Protective Effects of Astragalus Membranaceus and Ligustrazine on Rat Brain Microvascular Endothelial Cell Injury after Oxygen-Glucose Deprivation/ Reoxygenation by Suppressing the PKCδ/MARCKS Pathway.

AIM AND OBJECTIVE: Cell death is a main pathological change in brain ischemia. Astragalus membranaceus (Ast) and ligustrazine (Lig), as traditional Chinese herbs, have a protective effect against ischemia-reperfusion injury. We aim to find whether the underlying protective mechanism of Astragalus membranaceus and ligustrazine against Oxygen-glucose deprivation/reoxygenation (OGD/R) -induced injury in RBMECs is related to PKCδ/MARCKS pathway. MATERIALS AND METHODS: OGD/R preconditioning was instituted in rat brain microvascular endothelial cells (RBMECs). The survival and apoptosis of RBMECs were detected by a Cell Counting Kit-8 and TUNEL staining; PKCδ/MARCKS and MMP9 expression were examined by immunofluorescence, western blot and quantitative real-time PCR. RESULTS: OGD/R stimulation significantly increased RBMEC apoptosis, whereas Ast+Lig, Rottlerin or Ast+Lig+Rottlerin treatment evidently reduced cellular apoptosis and increased cell viability (P <0.05). Furthermore, Ast+Lig, Rottlerin or Ast+Lig+Rottlerin treatment significantly reduced mRNA expression levels of PKCδ/MARCKS and MMP9 (P <0.05), compared to OGD/R control group. Moreover, Ast+Lig, Rottlerin or Ast+Lig+Rottlerin treatment evidently reduced protein expression levels of PKCδ, MMP9, and MARCKS (P <0.05), compared to OGD/R control group, detected by western blotting or immunofluorescence. CONCLUSION: The administration of Astragalus membranaceus and ligustrazine protected RBMECs against OGD/R-induced apoptosis. PKCδ/MARCKS and MMP9 expression were significantly increased after OGD/R stimulation, while Astragalus membranaceus and ligustrazine treatment evidently suppressed. Collectively, Astragalus membranaceus and ligustrazine play protective effects against OGD/R-induced injury in RBMECs through regulating PKCδ/MARCKS pathway to inhibit MMP9 activation.

Molecular effects of lithium are partially mimicked by inositol-monophosphatase (IMPA)1 knockout mice in a brain region-dependent manner.

We have previously shown that homozygote knockout (KO) of inositol-monophosphatase1 (IMPA1) results in lithium (Li)-like behavior. We now aimed to find out whether Li-treated mice and IMPA1 KO mice exhibit neurochemical similarity at the gene- and protein-expression level. Hippocampal and frontal cortex B-cell lymphoma (Bcl-2), Bcl-2-associated X protein (BAX), P53, Perodoxin2 (PRDX2), myristoylated alanine-rich C kinase substrate (MARCKS) and neuropeptide Y (NPY) mRNA levels, and hippocampal, frontal cortex and hypothalamic cytokine levels, all previously reported to be affected by lithium treatment, were measured in three groups of mice: wildtype (WT) on regular-food (RF), WT on Li-supplemented food (Li-treated) and IMPA1-KOs. Hippocampal and frontal cortex Bcl-2 and MARCKS were the only genes commonly affected (downregulated) by Li and IMPA1 KO; Bcl-2 - by 28% and 19%, respectively; MARCKS - by about 20% in both regions. The effect of Li and of IMPA1 KO on cytokine levels differed among the three brain areas studied. Only in the hippocampus both interventions exerted similar effects. Frontal cortex cytokine levels were unaffected neither by Li nor by IMPA1 KO. Similar changes in Bcl-2 and MARCKS but not in PRDX2 and NPY following both Li-treatment and IMPA1 KO suggest a mechanism different than inositol-monophosphatase1 inhibition for Li׳s effect on the latter genes. The cytokine levels results suggest that the mechanism mediating Li׳s effect on the inflammatory system differs among brain regions. Only in the hippocampus the results favor the involvement of the phosphatidylinositol (PI) cycle.

Maternal dietary loads of α-tocopherol depress protein kinase C signaling and synaptic plasticity in rat postnatal developing hippocampus and promote permanent deficits in adult offspring.

