Multiple G-protein involvement in parathyroid hormone regulation of acid production by osteoclasts

The involvement of multiple G-proteins in parathyroid hormone regulation of acid production was demonstrated in a highly enriched osteoclast population. Osteoclasts were isolated from the endosteum of 2.5 to 3-week-old chicken tibia using sequential enzymatic digestion. Single cell analysis of acid production was accomplished using microscope photometry and vital staining with acridine orange, a hydrogen ion concentration sensitive fluorescent dye. Lithium chloride, an uncoupler of G-proteins from their respective receptors, blocked parathyroid hormone stimulated production of acid. Cholera toxin, which permanently activates G_s-proteins, mimicked PTH stimulation. Pertussis toxin, which prevents receptor interaction with G_i- and G_o-proteins, blocked both 10 ⁸ M and 10 ¹¹ M PTH stimulated acid production, suggesting that the pertussis toxin-sensitive G-protein is utilized at both PTH concentrations. Immunoblots of osteoclast plasma membrane proteins, using a panel of antibodies generated against specific G-protein α subunits, revealed a 48 kDa G_sα, a 41 G_oα, a 34 kDa G_iα-3, and a unique 68 kDa Gα subunit, with the 41 kDa and 34 kDa bands being the most intense. Immunoblots of osteoblast plasma membrane proteins had a substantially different profile with the most intense bands being a G_sα (48 kDa) and a G_oα (36 and 38 kDa). The studies suggest the utilization of at least two different G-proteins in the parathyroid hormone regulation of acid formation by osteoclasts, a G_s and a pertussis toxin-sensitive G-protein (G_o and/or G_iα-3). J. Cell. Biochem. 64:161–170. © 1997 Wiley-Liss, Inc.     

Actions of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP formation in chicken and rat osteoclasts

The effects of calcitonin, parathyroid hormone, and prostaglandin E2 on cyclic AMP production were studied in osteoclast-rich cultures derived from medullary bone of laying hens and from the long bones of newborn rats. Cyclic AMP was assayed biochemically in replicate cultures, and furthermore, changes in cytoplasmic fluorescence were sought by indirect immunofluorescence with rabbit anti-cyclic AMP and FITC-labelled goat anti-rabbit IgG. Treatment of rat osteoclasts with calcitonin increased cyclic AMP formation as measured biochemically, and this was confirmed by the immunofluorescence method. No such increase took place in chick osteoclasts. Prostaglandin E2 increased cyclic AMP production in both rat and chick osteoclasts as determined by both methods. Since the immunofluorescence method failed to detect a response to parathyroid hormone either in chick or rat osteoclasts, its variable biochemical effects were concluded to be due to actions on contaminating osteoblasts in the cultures. Thus it has been possible with a combined biochemical and immunocytochemical approach to define the cyclic AMP responses to the calcium-regulating hormones in rat and chick osteoclasts. The failure of calcitonin to increase cyclic AMP in chick osteoclasts identifies a need to investigate the nature of calcitonin action on avian osteoclasts, which may contribute to understanding of its actions on mammalian cells.     

Parathyroid hormone stimulates protein kinase C but not adenylate cyclase in mouse epidermal keratinocytes.

Intact human parathyroid hormone, hPTH [1-84], and the hPTH [1-34] fragment stimulated membrane-associated protein kinase C (PKC) activity in immortalized (but still differentiation-competent) murine BALB/MK-2 skin keratinocytes. Unexpectedly, the hormone and its fragment did not stimulate adenylate cyclase. The failure of PTH to stimulate adenylate cyclase activity was not due to the lack of a functioning receptor-cyclase coupling mechanism because the cells were stimulated to synthesize cyclic adenosine monophosphate (cyclic AMP) by the beta-adrenergic drug isoproterenol. Thus, skin keratinocytes seem to have an unconventional PTH receptor that is coupled to a PKC-activating mechanism but not to adenylate cyclase. These observations suggest that normal and neoplastic skin keratinocytes respond to the PTH-related peptide that they make and secrete.     

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: Further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDa protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells. we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 μM), on pepsinogen secretion and phosphorylation of the 72-kDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with ³²P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 μM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS “phosphorylation/calmodulin binding domain peptide” indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion. J. Cell. Biochem. 64:514–523. © 1997 Wiley-Liss, Inc.     

