Myristoylated Alanine-rich C Kinase Substrate Phosphorylation Research Articles

Restoration of MARCK enhances chemosensitivity in cancer

PurposeIncreased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1.MethodsCRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU).ResultsWe found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU.ConclusionsCells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.

MARCKS phosphorylation and amylase release in GLP-1-stimulated acini isolated from rat pancreas.

Little is known about the effects of glucagon-like peptide 1 (GLP-1) on the pancreatic exocrine gland. In the gland, secretagogues induce amylase release. That signal transduction is evoked mainly by an increase in intracellular Ca2+ levels and activation of protein kinase C (PKC). We previously demonstrated that myristoylated alanine-rich C kinase substrate (MARCKS), a PKC substrate, is involved in pancreatic amylase release. Here, we studied the effects of GLP-1 on MARCKS phosphorylation and amylase release in rat pancreatic acini. GLP-1 induced amylase release and MARCKS phosphorylation in isolated pancreatic acini. Inhibitors of cAMP-dependent protein kinase (PKA) suppressed those effects. Furthermore, a MARCKS-related peptide inhibited the GLP-1-induced amylase release. These findings suggest that GLP-1 induces amylase release through MARCKS phosphorylation via activation of PKA in isolated pancreatic acini.

The Role of Myristoylated, Alanine-rich C-kinase Substrate (MARCKS) in a Murine Model of Chronic Airway Inflammation

Abstract Severe asthma, a complicate chronic lung inflammation. Several chronic inflammatory cells that involved in the inflammatory responses, such as macrophages, neutrophils, and eosinophils, produce large amounts of ROS, the excess level of ROS would increase lung inflammation and lung injury. In fact, this inflammation is well controlled by steroid, but some subset of patients these glucocorticoids are less effective. Neutrophil recruitment is a prominent feature of acute exacerbations of chronic asthma. In patients with difficult-to-control asthma, neutrophilic inflammation rather than eosinophils often predominate. Myristoylated alanine-rich C kinase substrate (MARCKS), is a protein kinase C (PKC) substrate. MARCKS also plays a crucial role that phosphorylation of MARCKS by PKC leads its release from plasma membrane into the cytosol, participating in regulating cell migration such as macrophage and neutrophil. We found that increase of airway MARCKS phosphorylation in asthmatic patients and murine model of chronic steroid-resistance allergic asthma. In this report, our observations indicate that 1) targeting of MARCKS phosphorylation inhibited chronic asthmatic symptoms, such as eosinophil and neutrophil infiltrate into lung. MPS peptide inhibits mucus production, collagen deposition and airway hyperresponsiveness rather than steroid-resistance group; 2) targeting of MARCKS phosphorylation decreased IFN-r, IL-4, IL-17A, IL-33, eotaxin and KC production. MPS peptide also inhibit NADPH oxidase DUOX-1, 2 protein expression and end product 3-nitrotyrosine production; We found that MPS peptide shows a therapeutic potential for steroid-resistance allergic asthma.

Myristoylated alanine-rich C kinase substrate (MARCKS): a multirole signaling protein in cancers.

Emerging evidence implicates myristoylated alanine-rich C-kinase substrate (MARCKS), a major substrate of protein kinase C (PKC), in a critical role for cancer development and progression. MARCKS is tethered to the plasma membrane but can shuttle between the cytosol and plasma membrane via the myristoyl-electrostatic switch. Phosphorylation of MARCKS by PKC leads to its translocation from the plasma membrane to the cytosol where it functions in actin cytoskeletal remodeling, Ca2+ signaling through binding to calmodulin, and regulation of exocytic vesicle release in secretory cells such as neurons and airway goblet cells. Although the contribution of MARCKS to various cellular processes has been extensively studied, its roles in neoplastic disease have been conflicting. This review highlights the molecular and functional differences of MARCKS that exist between normal and tumor cells. We also discuss the recent advances in the potential roles of MARCKS in tumorigenesis, metastasis, and resistance to anti-cancer therapies, with a focus on addressing the inconsistent results regarding the function of MARCKS as a promoter or inhibitor of oncogenesis.