Vitamin E (α-tocopherol) supplementation has been tested as prophylaxis against gestational disorders associated with oxidative damage. However, recent evidence showing that high maternal α-tocopherol intake can adversely affect offspring development raises concerns on the safety of vitamin E extradosages during pregnancy. Besides acting as an antioxidant, α-tocopherol depresses cell proliferation and modulates cell signaling through inhibiting protein kinase C (PKC), a kinase that is deeply involved in neural maturation and plasticity. Possible effects of α-tocopherol loads in the maturing brain, where PKC dysregulation is associated to developmental dysfunctions, are poorly known. Here, supranutritional doses of α-tocopherol were fed to pregnant and lactating dams to evaluate the effects on PKC signaling and morphofunctional maturation in offspring hippocampus. Results showed that maternal supplementation potentiates hippocampal α-tocopherol incorporation in offspring and leads to marked decrease of PKC phosphorylation throughout postnatal maturation, accompanied by reduced phosphorylation of growth-associated protein-43 and myristoylated alanine-rich C kinase substrate, two PKC substrates involved in neural development and plasticity. Although processes of neuronal maturation, synapse formation and targeting appeared unaffected, offspring of supplemented mothers displayed a marked reduction of long-term synaptic plasticity in juvenile hippocampus. Interestingly, this impairment persisted in adulthood, when a deficit in hippocampus-dependent, long-lasting spatial memory was also revealed. In conclusion, maternal supplementation with elevated doses of α-tocopherol can influence cell signaling and synaptic plasticity in developing hippocampus and promotes permanent adverse effects in adult offspring. The present results emphasize the need to evaluate the safety of supranutritional maternal intake of α-tocopherol in humans.

Inhibition of myristoylated alanine-rich C kinase substrate (MARCKS) protein inhibits ozone-induced airway neutrophilia and inflammation.

Evidence suggests inhibition of leukocyte trafficking mitigates, in part, ozone-induced inflammation. In the present study, the authors postulated that inhibition of myristoylated alanine-rich C kinase substrate (MARCKS), an 82-kDa protein with multiple biological roles, could inhibit ozone-induced leukocyte trafficking and cytokine secretions. BALB/c mice (n = 5/cohort) were exposed to ozone (100 ppb) or forced air (FA) for 4 hours. MARCKS-inhibiting peptides, MANS, BIO-11000, BIO-11006, or scrambled control peptide RNS, were intratracheally administered prior to ozone exposure. Ozone selectively enhanced bronchoalveolar lavage (BAL) levels of killer cells (KCs; 6 +/- 0.9-fold), interleukin-6 (IL-6; 12.7 +/- 1.9-fold), and tumor necrosis factor (TNF; 2.1 +/- 0.5-fold) as compared to cohorts exposed to FA. Additionally, ozone increased BAL neutrophils by 21% +/- 2% with no significant (P > .05) changes in other cell types. MANS, BIO-11000, and BIO-11006 significantly reduced ozone-induced KC secretion by 66% +/- 14%, 47% +/- 15%, and 71.1% +/- 14%, and IL-6 secretion by 69% +/- 12%, 40% +/- 7%, and 86.1% +/- 11%, respectively. Ozone-mediated increases in BAL neutrophils were reduced by MANS (86% +/- 7%) and BIO-11006 (84% +/- 2.5%), but not BIO-11000. These studies identify for the first time the novel potential of MARCKS protein inhibitors in abrogating ozone-induced increases in neutrophils, cytokines, and chemokines in BAL fluid. BIO-11006 is being developed as a treatment for chronic obstructive pulmonary disorder (COPD) and is currently being evaluated in a phase 2 clinical study.

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

AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/INTERPRETATION: 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.

[Regulation function of Qingnao drop pilula to MARCKS mRNA express changes in acute cerebral ischemia hippocampus].

