Activity-dependent CREB phosphorylation: Convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway

The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is activated by phosphorylation on Ser-133. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. Previous work in neurons has indicated that physiologic synaptic stimulation recruits a fast calmodulin kinase IV (CaMKIV)-dependent pathway that dominates early signaling to CREB. Here we show in hippocampal neurons that the fast, CaMK-dependent pathway can be followed by a slower pathway that depends on Ras/mitogen-activated protein kinase (MAPK), along with CaMK. This pathway was blocked by dominant-negative Ras and was specifically recruited by depolarizations that produced strong intracellular Ca2+ transients. When both pathways were recruited, phosphorylated CREB (pCREB) formation was overwhelmingly dominated by the CaMK pathway between 0 and 10 min, and by the MAPK pathway at 60 min, whereas the two pathways acted in concert at 30 min. The Ca2+ signals that produced only rapid CaMK signaling to pCREB or both rapid CaMK and slow MAPK signaling deviated significantly for only ≈1 min, yet their differential impact on pCREB extended over a much longer period, between 20 and 60 min and beyond, which is of likely significance for gene expression. The CaMK-dependent MAPK pathway may inform the nucleus about stimulus amplitude. In contrast, the CaMKIV pathway may be well suited to conveying information on the precise timing of localized synaptic stimuli, befitting its greater speed and sensitivity, whereas the previously described calcineurin pathway may carry information about stimulus duration.

Characterization of Chlamydomonas reinhardtii Zygote-Specific cDNAs That Encode Novel Proteins Containing Ankyrin Repeats and WW Domains1

Genes that are expressed only in the young zygote are considered to be of great importance in the development of an isogamous green alga, Chlamydomonas reinhardtii. Clones representing the Zys3 gene were isolated from a cDNA library prepared using zygotes at 10 min after fertilization. Sequencing of Zys3 cDNA clones resulted in the isolation of two related molecular species. One of them encoded a protein that contained two kinds of protein-to-protein interaction motifs known as ankyrin repeats and WW domains. The other clone lacked the ankyrin repeats but was otherwise identical. These mRNA species began to accumulate simultaneously in cells beginning 10 min after fertilization, and reached maximum levels at about 4 h, after which time levels decreased markedly. Genomic DNA gel-blot analysis indicated that Zys3 was a single-copy gene. The Zys3 proteins exhibited parallel expression to the Zys3 mRNAs at first, appearing 2 h after mating, and reached maximum levels at more than 6 h, but persisted to at least 1 d. Immunocytochemical analysis revealed their localization in the endoplasmic reticulum, which suggests a role in the morphological changes of the endoplasmic reticulum or in the synthesis and transport of proteins to the Golgi apparatus or related vesicles.

Fluid shear stress inhibits TNF-α activation of JNK but not ERK1/2 or p38 in human umbilical vein endothelial cells: Inhibitory crosstalk among MAPK family members

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and IL-1 stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. TNF-α and IL-1 regulate gene expression in ECs, in part, by stimulating mitogen-activated protein kinases (MAPK), which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAPK in EC. To test this hypothesis, we determined the effects of flow (shear stress = 12 dynes/cm2) on TNF-α and IL-1-stimulated activity of three MAPK in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). Flow alone stimulated ERK1/2 and p38 activity but decreased JNK activity compared with static controls. TNF-α or IL-1 alone activated ERK1/2, p38, and JNK maximally at 15 min in HUVEC. Preexposing HUVEC for 10 min to flow inhibited TNF-α and IL-1 activation of JNK by 46% and 49%, respectively, but had no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, which inhibits flow-mediated ERK1/2 activation, prevented flow from inhibiting cytokine activation of JNK. Phorbol 12-myristate 13-acetate, which strongly activates ERK1/2, also inhibited TNF-α activation of JNK. These findings indicate that fluid shear stress inhibits TNF-α-mediated signaling events in HUVEC via the activation of the ERK1/2 signaling pathway. Inhibition of TNF-α signal transduction represents a mechanism by which steady laminar flow may exert atheroprotective effects on the endothelium.