Involvement of myristoylated alanine-rich C kinase substrate phosphorylation and translocation in cholecystokinin-induced amylase release in rat pancreatic acini.

Cholecystokinin (CCK) is a gastrointestinal hormone that induces exocytotic amylase release in pancreatic acinar cells. The activation of protein kinase C (PKC) is involved in the CCK-induced pancreatic amylase release. Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed substrate of PKC. MARCKS has been implicated in membrane trafficking in several cell types. The phosphorylation of MARCKS by PKC results in the translocation of MARCKS from the membrane to the cytosol. Here, we studied the involvement of MARCKS in the CCK-induced amylase release in rat pancreatic acini. Employing Western blotting, we detected MARCKS protein in the rat pancreatic acini. CCK induced MARCKS phosphorylation. A PKC-δ inhibitor, rottlerin, inhibited the CCK-induced MARCKS phosphorylation and amylase release. In the translocation assay, we also observed CCK-induced PKC-δ activation. An immunohistochemistry study showed that CCK induced MARCKS translocation from the membrane to the cytosol. When acini were lysed by a detergent, Triton X-100, CCK partially induced displacement of the MARCKS from the GM1a-rich detergent-resistant membrane fractions (DRMs) in which Syntaxin2 is distributed. A MARCKS-related peptide inhibited the CCK-induced amylase release. These findings suggest that MARCKS phosphorylation by PKC-δ and then MARCKS translocation from the GM1a-rich DRMs to the cytosol are involved in the CCK-induced amylase release in pancreatic acinar cells.

MARCKS is involved in methylmercury-induced decrease in cell viability and nitric oxide production in EA.hy926 cells.

Methylmercury (MeHg) is a persistent environmental contaminant that has been reported worldwide. MeHg exposure has been reported to lead to increased risk of cardiovascular diseases; however, the mechanisms underlying the toxic effects of MeHg on the cardiovascular system have not been well elucidated. We have previously reported that mice exposed to MeHg had increased blood pressure along with impaired endothelium-dependent vasodilation. In this study, we investigated the toxic effects of MeHg on a human endothelial cell line, EA.hy926. In addition, we have tried to elucidate the role of myristoylated alanine-rich C kinase substrate (MARCKS) in the MeHg toxicity mechanism in EA.hy926 cells. Cells exposed to MeHg (0.1–10 µM) for 24 hr showed decreased cell viability in a dose-dependent manner. Treatment with submaximal concentrations of MeHg decreased cell migration in the wound healing assay, tube formation on Matrigel and spontaneous nitric oxide (NO) production of EA.hy926 cells. MeHg exposure also elicited a decrease in MARCKS expression and an increase in MARCKS phosphorylation. MARCKS knockdown or MARCKS overexpression in EA.hy926 cells altered not only cell functions, such as migration, tube formation and NO production, but also MeHg-induced decrease in cell viability and NO production. These results suggest the broad role played by MARCKS in endothelial cell functions and the involvement of MARCKS in MeHg-induced toxicity.

Abstract 2769: PKC/MARCKS pathway is a novel therapeutic target associated with breast cancer malignancy potential and paclitaxel resistance