OBJECTIVE: To observe the contribution of Qingnao drop pilula to the alteration of myristoylated alanine-rich C kinase substrate (MARCKS) mRNA expression in acute multi-infarction hippocampus. METHOD: Rat models of acute multi-infarction were established by injecting the embolus of blood powder through the right external carotid arteryinto the internal carotid artery, rats were randomly divided into five groups (n = 12 in each): normal, sham operation, model, Chinese medicine treatment, and Western medicine treatment. Qingnao drop pilula (133.28 mg x kg(-1)), nimodipine (7.25 mg x kg(-1)) were administered respectively to Chinese medicine treatment group and Western medicine treatment group by gavage, equal volume of normal saline were given to three groups. Rats were treated with drugs starting at 3rd day before the operation, one time per day. Observing morphologic changes in hippocampus by optical microscope and electron microscope. Detecting expression level of MARCKS mRNA in hippocampus by semi-quantification PCR method. RESULT: Hippocampus cells arrange tidy, administrative levels were compactness in normal group, which cells differentially impaired in model group, Chinese medicine treatment group and Western medicine treatment group. Hippocampus cells damage of Chinese medicine treatment group have more reckless than the model group in histopathology. The MARCKS mRNA were expressioned in model group vs medication treatment groups, in Chinese medicine treatment group vs the model group. CONCLUSION: Qingnao drop pilula can alleciate histomorphology lesion of hippocampus when occurring acute multi-infarction, to turn slower MARCKS mRNA expression, may play a neuroprotective effect role through accommodating PKC-MARCKS signal transduction system.

Regulation function of Qingnao drop pilula to MARCKS mRNA express changes in acute cerebral ischemia hippocampus / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the contribution of Qingnao drop pilula to the alteration of myristoylated alanine-rich C kinase substrate (MARCKS) mRNA expression in acute multi-infarction hippocampus.</p><p><b>METHOD</b>Rat models of acute multi-infarction were established by injecting the embolus of blood powder through the right external carotid arteryinto the internal carotid artery, rats were randomly divided into five groups (n = 12 in each): normal, sham operation, model, Chinese medicine treatment, and Western medicine treatment. Qingnao drop pilula (133.28 mg x kg(-1)), nimodipine (7.25 mg x kg(-1)) were administered respectively to Chinese medicine treatment group and Western medicine treatment group by gavage, equal volume of normal saline were given to three groups. Rats were treated with drugs starting at 3rd day before the operation, one time per day. Observing morphologic changes in hippocampus by optical microscope and electron microscope. Detecting expression level of MARCKS mRNA in hippocampus by semi-quantification PCR method.</p><p><b>RESULT</b>Hippocampus cells arrange tidy, administrative levels were compactness in normal group, which cells differentially impaired in model group, Chinese medicine treatment group and Western medicine treatment group. Hippocampus cells damage of Chinese medicine treatment group have more reckless than the model group in histopathology. The MARCKS mRNA were expressioned in model group vs medication treatment groups, in Chinese medicine treatment group vs the model group.</p><p><b>CONCLUSION</b>Qingnao drop pilula can alleciate histomorphology lesion of hippocampus when occurring acute multi-infarction, to turn slower MARCKS mRNA expression, may play a neuroprotective effect role through accommodating PKC-MARCKS signal transduction system.</p>

MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein-mediated constitutive inhibition of calcium channels.

We have previously shown that the interaction of Ca2+/calmodulin with the metabotropic glutamate receptor type 7 (mGluR7) promotes the G-protein-mediated inhibition of voltage-sensitive Ca2+ channels (VSCCs) seen upon agonist activation. Here, we performed a yeast two-hybrid screen of a new-born rat brain cDNA library using the cytoplasmic C-terminal tail of mGluR7 as bait and identified macrophage myristoylated alanine-rich c-kinase substrate (MacMARCKS) as a binding protein. The interaction was confirmed in vitro and in vivo by pull-down assays, immunoprecipitation, and colocalization of mGluR7 and MacMARCKS in transfected HEK293 cells and cultured cerebellar granule cells. Binding of MacMARCKS to mGluR7 was antagonized by Ca2+/calmodulin. In neurons, cotransfection of MacMARCKS with mGluR7, but not mGluR7 mutants unable to bind MacMARCKS, reduced the G-protein-mediated tonic inhibition of VSCCs in the absence of mGluR7 agonist. These results suggest that competitive interactions of Ca2+/calmodulin and MacMARCKS with mGluR7 control the tonic inhibition of VSCCs by G-proteins.