Shifts of Intracellular pH Distribution as a Part of the Signal Mechanism Leading to the Elicitation of Benzophenanthridine Alkaloids1 : Phytoalexin Biosynthesis in Cultured Cells of Eschscholtzia californica

Cultured cells of Eschscholtzia californica (Californian poppy) respond to a yeast elicitor preparation or Penicillium cyclopium spores with the production of benzophenanthridine alkaloids, which are potent phytoalexins. Confocal pH mapping with the probe carboxy-seminaphthorhodafluor-1-acetoxymethylester revealed characteristic shifts of the pH distribution in challenged cells: within a few minutes after elicitor contact a transient acidification of cytoplasmic and nuclear areas occurred in parallel with an increase of the vacuolar pH. The change of proton concentration in the vacuole and in the extravacuolar area showed a nearly constant relation, indicating an efflux of vacuolar protons into the cytosol. A 10-min treatment with 2 mm butyric or pivalic acid caused a transient acidification of the cytoplasm comparable to that observed after elicitor contact and also induced alkaloid biosynthesis. Experimental depletion of the vacuolar proton pool reversibly prevented both the elicitor-triggered pH shifts and the induction of alkaloid biosynthesis. pH shifts and induction of alkaloid biosynthesis showed a similar dependence on the elicitor concentration. Net efflux of K+, alkalinization of the outer medium, and browning of the cells were evoked only at higher elicitor concentrations. We suggest that transient acidification of the cytoplasm via efflux of vacuolar protons is both a necessary and sufficient step in the signal path toward biosynthesis of benzophenanthridine alkaloids in Californian poppy cells.

N-Acylethanolamines: Formation and Molecular Composition of a New Class of Plant Lipids1

Recently, the biosynthesis of an unusual membrane phospholipid, N-acylphosphatidylethanolamine (NAPE), was found to increase in elicitor-treated tobacco (Nicotiana tabacum L.) cells (K.D. Chapman, A. Conyers-Hackson, R.A. Moreau, S. Tripathy [1995] Physiol Plant 95: 120–126). Here we report that before induction of NAPE biosynthesis, N-acylethanolamine (NAE) is released from NAPE in cultured tobacco cells 10 min after treatment with the fungal elicitor xylanase. In radiolabeling experiments [14C]NAE (labeled on the ethanolamine carbons) increased approximately 6-fold in the culture medium, whereas [14C]NAPE associated with cells decreased approximately 5-fold. Two predominant NAE molecular species, N-lauroylethanolamine and N-myristoylethanolamine, were specifically identified by gas chromatography-mass spectrometry in lipids extracted from culture medium, and both increased in concentration after elicitor treatment. NAEs were found to accumulate extracellularly only. A microsomal phospholipase D activity was discovered that formed NAE from NAPE; its activity in vitro was stimulated about 20-fold by mastoparan, suggesting that NAPE hydrolysis is highly regulated, perhaps by G-proteins. Furthermore, an NAE amidohydrolase activity that catalyzed the hydrolysis of NAE in vitro was detected in homogenates of tobacco cells. Collectively, these results characterize structurally a new class of plant lipids and identify the enzymatic machinery involved in its formation and inactivation in elicitor-treated tobacco cells. Recent evidence indicating a signaling role for NAPE metabolism in mammalian cells (H.H.O. Schmid, P.C. Schmid, V. Natarajan [1996] Chem Phys Lipids 80: 133–142) raises the possibility that a similar mechanism may operate in plant cells.

Rearrangement of Actin Microfilaments in Plant Root Hairs Responding to Rhizobium etli Nodulation Signals1

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.

Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells1

Auxin is transported across the plasma membrane of plant cells by diffusion and by two carriers operating in opposite directions, the influx and efflux carriers. Both carriers most likely play an important role in controlling auxin concentration and distribution in plants but little is known regarding their regulation. We describe the influence of modifications of the transmembrane pH gradient and the effect of agents interfering with protein synthesis, protein traffic, and protein phosphorylation on the activity of the auxin carriers in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Carrier-mediated influx and efflux were monitored independently by measuring the accumulation of [14C]2,4-dichlorophenoxyacetic acid and [3H]naphthylacetic acid, respectively. The activity of the influx carrier decreased on increasing external pH and on decreasing internal pH, whereas that of the efflux carrier was only impaired on internal acidification. The efflux carrier activity was inhibited by cycloheximide, brefeldin A, and the protein kinase inhibitors staurosporine and K252a, as shown by the increased capability of treated cells to accumulate [3H]naphthylacetic acid. Kinetics and reversibility of the effect of brefeldin A were consistent with one or several components of the efflux system being turned over at the plasma membrane with a half-time of less than 10 min. Inhibition of efflux by protein kinase inhibitors suggested that protein phosphorylation was essential to sustain the activity of the efflux carrier. On the contrary, the pharmacological agents used in this study failed to inhibit [14C]2,4-dichlorophenoxyacetic acid accumulation, suggesting that rapidly turned-over proteins or proteins activated by phosphorylation are not essential to carrier-mediated auxin influx. Our data support the idea that the efflux carrier in plants constitutes a complex system regulated at multiple levels, in marked contrast with the influx carrier. Physiological implications of the kinetic features of this regulation are discussed.

Fast Induction of High-Affinity HCO3− Transport in Cyanobacteria1

The induction of a high-affinity state of the CO2-concentration mechanism was investigated in two cyanobacterial species, Synechococcus sp. strain PCC7002 and Synechococcus sp. strain PCC7942. Cells grown at high CO2 concentrations were resuspended in low-CO2 buffer and illuminated in the presence of carbonic anhydrase for 4 to 10 min until the inorganic C compensation point was reached. Thereafter, more than 95% of a high-affinity CO2-concentration mechanism was induced in both species. Mass-spectrometric analysis of CO2 and HCO3− fluxes indicated that only the affinity of HCO3− transport increased during the fast-induction period, whereas maximum transport activities were not affected. The kinetic characteristics of CO2 uptake remained unchanged. Fast induction of high-affinity HCO3− transport was not inhibited by chloramphenicol, cantharidin, or okadaic acid. In contrast, fast induction of high-affinity HCO3− transport did not occur in the presence of K252a, staurosporine, or genistein, which are known inhibitors of protein kinases. These results show that induction of high-affinity HCO3− transport can occur within minutes of exposure to low-inorganic-C conditions and that fast induction may involve posttranslational phosphorylation of existing proteins rather than de novo synthesis of new protein components.

Cystic fibrosis transmembrane conductance regulator activation stimulates endosome fusion in vivo.

Previous studies have suggested a role for cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of intracellular vesicular trafficking. A quantitative fluorescence method was used to test the hypothesis that CFTR expression and activation affects endosome-endosome fusion in intact cells. Endosomes from CFTR-expressing and control (vector-transfected) Swiss 3T3 fibroblasts were labeled by internalization with 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene (Bodipy)-avidin, a fluid-phase marker whose fluorescence increases approximately 8-fold upon biotin binding. Cells were washed, chased, and then labeled with biotin-albumin or biotin-transferrin. The fraction of Bodipy-avidin-labeled endosomes that fused with biotin-containing endosomes (f(fusion)) was quantified by ratio imaging microfluorimetry. Endosome fusion in unstimulated CFTR-expressing cells was similar to that in control cells. However, in CFTR-expressing cells activated by forskolin, ffusion was increased by 1.30 +/- 0.18- and 2.65 +/- 0.17-fold for a 0 and 10 min chase time between avidin and biotin-albumin pulses; f(fusion) also increased (1.32 +/- 0.11-fold) when biotin-transferrin replaced biotin-albumin. The stimulation of endosome fusion was not due to differences in rates of endocytosis or endosomal acidification. Endosome fusion was not stimulated by forskolin in Cl--depleted CFTR-expressing cells, suggesting that the increase in endosome fusion is due to the CFTR chloride channel activity. These results provide evidence that CFTR is involved in the regulation of endosome fusion and, thus, a possible basis for the cellular defects associated with cystic fibrosis.