Abstract Increasing evidence had suggested important roles of protein kinase C (PKC)-mediated phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) in modulating various cellular processes, including cancer cell growth and metastasis. However, there is a lack of information regarding to the roles of MARCKS and its pSer 159/163 phosphorylated product in breast cancer cells and tissues. To tackle this concern, we recently initiated both immunohistochemical and western blot analyses on a series of clinical specimens and several breast cancer cell lines. We have observed a significant elevation of phospho-MARCKS associated with advanced-stage breast cancer tissues as compared with benign ones, particularly in triple-negative breast cancer (TNBC) tissues and cell lines. Knockdown of MARCKS expression resulted in suppressions of lamellipodia/filopodia formation and migration/invasion as well as down-regulation of Src activity in these TNBC cell lines. Of note, MARCKS-silenced TNBC cells grew much more slowly and more sensitive to paclitaxel than the control and parental cells. Treatment with paclitaxel was shown to induce MARCKS phosphorylation in a dose-dependent manner, which may be related to an enhanced paclitaxel resistance in some of TNBC cells. However, PKC inhibitors were able to abrogate paclitaxel-induced MARCKS phosphorylation. Consistent with this potential, treatments of TNBC cells with a MARCKS N-terminus sequence peptide, namely MANS, to down regulate PKC/MARCKS pathway not only attenuated the associated aggressive phenotype but also synergistically enhanced paclitaxel-induced cytotoxicity in vitro and in vivo. These results demonstrate an association of PKC-mediated MARCKS phosphorylation with breast cancer malignancy potential and MARCKS phosphorylation as a predictor of paclitaxel resistance in TNBC cells. It is suggestive that an inhibition of PKC/MARCKS pathway may serve as an alternative therapeutic strategy for enhancing efficacy of chemotherapy. Citation Format: Ching-Hsien Chen, Muhammad Arif, Wen-Hsin Chang, Yuan Yuan, Jing Zhai, David K. Ann, Reen Wu. PKC/MARCKS pathway is a novel therapeutic target associated with breast cancer malignancy potential and paclitaxel resistance. [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 2769. doi:10.1158/1538-7445.AM2014-2769

Alteration in MARCKS phosphorylation and expression by methylmercury in SH-SY5Y cells and rat brain

The molecular mechanisms mediating methylmercury (MeHg)-induced neurotoxicity are not completely understood. Because myristoylated alanine-rich C kinase substrate (MARCKS) plays an essential role in the differentiation and development of neuronal cells, we studied the alteration of MARCKS expression and phosphorylation in MeHg-induced neurotoxicity of neuroblastoma SH-SY5Y cells and in the rat brain. Exposure to MeHg induced a decrease in cell viability of SH-SY5Y cells, which was accompanied by a significant increase in phosphorylation and a reduction in MARCKS expression. Pretreatment of cells with a protein kinase C inhibitor or an extracellular Ca2+ chelator suppressed MeHg-induced MARCKS phosphorylation. In MARCKS knock-down cells, MeHg-induced cell death was significantly augmented in comparison to control siRNA. In brain tissue from MeHg-treated rats, MARCKS phosphorylation was enhanced in the olfactory bulb in comparison to control rats. The present study may indicate that alteration in MARCKS expression or phosphorylation has consequences for MeHg-induced neurotoxicity.

Abstract 4919: Myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation potentiates human lung cancer cell malignancy .

Abstract Myristoylated alanine-rich C kinase substrate (MARCKS), a substrate of protein kinase C (PKC), is a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. Phosphorylated MARCKS (p-MARCKS) has been reportedly to promote inflammatory cell migration in several lung diseases. However, the functionality of MARCKS and its related phosphorylation in lung cancer malignancy has not been characterized. This study demonstrated MARCKS expression and elevated p-MARCKS in various lung cancer cell lines with high malignancy, such as CL1-0-F3, CL1-5, PC9, A549 and several tyrosine kinase inhibitor (TKI) resistant cell lines (H1650 and H1975), as compared with normal human bronchial epithelial (NHBE) cells and low metastasis cancer cell line. siRNA knockdown MARCKS expression resulted in an inhibition of cell migration, accompanying with a decrease of Slug expression in these malignant lung cancer cell lines. Treatment with myristoylated 24-amino-acid MARCKS N-terminus sequence (MANS) peptide, but not the control myristoylated random N-terminal sequence (RNS) peptide, suppressed MARCKS phosphorylation and impaired cell migration and wound healing ability in these lung cancer cell lines. Importantly, MANS peptide treatment resulted in a suppression of lamellipodia/filopodia formation and an enhancement of cell-cell contacts with increased E-cadherin expression. These results confirmed a crucial role for MARCKS, specifically its phosphorylation, in potentiating lung cancer cell migration and malignancy. This study has also suggested a potential usage of MANS or the MARCKS-related peptides in the treatment of lung cancer metastasis. Citation Format: Ching-Hsien Chen, Phillip Thai, Reen Wu. Myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation potentiates human lung cancer cell malignancy . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4919. doi:10.1158/1538-7445.AM2013-4919