Myoblast attachment and spreading are regulated by different patterns by ubiquitous calpains.

The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced mu- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed.

80K-H as a new Ca2+ sensor regulating the activity of the epithelial Ca2+ channel transient receptor potential cation channel V5 (TRPV5).

The epithelial Ca(2+) channel transient receptor potential cation channel V5 (TRPV5) constitutes the apical Ca(2+) entry pathway in the process of active Ca(2+) reabsorption. Ca(2+) influx through TRPV5 is tightly controlled by modulators of Ca(2+) homeostasis, including 1,25-dihydroxyvitamin D(3) and dietary Ca(2+). However, little is known about intracellular proteins that interact with TRPV5 and directly regulate the activation of this channel. By the use of cDNA microarrays, the present study identified 80K-H as the first protein involved in the Ca(2+)-dependent control of the epithelial Ca(2+) channel TRPV5. 80K-H was initially identified as a protein kinase C substrate, but its biological function remains to be established. We demonstrated a specific interaction between 80K-H and TRPV5, co-localization of both proteins in the kidney, and similar transcriptional regulation by 1,25-dihydroxyvitamin D(3) and dietary Ca(2+). Furthermore, 80K-H directly bound Ca(2+), and inactivation of its two EF-hand structures totally abolished Ca(2+) binding. Electrophysiological studies using 80K-H mutants showed that three domains of 80K-H (the two EF-hand structures, the highly acidic glutamic stretch, and the His-Asp-Glu-Leu sequence) are critical determinants for TRPV5 activity. Importantly, inactivation of the EF-hand pair reduced the TRPV5-mediated Ca(2+) current and increased the TRPV5 sensitivity to intracellular Ca(2+), accelerating the feedback inhibition of the channel. None of the 80K-H mutants altered the TRPV5 plasma membrane localization nor the association of 80K-H with TRPV5, suggesting that 80K-H has a direct effect on TRPV5 activity. In conclusion, we report a novel function for 80K-H as a Ca(2+) sensor controlling TRPV5 channel activity.

Vacuolar system-associated protein-60: a protein characterized from bovine granulosa and luteal cells that is associated with intracellular vesicles and related to human 80K-H and murine beta-glucosidase II.

It has been suggested that proteins of molecular size 56-58 kDa play an important role in bovine ovarian follicular development and oocyte maturation. A polyclonal antibody was raised against a 56- to 58-kDa protein band purified from bovine granulosa cells and was used to screen granulosa or luteal cell cDNA expression libraries. This work resulted in the identification of a cDNA encoding for a protein of 60.1 kDa with a signal peptide of 13 residues. The bovine 60.1-kDa protein shared an overall 86.7% and 81.8% identity with, respectively, the human 80K-H protein and the mouse putative beta subunit of glucosidase II (beta-GII), and was named vacuolar system-associated protein-60 (VASAP-60). Marked differences in sequence identity were noted in a putative molecular adapter domain containing a tandem D and E amino acid stretch flanked by proline-rich sequences presenting the minimal PXXP SH3 motif. VASAP-60 was shown to be unglycosylated using endoglycosidase H treatment and was found mainly in a cellular membrane fraction of bovine corpus luteum. VASAP-60 was localized in a rat hepatic Golgi/endosome fraction and in wheat germ agglutinin (WGA) affinity chromatographic eluates, thereby suggesting the presence of interactions with membrane glycoproteins. A polyclonal antibody was raised against the putative adapter domain of the recombinant VASAP-60; this was shown to recognize a major 88-kDa and two minor 58-kDa and 50-kDa proteins, suggesting that the major 88-kDa protein band represents the complete VASAP-60 protein whereas the 58-kDa and the 50-kDa bands represent its proteolytic fragments. Northern blot analysis demonstrated the presence of a single 2.3-kilobase transcript in all the bovine tissues analyzed with variation in the steady state level between tissues. Immunohistochemical observations showed that VASAP-60 was widely distributed in bovine tissues and was localized in pericytoplasmic and perinuclear membranes. In epithelial cells, the staining presented a basolateral or apical polarity associated with intracellular vacuoles. In conclusion, we have characterized a novel acidic membrane protein, associated with organelles of the vacuolar system, that is widely and histospecifically expressed in bovine tissues. VASAP-60 represents either the bovine ortholog or a new family member of the previously characterized human 80K-H and murine beta-GII proteins. Our results suggest that VASAP-60 presents characteristics of a molecular adaptor protein with functions in membrane-trafficking events.