Effects of inefficient cleavage of the signal sequence of HIV-1 gp 120 on its association with calnexin, folding, and intracellular transport.

The HIV-1 envelope glycoprotein gp120 displays inefficient intracellular transport, which is caused by its retention in the endoplasmic reticulum. Coexpression in insect cells (Sf9) of HIV-1 gp120 with calnexin has shown that their interaction was modulated by the signal sequence of HIV-1 gp120. gp120, with its natural signal sequence, showed a prolonged association with calnexin with a t1/2 of greater than 20 min. Replacement of the natural signal sequence with the signal sequence from mellitin led to a decreased time of association of gp120 with calnexin (t1/2 < 10 min). These different times of calnexin association coincided both with the folding of gp120 as measured by the ability of bind CD4 and with endoplasmic reticulum to Golgi transport as analyzed by the acquisition of partial endoglycosidase H resistance. Using a monospecific antibody to the HIV-1 gp120 natural signal peptide, we showed that calnexin associated with N-glycosylated but uncleaved gp120. Only after dissociation from calnexin was gp120 cleaved, but very inefficiently. Only the small proportion of signal-cleaved gp120 molecules acquired transport competence and were secreted. This is the first report demonstrating the effect of the signal sequence on calnexin association.

Factor XII-induced mitogenesis is mediated via a distinct signal transduction pathway that activates a mitogen-activated protein kinase.

Clotting factor XII (Hageman factor) contains epidermal growth factor (EGF)-homologous domains and is reported to be a potent mitogen for human hepatoma (HepG2) cells. In this study, we tested whether factor XII exhibits growth factor activity on several other EGF-sensitive target cells, including fetal hepatocytes, endothelial cells, alveolar type II cells, and aortic smooth muscle cells. We found that factor XII significantly enhanced [3H]thymidine incorporation in aortic smooth muscle cells (SMCs) and all other cells tested. Tyrphostin, a growth factor receptor/tyrosine kinase antagonist, inhibited both EGF- and factor XII-induced responses. However, differences in the levels of magnitude of DNA synthesis, the observed synergism between EGF and factor XII, and the differential sensitivity to tyrphostin suggest that the EGF receptor and the factor XII receptor may be nonidentical. The factor XII-induced mitogenic response was achieved at concentrations that were 1/10th the physiologic range for the circulating factor and was reduced by popcorn inhibitor, a specific factor XII protease inhibitor. Treatment of aortic SMCs with factor XII, as well as activated factor XII, resulted in a rapid and transient activation of a mitogen-activated/extracellular signal-regulated protein kinase with peak activity/tyrosine phosphorylation observed at 5 to 10 min of exposure. Taken together, these data (i) confirm that clotting factor XII functions as a mitogenic growth factor and (ii) demonstrate that factor XII activates a signal transduction pathway, which includes a mitogen-activated protein kinase.

Cooperative interactions among afferents govern the induction of homosynaptic long-term depression in the hippocampus.

Prolonged periods of low-frequency stimulation have been shown to produce a robust, long-term synaptic depression (LTD) in both hippocampus and visual cortex. In the present study we have examined the extent to which interactions among afferents govern the induction of homosynaptic LTD in young-adult rats in hippocampal region CA1 in vitro. Field excitatory postsynaptic potentials were assessed before and after conditioning stimulation consisting of two 10-min trains of low-frequency stimulation (LFS; 1 Hz) of the Schaffer collateral/commissural pathway. LFS at an intensity producing a 0.5-mV response did not produce significant synaptic depression. However, LFS administered at a higher intensity resulted in significant input-specific LTD of a 0.5-mV test response. Picrotoxin, which also facilitates depolarization of CA1 neurons, significantly enhanced the magnitude of LTD after LFS at 0.5 mV. In addition, LFS at 0.5 mV in normal perfusion medium (no picrotoxin) produced only small changes in synaptic efficacy when either of two converging pathways was conditioned separately but produced a robust LTD when both pathways were conditioned simultaneously. This cooperative LTD was reversibly blocked by prior administration of 100 microM DL-aminophosphonovaleric acid but not by 20 microM nimodipine. Taken together, these results suggest that cooperative interactions among afferents contribute to voltage-dependent processes underlying the induction of homosynaptic LTD.