Abstract 5290: Motility of B16 mouse melanoma cells is driven by phospho-MARCKS

Abstract Phosphorylation of Myristoylated Alanine-rich C-Kinase Substrate (MARCKS) by protein kinase Cα (PKCα) is known to trigger its release from the plasma membrane/cytoskeleton into the cytoplasm, thereby promoting actin reorganization during migration. This study shows that cytoplasmic phosphoMARCKS directly promotes motile behavior of melanoma cells. Aggressively motile B16 F10 mouse melanoma cells express high levels of phosphoMARCKS, whereas weakly motile F1 cells express negligible levels. When treated with okadaic acid (OA) (a protein phosphatase inhibitor), F1 cells exhibit a dramatic increase in phosphoMARCKS coincident with a 5-fold increase in motility. This motility is lost if OA-treated cells are also treated with a shRNA reagent targeted to MARCKS. Pre-treatment of cells with calphostin C (a PKC inhibitor) prevents OA-mediated elevation of phosphoMARCKS. These findings imply that PKC is highly active in F1 cells but that its phosphorylation of MARCKS is efficiently reversed by protein phosphatases. The mechanistic significance of phosphoMARCKS to motility is established with a pseudo-phosphorylated mutant of GFP-MARCKS in which Asp residues replaced Ser residues known to be phosphorylated by PKCα. This mutant localizes to the cytoplasm and engenders three-fold higher motility in F1 cells. However, when the mutant is fused to a nuclear localization signal, motility is substantially decreased. Expression of an unmyristoylated, phosphorylation-resistant MARCKS mutant that localizes to the cytoplasm, blocks motility by 40-50% of both OA-stimulated F1 cells and intrinsically motile F10 cells. These findings demonstrate the importance of phosphoMARCKS to the metastatic potential of melanoma cells, and reveal a previously undocumented cytoplasmic role for this phospho-protein. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5290.

PhosphoMARCKS drives motility of mouse melanoma cells

Phosphorylation of myristoylated alanine-rich C- kinase substrate (MARCKS) by protein kinase Cα (PKCα) is known to trigger its release from the plasma membrane/cytoskeleton into the cytoplasm, thereby promoting actin reorganization during migration. This study shows that once released into the cytoplasm, phosphoMARCKS directly promotes motility of melanoma cells. Aggressively motile B16 F10 mouse melanoma cells express high levels of phosphoMARCKS, whereas in weakly motile B16 F1 cells it is undetectable. Following treatment with okadaic acid (OA) (a protein phosphatase inhibitor), F1 cells exhibited a dramatic increase in phosphoMARCKS that was co-incident with a 5-fold increase in motility. Both MARCKS phosphorylation and motility were substantially decreased when prior to OA addition, MARCKS expression was knocked out by a MARCKS-specific shRNA, thereby implicating MARCKS as a major component of the motility pathway. Decreased motility and phosphoMARCKS levels in OA-treated cells were observed with a PKC inhibitor (calphostin C), thus indicating that PKC actively phosphorylates MARCKS in F1 cells but that this reaction is efficiently reversed by protein phosphatases. The mechanistic significance of phosphoMARCKS to motility was further established with a pseudo-phosphorylated mutant of MARCKS-GFP in which Asp residues replaced Ser residues known to be phosphorylated by PKCα. This mutant localized to the cytoplasm and engendered three-fold higher motility in F1 cells. Expression of an unmyristoylated, phosphorylation-resistant MARCKS mutant that localized to the cytoplasm, blocked motility by 40–50% of both OA-stimulated F1 cells and intrinsically motile F10 cells. These results demonstrate that phosphoMARCKS contributes to the metastatic potential of melanoma cells, and reveal a previously undocumented signaling role for this cytoplasmic phospho-protein.

