantial promise in inhibiting metastasis of lung cancer cells both in vitro and in vivo. Citation Format: Walker A. Long, Indu Parikh, Qi Yen, Shijing Fang, Anne L. Crews, Kenneth B. Adler. MARCKS Protein inhibitors attenuate cancer cell migration/metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4042. doi:10.1158/1538-7445.AM2014-4042
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Abstract 4042: MARCKS Protein inhibitors attenuate cancer cell migration/metastasis
Tumor Biology, 2014Co-Authors: Walker Long, Anne L. Crews, Shijing Fang, Indu Parikh, Qi Yen, Kenneth B AdlerAbstract:Background: Metastasis causes most deaths from cancer. In a number of studies, high expressions of the MARCKS Protein (Myristoylated Alanine-Rich C Kinase Substrate, a 332-amino acid Protein known to play a key role in normal cell motion) in cancer cells have been associated with higher rates of metastasis in breast, lung, prostate, brain, skin and other neoplasias. Recently, Chen et al. showed that the 24-amino acid N-terminal sequence of MARCKS, called the MANS peptide, blocked metastasis of lung cancer cells orthotopically injected into mouse lungs (http://www.ncbi.nlm.nih.gov/pubmed/23955080). Unfortunately, for various reasons, the MANS peptide is not practical as an anti-cancer drug candidate. We have identified shorter and far more soluble N-terminal peptide inhibitors of MARCKS that appear to block migration of cancer cells in vitro and metastasis in mice in-vivo at lower concentrations than the MANS peptide, and when administered either IP or as an inhaled aerosol. Methods: For the in vitro cancer cell migration assays, 1 X 105 of three separate human lung cancer cell lines (A549, PC-9, and CL1-5) were cultured in Transwell ® plates (24-well, 8-um pore size). Either PBS (control), MANS, or 3 different N-terminal peptide inhibitor analogs of MANS; BIO-11006, BIO-11002, and BIO-10901 were incubated with the cells for 12 hrs, and cells that migrated from the upper to lower surface of the chambers were stained with hematoxylin and counted. For the in vivo metastasis evaluations, PC-9 cells were orthotopically-injected into the lungs of NOD-SCID mice with or without preincubation with either MANS or BIO-11006, and animals were given MANS or BIO-11006 IP after 7 days and every 3 days up until day 25, when they were harvested and examined. Another group of orthotopically injected animals received treatment with BIO-11006 as an inhaled aerosol starting at day 4 and every day thereafter until day 25. Animals were euthanized at day 26; locations and magnitudes of systemic metastases were assessed macroscopically and microscopically. Results: All of the N-terminal peptide inhibitors showed concentration - dependent attenuation of in vitro migration of each of the cancer cell lines, with an inhibition of close to 75% at the highest concentration (100μM). The smaller MANS analogs appeared to be the most effective at the lower concentrations. IP injection of MANS or BIO-11006 dramatically inhibited metastasis of the primary tumor to other sites in the lung, heart and diaphragm, with BIO-11006 showing potent inhibition of metastasis even at a concentration an order of magnitude lower than MANS. Administration of 50 uM BIO-11006daily via inhaled aerosol had the same inhibitory effect on metastasis as IP injection of 50 uM every three days. Conclusion: N-terminal peptide inhibitors of MARCKS suitable for development as potential new anti-cancer drugs show substantial promise in inhibiting metastasis of lung cancer cells both in vitro and in vivo. Citation Format: Walker A. Long, Indu Parikh, Qi Yen, Shijing Fang, Anne L. Crews, Kenneth B. Adler. MARCKS Protein inhibitors attenuate cancer cell migration/metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4042. doi:10.1158/1538-7445.AM2014-4042
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Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein.
PloS one, 2013Co-Authors: Laura E. Ott, Kenneth B Adler, Mary K. Sheats, Eui Jae Sung, Adam T. Melvin, Jason M. Haugh, Samuel L. JonesAbstract:Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of Protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino-terminus. Expression of either MANS or unmyristoylated MANS (UMANS) C-terminal EGFP fusion Proteins resulted in similar levels of attenuated cell migration as observed with MANS peptide treatment. These data demonstrate that MARCKS regulates cell migration and suggests that MARCKS-mediated regulation of fibroblast migration involves the MARCKS amino-terminus. Further, this data demonstrates that MANS peptide treatment inhibits MARCKS function during fibroblast migration and that MANS mediated inhibition occurs independent of myristoylation.
David A. Foster - One of the best experts on this subject based on the ideXlab platform.