Specifying protein kinase C functions in melanoma

The protein kinase C (PKC) family of serine/threonine protein kinases is a heterogeneous group of enzymes receiving and integrating signals involved in both normal melanocyte biology and melanoma pathology. Alterations in PKC enzyme expression and activation contribute to the malignant phenotype of melanoma in both oncogenic and tumor suppressive roles. Delineating the diverse and often context-dependent functions of PKC enzymes in melanocyte/melanoma biology is key to capitalize on these kinases as drug targets. This review summarizes several of the diverse functions of PKC in melanocyte and melanoma biology with a focus on PKC enzyme regulation and function.     

Physiological stress and nerve growth factor treatment regulate stress-activated protein kinase activity in PC12 cells

The stress-activated protein kinases (SAPKs) are differentially activated by a variety of cellular stressors in PC12 cells. SAPK activation has been linked to the induction of apoptotic cell death upon serum withdrawal from undifferentiated cells or following nerve growth factor (NGF) withdrawal of neuronally differentiated PC12 cells. However, withdrawal of trophic support from differentiated cells led to only a very modest elevation of SAPK activity and led us to investigate the basis of the relative insensitivity of these enzymes to stressors. NGF-stimulated differentiation of the cells resulted in the elevation of basal SAPK activity to levels four- to sevenfold greater than in untreated cells, which was correlated with an approximate fivefold increase in SAPK protein levels. Paradoxically, in NGF-differentiated PC12 cells, exposure to cellular stressors provoked a proportionately smaller stimulation of SAPK activity than that observed in naive cells, despite the presence of much higher levels of SAPK protein. The insensitivity of SAPK to activation by stressors was reflective of the activity of the SAPK activator SEK, whose activation was also diminished following NGF differentiation of the cells. The data demonstrate that SAPKs are subject to complex controls through both induction of SAPK expression and the regulation mediated by upstream elements within this pathway. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 537–549, 1998     

Cellular distribution of gastric chief cell protein kinase C activity: differential effects of diacylglycerol, phorbol esters, carbachol, and cholecystokinin.

Stimulation of chief cells with carbachol or cholecystokinin (CCK) results in the production of inositol trisphosphate (IP3) and diacylglycerol (DAG). Although IP3 increases cell calcium concentration, thereby stimulating pepsinogen secretion, the role of DAG and its target, protein kinase C (PKC), is less clear. To examine the relation between the cellular distribution of PKC activity and pepsinogen secretion, we determined PKC activity in cytosolic and membrane fractions from dispersed chief cells from guinea pig stomach. To validate our assay, we studied the actions of the phorbol ester PMA. PMA caused a rapid, dose-dependent, 6-fold increase in pepsinogen secretion and membrane-associated PKC activity. Similarly, dose-response curves for pepsinogen secretion and the increase in membrane-associated PKC activity induced by a membrane-permeant DAG (1-oleoyl-2-acetylglycerol) were superimposable. In contrast, CCK (0.1 nM to 1.0 microM) and carbachol (0.1 microM to 1.0 mM) caused a 4-fold increase in pepsinogen secretion, but did not alter the distribution of PKC activity. These results indicate that in gastric chief cells, PMA- and DAG-induced pepsinogen secretion is accompanied by increased membrane-associated PKC activity. However, the cellular distribution of PKC activity is not altered by CCK or carbachol.     

Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.     

Dual coupling of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same Gi protein.

The coloned 5-HT_1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects of 5-hydroxytryptamine (5-HT) to inhibit cAMP formation and to stimulate the hydrolysis of phosphatidylinositol. Both responses were found to be pertussis toxin sensitive. We have examined these two responses in membranes derived from these cells and show that the 5-HT_1A receptor can directly regulate the activity of adenylyl cyclase and phospholipase C in response to agonist. In order to examine whether the same or distinct guanine nucleotide-binding regulatory protein(s) (G protein) are involved in these two signal transduction pathways, we used anti-peptide antibodies recognizing the -subunits of G_i1, G_i2, G_i3 as specific tools, since these pertussis toxin substrates are expressed in HeLa cells. These antibodies have previously been shown to prevent receptor-G protein coupling by binding to the regions of G proteins which are putatively involved in interaction with receptors. Our results indicate that the G_i proteins, but preferentially G₃, mediate the effects of 5-HT both to inhibit adenylyl cyclase and to stimulate phospholipase C. These findings demonstrate that the same receptor interacting with the same C protein can regulate several distinct effector molecules.     