Assembly of regularly spaced nucleosome arrays by Drosophila chromatin assembly factor 1 and a 56-kDa histone-binding protein.

To ascertain the mechanism by which nucleosomes are assembled by factors derived from Drosophila embryos, two proteins termed Drosophila chromatin assembly factors (CAFs) 1 and 4 (dCAF-1 and dCAF-4) were fractionated and purified from a Drosophila embryo extract. The assembly of chromatin by dCAF-1, dCAF-4, purified histones, ATP, and DNA is a process that generates regularly spaced nucleosomal arrays with a repeat length that resembles that of bulk native Drosophila chromatin and is not obligatorily coupled to DNA replication. The assembly of chromatin by dCAF-1 and dCAF-4 is nearly complete within 10 min. The dCAF-1 activity copurified with the Drosophila version of chromatin assembly factor-1 (CAF-1), a factor that has been found to be required for the assembly of chromatin during large tumor (T) antigen-mediated, simian virus 40 (SV40) origin-dependent DNA replication. The dCAF-4 activity copurified with a 56-kDa core-histone-binding protein that was purified to > 90% homogeneity.

Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells.

We investigated the effect of cell shrinkage on whole-cell currents of M-1 mouse cortical collecting duct cells. Addition of 100 mM sucrose to an isotonic NaCl bath solution induced cell shrinkage and increased whole-cell currents within 5-10 min by approximately 12-fold. The effect was reversible upon return to isotonic solution and could also be elicited by adding 100 mM urea or 50 mM NaCl. Replacement of bath Na+ by K+, Cs+, Li+, or Rb+ did not significantly affect the stimulated inward current, but replacement by N-methyl-D-glucamine reduced it by 88.1 +/- 1.3% (n = 34); this demonstrates that hypertonicity activates a nonselective alkali cation conductance. The activation was independent of extra- and intracellular Ca2+, but 1 or 10 mM ATP in the pipette suppressed it in a concentration-dependent manner, indicating that intracellular ATP levels may modulate the degree of channel activation. Flufenamic acid (0.1 mM) and gadolinium (0.1 mM) inhibited the stimulated current by 68.7 +/- 5.9% (n = 9) and 32.4 +/- 11.7% (n = 6), respectively, whereas 0.1 mM amiloride had no significant effect. During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 30 and more individual channels with a single-channel conductance of 26.7 +/- 0.4 pS (n = 29) could be resolved. Thus, we identified the nonselective cation channel underlying the shrinkage-induced whole-cell conductance that may play a role in volume regulation.

Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.

The aquaporins (AQPs) are a family of homologous water-channel proteins that can be inserted into epithelial cell plasma membranes either constitutively (AQP1) or by regulated exocytosis following vasopressin stimulation (AQP2). LLC-PK1 porcine renal epithelial cells were stably transfected with cDNA encoding AQP2 (tagged with a C-terminal c-Myc epitope) or rat kidney AQP1 cDNA in an expression vector containing a cytomegalovirus promoter. Immunofluorescence staining revealed that AQP1 was mainly localized to the plasma membrane, whereas AQP2 was predominantly located on intracellular vesicles. After treatment with vasopressin or forskolin for 10 min, AQP2 was relocated to the plasma membrane, indicating that this relocation was induced by cAMP. The location of AQP1 did not change. The basal water permeability of AQP1-transfected cells was 2-fold greater than that of nontransfected cells, whereas the permeability of AQP2-transfected cells increased significantly only after vasopressin treatment. Endocytotic uptake of fluorescein isothiocyanate-coupled dextran was stimulated 6-fold by vasopressin in AQP2-transfected cells but was only slightly increased in wild-type or AQP1-transfected cells. This vasopressin-induced endocytosis was inhibited in low-K+ medium, which selectively affects clathrin-mediated endocytosis. These water channel-transfected cells represent an in vitro system that will allow the detailed dissection of mechanisms involved in the processing, targeting, and trafficking of proteins via constitutive versus regulated intracellular transport pathways.