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MARCKS Protein is transcriptionally down-regulated in v-Src-transformed BALB/c 3T3 cells.
The Journal of biological chemistry, 1992Co-Authors: C K Joseph, S A Qureshi, D J Wallace, David A. FosterAbstract:Abstract Activation of Protein kinase C (PKC) by tumor-promoting phorbol esters leads to the phosphorylation of an 80-kilodalton PKC substrate (known as MARCKS) in murine fibroblasts. In BALB/c 3T3 cells stably transformed by v-Src, phorbol esters were unable to induce phosphorylation of MARCKS. Western blot analysis and in vitro kinase assays showed that both PKC Protein levels and kinase activity were unchanged in v-Src-transformed relative to the parental nontransformed BALB/c 3T3 cells. However, MARCKS Protein levels were reduced in v-Src-transformed cells relative to nontransformed cells. MARCKS RNA levels were also correspondingly reduced in v-Src-transformed cells. Nuclear "run-on" assays showed decreased transcription of MARCKS in v-Src-transformed cells. Thus, the absence of MARCKS in v-Src-transformed cells could be explained by a down-regulation of MARCKS transcription. Inhibiting the Protein tyrosine kinase activity of v-Src with herbimycin A restored MARCKS RNA levels, MARCKS transcription, and MARCKS Protein, suggesting that down-regulation of MARCKS in v-Src-transformed BALB/c 3T3 cells is a direct effect of v-Src.
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MARCKS Protein is transcriptionally down regulated in v src transformed balb c 3t3 cells
Journal of Biological Chemistry, 1992Co-Authors: C K Joseph, S A Qureshi, D J Wallace, David A. FosterAbstract:Abstract Activation of Protein kinase C (PKC) by tumor-promoting phorbol esters leads to the phosphorylation of an 80-kilodalton PKC substrate (known as MARCKS) in murine fibroblasts. In BALB/c 3T3 cells stably transformed by v-Src, phorbol esters were unable to induce phosphorylation of MARCKS. Western blot analysis and in vitro kinase assays showed that both PKC Protein levels and kinase activity were unchanged in v-Src-transformed relative to the parental nontransformed BALB/c 3T3 cells. However, MARCKS Protein levels were reduced in v-Src-transformed cells relative to nontransformed cells. MARCKS RNA levels were also correspondingly reduced in v-Src-transformed cells. Nuclear "run-on" assays showed decreased transcription of MARCKS in v-Src-transformed cells. Thus, the absence of MARCKS in v-Src-transformed cells could be explained by a down-regulation of MARCKS transcription. Inhibiting the Protein tyrosine kinase activity of v-Src with herbimycin A restored MARCKS RNA levels, MARCKS transcription, and MARCKS Protein, suggesting that down-regulation of MARCKS in v-Src-transformed BALB/c 3T3 cells is a direct effect of v-Src.
Jon P. Durkin - One of the best experts on this subject based on the ideXlab platform.
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The Role of Protein Kinase C and MARCKS Protein Phosphorylation in Rat Cerebromicrovascular Endothelial Cell Proliferation Induced by Astrocyte-Derived Factors
Biology and Physiology of the Blood-Brain Barrier, 1996Co-Authors: Danica Stanimirovic, Rita Ball, Josée Wong, Jon P. DurkinAbstract:Serum-free medium conditioned by rat cortical astrocytes was found to prevent apoptosis induced by growth factor-deprivation, accelerate DNA synthesis, induce transient activation of Protein kinase C (PKC), and increase the endogenous phosphorylation of the PKC-specific substrate, the 85 kD MARCKS Protein, in rat cerebromicrovascular endothelial cells (RCEC). The trophic and stimulatory factor(s) in astrocyte conditioned media (ACM) were heat-and trypsin-sensitive and found to have an apparent molecular weight greater than 10 kD. The potent PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), also stimulated RCEC proliferation, whereas the inhibition of PKC by staurosporine caused a concomitant loss in ACM-induced PKC translocation, MARCKS Protein phosphorylation and DNA synthesis. These findings implicate PKC activation as a critical early event in cerebral endothelial cell proliferation triggered by astrocyte-derived mitogen(s).