Protein kinase C and a viral K-RAS protein cooperatively enhance the response of adenylate cyclase to stimulators

The protein kinase C stimulator TPA (12-O-tetradecanoyl phorbol-13-acetate) enhanced the responsiveness of adenylate cyclase to IPR (isoproterenol) and PGE1 (prostaglandin E1) in quiescent tsKSV-NRK cells at the nonpermissive 41 degrees C. Reactivating the thermolabile mitogenic/oncogenic K-ras protein in tsKSV-NRK cells by dropping the temperature to 36 degrees C also enhanced the responsiveness of adenylate cyclase to IPR and PGE1. The enhancement was transient and peaked at 6 hours after the temperature shift. This enhanced responsiveness was specifically due to the reactivated viral K-ras protein rather than the temperature shift because the same temperature shift did not affect adenylate cyclase responsiveness in uninfected NRK cells, nor was it a result of the mitogenic stimulus since reacting the mitogenic pp60v-src protein in tsASV-NRK cells did not affect adenylate cyclase responsiveness. The increased responsiveness of adenylate cyclase at 6 hours after the temperature shift was not a result of elevated membrane-associated PKC activity. However, the reactivated viral K-ras protein strongly increased the stimulability of membrane-associated PKC by TPA and it further increased TPA's ability to enhance the responsiveness of adenylate cyclase to IPR and PGE1. Thus, a viral K-ras protein and membrane-associated protein kinase C can cooperate to increase the responsiveness of adenylate cyclase to agonists.     

植物MAPK级联途径参与调控ABA信号转导

促分裂原活化蛋白激酶(MAPK)级联途径信号通路在真核生物细胞信号的转换和放大过程中起重要作用。MAPK级联途径由三个成员组成,分别是MAPK、MAPKK及MAPKKK,此三个信号组分按照MAPKKK-MAPKK-MAPK的方式依次磷酸化将外源信号级联放大向下传递。大量研究表明,植物MAPK级联途径参与调控脱落酸(ABA)信号转导。因此,该文就ABA和MAPK的生物学功能、ABA信号转导中的磷酸化与去磷酸化以及MAPK级联途径与ABA信号转导之间的关系等方面的研究进展进行综述,以便进一步认识MAPK和ABA信号转导的分子机制。     

Platelet-derived growth factor-BB and lysophosphatidic acid distinctly regulate hepatic myofibroblast migration through focal adhesion kinase

Although hepatic myofibroblast (HMF) migration contributes to the development of fibrosis, the mechanisms coordinating this movement are uncertain. We determined the effects of lysophosphatidic acid (LPA) and platelet-derived growth factor-BB (PDGF) on actin polymerization, FAK tyrosine phosphorylation, and migration of cultured human HMFs. LPA (0.4-100 microM) stimulated migration, FAK tyrosine phosphorylation, and stress fiber assembly with a sigmoidal dose response. PDGF (1-250 ng/ml) stimulated migration, FAK tyrosine phosphorylation, and actin polymerization with a bell-shape dose-response characterized by a maximum at 10-25 ng/ml. Concentrations of cytochalasin D, which abolished FAK tyrosine phosphorylation, also blocked LPA- and PDGF-induced migration. A dose of 1-10 ng/ml PDGF acted synergistically with LPA (10 microM) to stimulate FAK tyrosine phosphorylation and migration, whereas higher concentrations of PDGF (100-250 ng/ml) inhibited FAK tyrosine phosphorylation and migration in response to LPA (10 microM). These data indicate that PDGF and LPA coordinately govern the migration of HMFs by differentially regulating FAK and suggest a novel model in which PDGF, acting as an amplifier/attenuator of LPA-induced signaling, facilitates HMF accumulation within injured areas of the liver.     

Overexpression of protein kinase C betaII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis.

Protein kinase C betaII (PKC betaII) has been implicated in proliferation of the intestinal epithelium. To investigate PKC betaII function in vivo, we generated transgenic mice that overexpress PKC betaII in the intestinal epithelium. Transgenic PKC betaII mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC betaII mice exhibit elevated colonic beta-catenin levels and decreased glycogen synthase kinase 3beta activity, indicating that PKC betaII stimulates the Wnt/adenomatous polyposis coli (APC)/beta-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC betaII in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/beta-catenin signaling pathway.     