Role of protein kinase C in cAMP-dependent exocytosis in parotid acinar cells

Summary It is well known that β-adrenergic receptor activation in parotid acinar cells results in increased intracellular cAMP levels, and consequently induces exocytotic amylase release. However, protein kinase C (PKC) has also been considered to contribute to amylase release. In this paper, we review the role of PKC in cAMP signaling and amylase release. PKCδ, a so-called ‘novel PKC', has been shown to be activated by β-adrenergic receptor stimulation. Myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate for PKC, is detected in parotid acinar cells, and MARCKS phosphorylation and translocation from the membrane to the cytosol are induced by β-adrenergic receptor stimulation. cAMP-dependent protein kinase (PKA) inhibitor inhibits β-agonist-induced PKC activation and MARCKS phosphorylation. The β-agonist-induced amylase release is inhibited by inhibitors of PKCδ or by a MARCKS-related peptide. These findings suggest that MARCKS phosphorylation via PKCδ activation, which is downstream of PKA activation, is involved in the cAMP-dependent amylase release in parotid acinar cells.

Dominant role of lipid rafts L‐type calcium channel in activity‐dependent potentiation of large dense‐core vesicle exocytosis

Calcium influx triggers exocytosis by promoting vesicle fusion with the plasma membrane. However, different subtypes of voltage-gated calcium channel (VGCC) have distinct roles in exocytosis. We previously reported that repetitive stimulation induces activity-dependent potentiation (ADP) which represents the increase of neurotransmitter release. Here, we show that L-type VGCC have a dominant role in ADP of large dense-core vesicle (LDCV) exocytosis. Repetitive stimulation activating VGCC can induce ADP, whereas activation of bradykinin (BK) G protein-coupled receptors or purinergic P2X cation channels can not. L-type VGCC has the dominant role in ADP of LDCV exocytosis by regulating Protein Kinase C (PKC)-epsilon translocation and phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), a target molecule of PKC-epsilon. We provide evidence that L-type VGCC, PKC-epsilon, and MARCKS, but not Q-type VGCC, are selectively located in lipid rafts. Also, PKC-epsilon translocation induced by L-type VGCC activation occurs in lipid rafts. Disruption of lipid rafts abolishes ADP of LDCV exocytosis and changes the fusion pore kinetics without affecting the first stimulation-induced exocytosis, showing that lipid rafts are involved in the potentiation process. Taken together, we suggest that L-type VGCC in lipid rafts selectively mediates ADP of LDCV exocytosis by regulating PKC-epsilon translocation and MARCKS phosphorylation.

Cortical F-Actin, the Exocytic Mode, and Neuropeptide Release in Mouse Chromaffin Cells Is Regulated by Myristoylated Alanine-rich C-Kinase Substrate and Myosin II

Adrenal medullary chromaffin cells are innervated by the sympathetic splanchnic nerve and translate graded sympathetic firing into a differential hormonal exocytosis. Basal sympathetic firing elicits a transient kiss-and-run mode of exocytosis and modest catecholamine release, whereas elevated firing under the sympathetic stress response results in full granule collapse to release catecholamine and peptide transmitters into the circulation. Previous studies have shown that rearrangement of the cell actin cortex regulates the mode of exocytosis. An intact cortex favors kiss-and-run exocytosis, whereas disrupting the cortex favors the full granule collapse mode. Here, we investigate the specific roles of two actin-associated proteins, myosin II and myristoylated alanine-rich C-kinase substrate (MARCKS) in this process. Our data demonstrate that MARCKS phosphorylation under elevated cell firing is required for cortical actin disruption but is not sufficient to elicit peptide transmitter exocytosis. Our data also demonstrate that myosin II is phospho-activated under high stimulation conditions. Inhibiting myosin II activity prevented disruption of the actin cortex, full granule collapse, and peptide transmitter release. These results suggest that phosphorylation of both MARCKS and myosin II lead to disruption of the actin cortex. However, myosin II, but not MARCKS, is required for the activity-dependent exocytosis of the peptide transmitters.