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Calcium and Protein Kinase C Signaling in Response to Vasoactive Peptides in Human Cerebromicrovascular Endothelial Cells
Biology and Physiology of the Blood-Brain Barrier, 1996Co-Authors: Danica Stanimirovic, Rita Ball, Paul Morley, Edith Hamel, Geoff Mealing, Jon P. DurkinAbstract:Vasoactive peptides endothelin-1 (ET-1) and bradykinin (BK) were shown to induce immediate increases in intracellular calcium concentrations, [Ca2+]i, and endogenous phosphorylation of the Protein kinase C (PKC)-specific substrate, 85 kD MARCKS Protein, in human cerebromicrovascular endothelial cells (HBEC). The peptides-induced [Ca2+]i surges were not affected by incubating the cells in Ca2+-free medium or by pretreating them with Ca2+-channel blocker D600 (50 µ;M). BK-stimulated [Ca2+]i increases were completely inhibited, whereas ET-1-induced [Ca2+]i, increases were insensitive to ADP-ribosylation of G-Proteins by pertussis toxin. Both peptides-triggered [Ca2+]i, surges and MARCKS Protein phosphorylation were abolished by the inhibitor of inositol phospholipid hydrolysis, U73122 (2.5–5 µM).
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Evidence for the role of Protein kinase C in astrocyte-induced proliferation of rat cerebromicrovascular endothelial cells
Neuroscience letters, 1995Co-Authors: Danica Stanimirovic, Rita Ball, Jon P. DurkinAbstract:The proliferation of cerebral endothelial cells is a crucial step in neural angiogenesis and is a process responsive to changes in the surrounding environment. Serum-free medium conditioned by rat cortical astrocytes was found to accelerate DNA synthesis, induce transient activation of Protein kinase C (PKC), and increase the endogenous phosphorylation of the PKC-specific substrate, the 85 kDa MARCKS Protein, in rat cerebromicrovascular endothelial cells (RCEC). The stimulatory factor(s) in astrocyte conditioned media (ACM) were heat- and trypsin-sensitive and found to have an apparent molecular weight greater than 10 kDa. The potent PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), also stimulated RCEC proliferation, whereas the inhibition of PKC by staurosporine caused a concomitant loss in ACM-induced PKC translocation, MARCKS Protein phosphorylation and DNA synthesis. These findings implicate PKC activation as a critical early event in cerebral endothelial cell proliferation triggered by astrocyte-derived mitogen(s).
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Stimulation of Protein Kinase C during Ca-induced Keratinocyte Differentiation SELECTIVE BLOCKADE OF MARCKS PHOSPHORYLATION BY CALMODULIN
The Journal of biological chemistry, 1995Co-Authors: Balu Chakravarthy, Jon P. Durkin, Paul Morley, R. J. Isaacs, James F. WhitfieldAbstract:Abstract Raising the external Ca concentration from 0.05 to 1.8 mM stimulated membrane-associated Protein kinase Cs (PKCs) activity as strongly as the specific PKCs activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) in BALB/MK mouse keratinocytes. This was indicated by the increased phosphorylation of a PKC-selective peptide substrate, Ac-FKKSFKL-NH2, by membranes isolated from the Ca- or TPA-stimulated keratinocytes. Raising the external Ca concentration to 1.8 mM also triggered a 4-fold rise in the intracellular free Ca concentration. As reported elsewhere (Moscat, J. Fleming, T. P., Molloy, C. J. LopezBarahona, M., and Aaronson, S. A.(1989) J. Biol. Chem. 264, 11228-11235), TPA stimulated the phosphorylation of the PKCs substrate, the 85-kDa myristoylated alanine-rich kinase C substrate (MARCKS) Protein, in intact keratinocytes, but Ca did not. Furthermore, Ca-pretreatment reduced the TPA-induced phosphorylation of the 85-kDa Protein in intact cells. There was no significant increase in MARCKS phosphorylation when keratinocytes were treated with a Ca•CaM-dependent phosphatase inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca. Ca•calmodulin suppressed the ability of isolated membranes to phosphorylate the 85-kDa MARCKS holoProtein in vitro in the presence of phosphatase inhibitors such as fluoride, pyrophosphate, and vanadate, and this inhibition was overcome by a calmodulin antagonist, the calmodulin-binding domain peptide. Thus, the ability of 1.8 mM Ca to strongly stimulate the membrane PKCs activity without stimulating the phosphorylation of the MARCKS Protein in keratinocytes is consistent with the possibility of Ca•calmodulin complexes, formed by the internal Ca surge, binding to, and blocking the phosphorylation of, this PKC Protein substrate.
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The role of intracellular calcium and Protein kinase C in endothelin-stimulated proliferation of rat type I astrocytes.