Thrombin-induced phosphorylation of MARCKS does not alter its interactions with calmodulin or actin.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a calmodulin (CaM)- and actin-binding protein and prominent protein kinase C (PKC) substrate. In vitro phosphorylation of MARCKS by PKC has been shown to induce the release of both CaM and actin, leading to the suggestion that MARCKS may regulate CaM availability during agonist-induced signalling. In support of this hypothesis we previously demonstrated that thrombin-induced MARCKS phosphorylation in endothelial cells (EC) parallels activation of myosin light chain kinase, a CaM-dependent enzyme. To test this theory further, we transfected CHO cells, which normally do not express significant levels of MARCKS, with a MARCKS cDNA. The thrombin-stimulated phosphorylation of myosin light chains and the sensitivity to CaM antagonists in the MARCKS overexpressing cells was the same as that in control CHO cells. MARCKS associated with the actin cytoskeleton in EC was markedly increased upon treatment with the PKC activator, PMA, but only modestly enhanced by thrombin treatment. Similarly, colocalisation of MARCKS with actin was enhanced when the EC were challenged with PMA but not thrombin. These data may be partially explained by PKC-independent phosphorylation of MARCKS in response to thrombin stimulation.

Generation of a novel A kinase anchor protein and a myristoylated alanine-rich C kinase substrate-like analog from a single gene.

A unique Drosophila gene encodes two novel signaling proteins. Drosophila A kinase anchor protein 200 (DAKAP200) (753 amino acids) binds regulatory subunits of protein kinase AII (PKAII) isoforms in vitro and in intact cells. The acidic DAKAP200 polypeptide (pI approximately 3.8) contains an optimal N-terminal myristoylation site and a positively charged domain that resembles the multifunctional phosphorylation site domain of vertebrate myristoylated alanine-rich C kinase substrate proteins. The 15-kilobase pair DAKAP200 gene contains six exons and encodes a second protein, DeltaDAKAP200. DeltaDAKAP200 is derived from DAKAP200 transcripts by excision of exon 5 (381 codons), which encodes the PKAII binding region and a Pro-rich sequence. DeltaDAKAP200 appears to be a myristoylated alanine-rich C kinase substrate analog. DAKAP200 and DeltaDAKAP200 are evident in vivo at all stages of Drosophila development. Thus, both proteins may play important physiological roles throughout the life span of the organism. Nevertheless, DAKAP200 gene expression is regulated. Maximal levels of DAKAP200 are detected in the pupal phase of development; DeltaDAKAP200 content is elevated 7-fold in adult head (brain) relative to other body parts. Enhancement or suppression of exon 5 excision during DAKAP200 pre-mRNA processing provides potential mechanisms for regulating anchoring of PKAII and targeting of cAMP signals to effector sites in cytoskeleton and/or organelles.

Overexpression of MARCKS, but not protein kinase C-alpha, increases phorbol ester-stimulated synthesis of phosphatidylcholine in human SK-N-MC neuroblastoma cells.

To investigate the regulation of phorbol ester-stimulated synthesis of phosphatidylcholine (PtdCho), myristoylated alanine-rich protein kinase C substrate (MARCKS) and the alpha-isoform of protein kinase C (PKC-alpha) were overexpressed in a human neuroblastoma (SK-N-MC) cell line that does not increase PtdCho synthesis in response to 4beta-12-O-tetradecanoylphorbol 13-acetate (TPA). In five clones with a less than fivefold increase in MARCKS protein level, the synthesis of PtdCho from [methyl-3H] choline was stimulated 1.88-2.34-fold in the presence of 100-200 nM TPA. In clones overexpressing PKC-alpha (30-40-fold increased level of protein) or in mock-transfected vector controls, TPA had much less of a stimulatory effect (1.04-1.43 fold) on PtdCho synthesis. TPA caused translocation of PKC-alpha and increased phosphorylation of MARCKS, indicating that both overexpressed proteins responded to stimulation. Thus, in SK-N-MC cells, MARCKS is required for TPA-stimulated synthesis of PtdCho and PKC-alpha alone is insufficient for supporting enhanced synthesis.