Functional involvement of protein kinase C-βII and its substrate, myristoylated alanine-rich C-kinase substrate (MARCKS), in insulin-stimulated glucose transport in L6 rat skeletal muscle cells

Insulin stimulates phosphorylation cascades, including phosphatidylinositol-3-kinase (PI3K), phosphatidylinositol-dependent kinase (PDK1), Akt, and protein kinase C (PKC). Myristoylated alanine-rich C-kinase substrate (MARCKS), a PKCbetaII substrate, could link the effects of insulin to insulin-stimulated glucose transport (ISGT) via phosphorylation of its effector domain since MARCKS has a role in cytoskeletal rearrangements. We examined phosphoPKCbetaII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCbetaII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCbetaII and calmodulin inhibitors, respectively, before insulin treatment, using western blots. ISGT was examined after cells had been treated with inhibitors, small inhibitory RNA (siRNA) for MARCKS, or transfection with MARCKS mutated at a PKC site. MARCKS, PKCbetaII, GLUT4 and insulin receptor were immunoblotted in subcellular fractions with F-actin antibody immunoprecipitates to demonstrate changes following insulin treatment. GLUT4 membrane insertion was followed after insulin with or without CG53353. Insulin increased phosphoPKCbetaII(Ser660 and Thr641); LY294002 blocked this, indicating its activation by PI3K. Insulin treatment increased cytosolic phosphoMARCKS, decreased membrane MARCKS and increased membrane phospholipase D1 (PLD1), a protein regulating glucose transporter vesicle fusion resulted. PhosphoMARCKS was attenuated by CG53353 or MARCKS siRNA. MARCKS siRNA blocked ISGT. Association of PKCbetaII and GLUT4 with membrane F-actin was enhanced by insulin, as was that of cytosolic and membrane MARCKS. ISGT was attenuated in myocytes transfected with mutated MARCKS (Ser152Ala), whereas overproduction of wild-type MARCKS enhanced ISGT. CG53353 blocked insertion of GLUT4 into membranes of insulin treated cells. The results suggest that PKCbetaII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.

Myristoylated alanine-rich C kinase substrate, but not Ca2+/calmodulin-dependent protein kinase II, is the mediator in cortical granules exocytosis

Sperm-egg fusion induces cortical granules exocytosis (CGE), a process that ensures the block to polyspermy. CGE can be induced independently by either a rise in intracellular calcium concentration or protein kinase C (PKC) activation. We have previously shown that myristoylated alanine-rich C kinase substrate (MARCKS) cross-links filamentous actin (F-actin) and regulates its reorganization. This activity is reduced either by PKC-induced MARCKS phosphorylation (PKC pathway) or by its direct binding to calmodulin (CaM; CaM pathway), both inducing MARCKS translocation, F-actin reorganization, and CGE. Currently, we examine the involvement of Ca(2)(+)/CaM-dependent protein kinase II (CaMKII) and MARCKS in promoting CGE and show that PKC pathway can compensate for lack of Ca(2)(+)/CaM pathway. Microinjecting eggs with either overexpressed protein or complementary RNA of constitutively active alphaCaMKII triggered resumption of second meiotic division, but induced CGE of an insignificant magnitude compared with CGE induced by wt alphaCaMKII. Microinjecting eggs with mutant-unphosphorylatable MARCKS reduced the intensity of 12-O-tetradecanoylphorbol 13-acetate or ionomycin-induced CGE by 50%, indicating that phosphorylation of MARCKS by novel and/or conventional PKCs (n/cPKCs) is a pivotal event associated with CGE. Moreover, we were able to demonstrate cPKCs involvement in ionomycin-induced MARCKS translocation and CGE. These results led us to propose that MARCKS, rather than CaMKII, as a key mediator of CGE.

Protein Kinase Cδ Regulates Airway Mucin Secretion via Phosphorylation of MARCKS Protein

Mucin hypersecretion is a major pathological feature of many respiratory diseases, yet cellular mechanisms regulating secretion of mucin have not been fully elucidated. Previously, we reported that mucin hypersecretion induced by human neutrophil elastase involves activation of protein kinase C (PKC), specifically the delta-isoform (PKC delta). Here, we further investigated the role of PKC delta in mucin hypersecretion using both primary human bronchial epithelial cells and the human bronchial epithelial 1 cell line as in vitro model systems. Phorbol-12-myristate-13-acetate (PMA)-induced mucin hypersecretion was significantly attenuated by rottlerin, a PKC delta-selective inhibitor. Rottlerin also reduced PMA- or human neutrophil elastase-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in these cells. Both secretion and MARCKS phosphorylation were significantly enhanced by the PKC delta activator bryostatin 1. A dominant-negative PKC delta construct (pEGFP-N1/PKC delta K376R) transfected into human bronchial epithelial 1 cells significantly attenuated both PMA-induced mucin secretion and phosphorylation of MARCKS, whereas transfection of a wild-type construct increased PKC delta and enhanced mucin secretion and MARCKS phosphorylation. Similar transfections of a dominant-negative or wild-type PKC epsilon construct did not affect either mucin secretion or MARCKS phosphorylation. The results suggest that PKC delta plays an important role in mucin secretion by airway epithelium via regulation of MARCKS phosphorylation.