Glia, 1995Co-Authors: Danica Stanimirovic, Rita Ball, Paul Morley, Geoff Mealing, Jon P. DurkinAbstract:The increased expression of immunoreactive endothelin-1 (ET-1) in reactive astrocytes and its mitogenic effects on astrocytes and glioma cell lines, have implicated endothelins in the development of reactive gliosis. In this study, an increase in DNA synthesis in rat type I astrocytes was observed after cultures were transiently exposed to ET-1 for 15 min, suggesting that early signal transduction events are essential and sufficient for the propagation of the ET-1-induced mitogenic signal. Prompt increases in inositol triphosphate (IP 3 ) formation and [Ca 2+ ] i were observed upon the addition of ET-1 to these cells. The ET-1-evoked increase in [Ca 2+ ] i consisted of an initial peak which was preserved in Ca 2+ -free medium, and a sustained phase which was abolished in Ca 2+ -free medium and partly attenuated by nifedipine. ET-1 also increased the activity of membrane-associated Protein kinase C (PKC) and induced the in vivo phosphorylation of the 85 kD MARCKS Protein, an endogenous PKC-specific substrate. The ET-1-evoked increases in DNA synthesis, IP 3 , [Ca 2+ ] i , membrane PKC, and 85 kD MARCKS Protein phosphorylation in rat cortical astrocytes were prevented by either the selective endothelin ET A receptor antagonist, BQ-123, or the phospholipase C (PLC)-specific inhibitor, U-73122. However, the inhibition of PKC activity did not affect ET-1-induced DNA synthesis in rat cortical astrocytes. These results suggest that ET-1-induced IP3 and/or [CA 2+ ] i responses, but not the activation of PKC, are essential for the growth-factor like actions of ET-1 in rat cortical astrocytes.
C K Joseph - One of the best experts on this subject based on the ideXlab platform.
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MARCKS Protein is transcriptionally down-regulated in v-Src-transformed BALB/c 3T3 cells.
The Journal of biological chemistry, 1992Co-Authors: C K Joseph, S A Qureshi, D J Wallace, David A. FosterAbstract:Abstract Activation of Protein kinase C (PKC) by tumor-promoting phorbol esters leads to the phosphorylation of an 80-kilodalton PKC substrate (known as MARCKS) in murine fibroblasts. In BALB/c 3T3 cells stably transformed by v-Src, phorbol esters were unable to induce phosphorylation of MARCKS. Western blot analysis and in vitro kinase assays showed that both PKC Protein levels and kinase activity were unchanged in v-Src-transformed relative to the parental nontransformed BALB/c 3T3 cells. However, MARCKS Protein levels were reduced in v-Src-transformed cells relative to nontransformed cells. MARCKS RNA levels were also correspondingly reduced in v-Src-transformed cells. Nuclear "run-on" assays showed decreased transcription of MARCKS in v-Src-transformed cells. Thus, the absence of MARCKS in v-Src-transformed cells could be explained by a down-regulation of MARCKS transcription. Inhibiting the Protein tyrosine kinase activity of v-Src with herbimycin A restored MARCKS RNA levels, MARCKS transcription, and MARCKS Protein, suggesting that down-regulation of MARCKS in v-Src-transformed BALB/c 3T3 cells is a direct effect of v-Src.
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MARCKS Protein is transcriptionally down regulated in v src transformed balb c 3t3 cells
Journal of Biological Chemistry, 1992Co-Authors: C K Joseph, S A Qureshi, D J Wallace, David A. FosterAbstract:Abstract Activation of Protein kinase C (PKC) by tumor-promoting phorbol esters leads to the phosphorylation of an 80-kilodalton PKC substrate (known as MARCKS) in murine fibroblasts. In BALB/c 3T3 cells stably transformed by v-Src, phorbol esters were unable to induce phosphorylation of MARCKS. Western blot analysis and in vitro kinase assays showed that both PKC Protein levels and kinase activity were unchanged in v-Src-transformed relative to the parental nontransformed BALB/c 3T3 cells. However, MARCKS Protein levels were reduced in v-Src-transformed cells relative to nontransformed cells. MARCKS RNA levels were also correspondingly reduced in v-Src-transformed cells. Nuclear "run-on" assays showed decreased transcription of MARCKS in v-Src-transformed cells. Thus, the absence of MARCKS in v-Src-transformed cells could be explained by a down-regulation of MARCKS transcription. Inhibiting the Protein tyrosine kinase activity of v-Src with herbimycin A restored MARCKS RNA levels, MARCKS transcription, and MARCKS Protein, suggesting that down-regulation of MARCKS in v-Src-transformed BALB/c 3T3 cells is a direct effect of v-Src.