Alterations in protein kinase C isoenzyme expression and autophosphorylation during the progression of pressure overload-induced left ventricular hypertrophy

Cardiomyocytes express several isoenzymes of protein kinase C (PKC), which as a group have been implicated in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. Individual PKC isoenzymes also require transphosphorylation and autophosphorylation for enzymatic activity. To determine whether PKC isoenzyme expression and autophosphorylation are altered during LVH progression in vivo, suprarenal abdominal aortic coarctation was performed Sprague-Dawley rats. Quantitative Western blotting was performed on LV tissue 1, 8 and 24 weeks after aortic banding, using antibodies specific for total PKC, PKC and PKC, and their C-terminal autophosphorylation sites. Aortic banding produced sustained hypertension and gradually developing LVH that progressed to diastolic heart failure over time. PKC levels and autophosphorylation were not significantly different from sham-operated controls during any stage of LVH progression. PKC expression levels were also unaffected during the induction of LVH, but increased 3.2 ± 0.8 fold during the transition to heart failure. In addition, there was a high degree of correlation between PKC levels and the degree of LVH in 24 week banded animals. However, autophosphorylated PKC was not increased at any time point. In contrast, PKC autophosphorylation was increased prior to the development of LVH, and also during the transition to heart failure. The increased PKC autophosphorylation in 1 week banded rats was not accompanied by an increase in total PKC, whereas total PKC levels were markedly increased (6.0 ± 1.7 fold) in 24 week banded animals. Furthermore, both phosphorylated and total PKC levels were highly correlated with the degree of LVH in 24 week banded rats. In summary, we provide indirect evidence to indicate that PKC may be involved in the induction of pressure overload LVH, whereas both PKC and PKC may be involved in the transition to heart failure.     

植物蛋白激酶介导的非生物胁迫和激素信号转导途径的研究进展

干旱、盐渍、低温和高温等非生物胁迫严重影响植物的生长发育和作物的产量。在长期的进化过程中,植物逐渐形成了对外部刺激快速感知和主动适应的能力,其中植物体内逆境信号的传递在植物快速感知外部刺激和主动适应非生物胁迫过程中起着非常重要的作用。蛋白激酶和蛋白磷酸酶催化的蛋白质磷酸化和去磷酸化是植物体内存在的最普遍且最重要的信号转导调节方式。其中,蛋白激酶的主要作用是将ATP或GTP上的γ磷酸基团转移到特定的底物蛋白上,使蛋白磷酸化,被磷酸化的蛋白发挥相应的生理功能。近年来,利用生物技术和基因工程等手段从细胞、分子水平上研究有关蛋白激酶的抗逆机理,通过基因沉默、基因过表达等策略提高植物的抗逆性成为国内外抗逆分子生物学与分子育种学研究的热点。本文主要对植物蛋白激酶在介导非生物胁迫和激素信号通路中的作用进行综述,为进一步研究植物蛋白激酶功能提供有价值的信息。     

Stimulation of a histone H4 protein kinase in Triton X-100 lysates of rabbit peritoneal neutrophils pretreated with chemotactic factors: lack of requirements of calcium mobilization and protein kinase C activation

The characteristics of the activation of a histone H4 kinase activity in Triton X-100 lysates of rabbit peritoneal neutrophils pretreated with fMet-Leu-Phe were studied: The activation of the kinase was a) inhibited by the antagonist of formylpeptide, t-Boc-(Phe-Leu)2(-)-Phe, b) completely inhibited by pertussis toxin pretreatment, c) not affected by the pretreatment of neutrophils with an activator of protein kinase C, phorbol-12-myristate-13-acetate, or an inhibitor of protein kinase C, 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine, and d) not inhibited in the cells preloaded with the intracellular calcium chelators, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra acetic acid acetoxymethyl-ester (BAPTA/AM). These results suggest that the stimulus-induced activation of H4 kinase requires functional receptor and GTP-binding protein but neither calcium mobilization nor protein kinase C activation.     

Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2

We report the cloning of both the cDNA and the corresponding genomic sequence of a new PP2C from Arabidopsis thaliana, named AtP2C-HA (for homology to ABI1/ABI2). The AtP2C-HA cDNA contains an open reading frame of 1536 bp and encodes a putative protein of 511 amino acids with a predicted molecular mass of 55.7 kDa. The AtP2C-HA protein is composed of two domains, a C-terminal PP2C catalytic domain and a N-terminal extension of ca. 180 amino acid residues. The deduced amino acid sequence is 55% and 54% identical to ABI1 and ABI2, respectively. Comparison of the genomic structure of the ABI1, ABI2 and AtP2C-HA genes suggests that they belong to a multigene family. The expression of the AtP2C-HA gene is up-regulated by abscisic acid (ABA) treatment.