Peptides Derived from the C2 Domain of Protein Kinase Cϵ (ϵPKC) Modulate ϵPKC Activity and Identify Potential Protein-Protein Interaction Surfaces

Peptides Derived from the C2 Domain of Protein Kinase Cϵ (ϵPKC) Modulate ϵPKC Activity and Identify Potential Protein-Protein Interaction Surfaces

Myristoylated, Alanine-rich C-Kinase Substrate Phosphorylation Regulates Growth Cone Adhesion and Pathfinding

Repellents evoke growth cone turning by eliciting asymmetric, localized loss of actin cytoskeleton together with changes in substratum attachment. We have demonstrated that semaphorin-3A (Sema3A)-induced growth cone detachment and collapse require eicosanoid-mediated activation of protein kinase C epsilon (PKC epsilon) and that the major PKC epsilon target is the myristoylated, alanine-rich C-kinase substrate (MARCKS). Here, we show that PKC activation is necessary for growth cone turning and that MARCKS, while at the membrane, colocalizes with alpha3-integrin in a peripheral adhesive zone of the growth cone. Phosphorylation of MARCKS causes its translocation from the membrane to the cytosol. Silencing MARCKS expression dramatically reduces growth cone spread, whereas overexpression of wild-type MARCKS inhibits growth cone collapse triggered by PKC activation. Expression of phosphorylation-deficient, mutant MARCKS greatly expands growth cone adhesion, and this is characterized by extensive colocalization of MARCKS and alpha3-integrin, resistance to eicosanoid-triggered detachment and collapse, and reversal of Sema3A-induced repulsion into attraction. We conclude that MARCKS is involved in regulating growth cone adhesion as follows: its nonphosphorylated form stabilizes integrin-mediated adhesions, and its phosphorylation-triggered release from adhesions causes localized growth cone detachment critical for turning and collapse.

Involvement of Protein Kinase C-ε in Activity-Dependent Potentiation of Large Dense-Core Vesicle Exocytosis in Chromaffin Cells

Neurotransmitter release is modulated in an activity-dependent manner. We showed previously that repetitive stimulation of nicotinic acetylcholine receptor (nAChR) induced activity-dependent potentiation (ADP) of large dense-core vesicle (LDCV) exocytosis in chromaffin cells. Here we report that protein kinase C (PKC)-epsilon is critically involved in ADP. Stimulation of nAChR induced activation of PKC-epsilon, and inhibition of PKC-epsilon by expression of the dominant-negative mutant of PKC-epsilon (DN-PKC-epsilon) or short interfering (siRNA) against PKC-epsilon abolished ADP via decreasing the frequency and quantal size of fused vesicles without affecting basal exocytosis, suggesting that PKC-epsilon is specifically involved in ADP. Electron microscopy revealed that inhibition of PKC-epsilon disrupts activity-induced vesicle translocation required for ADP. We also suggest the involvement of myristoylated alanine-rich C kinase substrate (MARCKS), which is known as a downstream target of PKC-epsilon, in ADP of LDCV exocytosis. The level of phospho-MARCKS correlated with the time course of ADP and was reduced by transfection with DN-PKC-epsilon. Actin filament disassembly induced by MARCKS phosphorylation was also significantly blocked by transfection of DN-PKC-epsilon. Furthermore, knockdown of MARCKS by siRNA resulted in inhibition of ADP and reduction of the number of fused vesicles. Together, we provide evidence that ADP of LDCV exocytosis is regulated by PKC-epsilon and its downstream target MARCKS via modulating vesicle translocation.