A 3-Mb region for the familial hemiplegic migraine locus on 19p13.1-p13.2: exclusion of PRKCSH as a candidate gene. Dutch Migraine Genetic Research Group.

Familial hemiplegic migraine (FHM) is an autosomal domianant subtype of migraine with attacks, associated with transient episodes of hemiparesis. One of the genes for FHM has been assigned to chromosome 19p13. Detailed analysis of critical recombinants from two different chromosome 19-linked FHM families, using new markers indicated a 6-cM candidate region on 19p13.1-p13.2 flanked by loci D19S394 and D19S226. Another paroxysmal neurological disorder, episodic ataxia type 2 (EA-2), has also been linked to the same chromosomal region. Most of the interval was completely covered by YAC and cosmid contigs; the physical map yielded approximately 3 Mb encompassing several genes including the protein kinase substrate 80K-H (PRKCSH) gene. Since PRKCSH is involved in neuronal signal transduction, it was considered to be an FHM candidate gene. The genomic structure of this gene was established and mutation analysis for all exon and flanking intron sequences was performed in FHM- and EA-2-affected individuals. Five polymorphisms were identified, including a trinucleotide repeat length variation in the coding sequence. However, no potential disease causing mutation was found and therefore the PRKCSH gene can be excluded for both FHM and EA-2.

Phorbol esters downregulate expression of the sodium/calcium exchanger in renal epithelial cells.

The Na+/Ca2+ exchanger (NCE) contributes to Ca2+ reabsorption by connecting tubules of the nephron. A line of renal epithelial cells from monkey kidney (LLC-MK2) was used to investigate the regulation of NCE expression. After the activation of protein kinase C (PKC) by phorbol myristate acetate (PMA), NCE activity decreased exponentially by 75% in 48 h (half time approximately 19 h). PMA decreased NCE mRNA by 85% in 24 h. The decrease in NCE transcript preceded the downregulation of NCE activity. NCE protein was quantified with a monoclonal antibody to cardiac NCE. PMA decreased the binding of 3H-labeled antibody to cell sonicates by 40% in 24 h. Immunoblots show that PMA produced a marked and extended increase in membrane-associated PKC-alpha, although PMA depleted total PKC-alpha by 65% in 24 h. In vivo 32P labeling of myristolated alanine-rich C kinase substrate, a specific PKC substrate, confirmed that PMA produced a rapid and extended activation of PKC. 4 alpha-PMA, a stereoisomer of PMA that neither binds nor activates PKC, had no effect on NCE activity or transcript. These findings indicate that activation of PKC with phorbol esters downregulates NCE mRNA, protein, and activity in renal epithelial cells.

Radiolabelling of bovine myristoylated alanine-rich protein kinase C substrate (MARCKS) in an ADP-ribosylation reaction.

In an ADP-ribosylation reaction, we have observed the radiolabelling of a protein in a crude bovine brain homogenate, which upon two-dimensional gel electrophoresis migrated with an acidic pI (< 4.5) and an apparent molecular mass (80-90 kDa) consistent with the properties of the myristoylated, alanine-rich, protein kinase C substrate protein termed MARCKS. To establish the identity of this radiolabelled constituent in brain homogenates, we first purified bovine brain MARCKS using calmodulin-Sepharose affinity chromatography and we then supplemented the crude ADP-ribosylation reaction mixture with this purified MARCKS fraction. Concordant increases in radiolabelling and silver staining of the same protein component from the MARCKS-supplemented ADP-ribosylation reaction, as compared with the ADP-ribosylated crude homogenate, established the identity of this constituent as MARCKS. The radiolabelling of MARCKS was lower in comparison with the ADP-ribosylation of the related neuronal protein B-50/GAP-43 under identical reaction conditions. The potential functional consequences of the ADP-ribosylation of MARCKS are discussed and the possibility is raised that other members of the MARCKS family, such as the F52/MacMARCKS/MRP protein, may also be subject to ADP-ribosylation.