Triadins modulate intracellular Ca(2+) homeostasis but are not essential for excitation-contraction coupling in skeletal muscle

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca(2+) imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca(2+) transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca(2+).[(3)H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca(2+) activation or Ca(2+) and Mg(2+) inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca(2+) homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.     

Swelling of mitochondria in cultured rat hippocampal astrocytes is induced by high cytosolic Ca(2+) load,but not by mitochondrial depolarization

The influence of cytosolic Ca(2+) load and of mitochondrial membrane potential change on mitochondrial morphology was investigated in cultured rat hippocampal astrocytes. The uncoupler FCCP, applied together with oligomycin, depolarized mitochondria rapidly but did not change their morphology. Depolarization was associated with a moderate cytosolic [Ca(2+)](i) rise of up to 0.3 microM. Only high cytosolic Ca(2+) load (above a threshold of 50 microM), which was evoked by application of the ionophore 4-Br-A23187 in Ca(2+)-containing medium, caused drastic change of mitochondrial morphology. The shape change from the typical rod-like to a spherical shape, indicating mitochondrial swelling, was associated with depolarization. Cyclosporin A sensitivity suggests involvement of permeability transition. Thus, a dramatic cytosolic [Ca(2+)](i) rise is required to induce mitochondrial swelling and depolarization. A large but still moderate [Ca(2+)](i) rise evoked by physiological stimulation, however, has no comparable effect.     

Actin-dependent chloroplast anchoring is regulated by Ca(2+)-calmodulin in spinach mesophyll cells

Chloroplasts are actively anchored at the appropriate intracellular regions to maintain advantageous distribution patterns under specific environmental conditions. Redistribution of chloroplasts is accompanied by their de-anchoring and re-anchoring, respectively, from and to the cortical cytoplasm. In spinach mesophyll cells, high-intensity blue light and Ca(2+) treatment induced the disappearance of the meshwork-like array of actin filaments surrounding chloroplasts, which was suppressed by a calmodulin antagonist. Regulatory mechanisms of chloroplast anchoring were investigated using plasma membrane (PM) ghosts, on which the cortical cytoplasm underlying the PM was exposed. Addition of an actin-depolymerizing reagent or > 1 μM Ca(2+) induced detachment of a substantial number of chloroplasts from the PM ghosts concomitant with disordered actin organization. Calmodulin antagonists and anti-calmodulin antibodies negated the effects of Ca(2+). In addition, Ca(2+)-induced detachment of chloroplasts was no longer evident on the calmodulin-depleted PM ghosts. We propose that chloroplasts are anchored onto the cortical cytoplasm through interaction with the actin cytoskeleton, and that Ca(2+)-calmodulin-sensitized de-anchoring of chloroplasts is a critical early step in chloroplast redistribution induced by environmental stimuli.     

Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state

The bidentate complex of ATP with Cr(3+), CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca(2+)-ATPase and the Na(+),K(+)-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca(2+) and Na(+), respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca(2+)-ATPase. The complex inhibited with similar efficiency the Ca(2+)-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T(1/2)=30 min at 37 degrees C) with a K(i)=28+/-9 microM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg(2+) but unaltered when Ca(2+) was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca(2+) occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La(3+) with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca(2+) at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca(2+) promoted by the plasma membrane Ca(2+)-ATPase goes through an enzymatic phospho-intermediate that maintains Ca(2+) ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.     

Nucleoplasmic Ca(2+)loading is regulated by mobilization of perinuclear Ca(2+)

Regulation of nucleoplasmic calcium (Ca(2+)) concentration may occur by the mobilization of perinuclear luminal Ca(2+)pools involving specific Ca(2+)pumps and channels of both inner and outer perinuclear membranes. To determine the role of perinuclear luminal Ca(2+), we examined freshly cultured 10 day-old embryonic chick ventricular cardiomyocytes. We obtained evidence suggesting the existence of the molecular machinery required for the bi-directional Ca(2+)fluxes using confocal imaging techniques. Embryonic cardiomyocytes were probed with antibodies specific for ryanodine-sensitive Ca(2+)channels (RyR2), sarco/endoplasmic reticulum Ca(2+)ATPase (SERCA2)-pumps, and fluorescent BODIPY derivatives of ryanodine and thapsigargin. Using immunocytochemistry techniques, confocal imaging showed the presence of RyR2 Ca(2+)channels and SERCA2-pumps highly localized to regions surrounding the nucleus, referable to the nuclear envelope. Results obtained from Fluo-3, AM loaded ionomycin-perforated embryonic cardiomyocytes demonstrated that gradual increases of extranuclear Ca(2+)from 100 to 1600 nM Ca(2+)was localized to the nucleus. SERCA2-pump inhibitors thapsigargin and cyclopiazonic acid showed a concentration-dependent inhibition of nuclear Ca(2+)loading. Furthermore, ryanodine demonstrated a biphasic concentration-dependence upon active nuclear Ca(2+)loading. The concomitant addition of thapsigargin or cyclopiazonic acid with ryanodine at inhibitory concentrations caused an significant increase in nuclear Ca(2+)loading at low concentrations of extranuclear added Ca(2+). Our results show that the perinuclear lumen in embryonic chick ventricular cardiomyocytes is capable of autonomously regulating nucleoplasmic Ca(2+)fluxes.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Mitochondria fine-tune the slow Ca(2+) transients induced by electrical stimulation of skeletal myotubes

Mitochondria sense cytoplasmic Ca(2+) signals in many cell types. In mammalian skeletal myotubes, depolarizing stimuli induce two independent cytoplasmic Ca(2+) signals: a fast signal associated with contraction and a slow signal that propagates to the nucleus and regulates gene expression. How mitochondria sense and possibly affect these cytoplasmic Ca(2+) signals has not been reported. We investigated here (a) the emergence of mitochondrial Ca(2+) signals in response to electrical stimulation of myotubes, (b) the contribution of mitochondrial Ca(2+) transients to ATP generation and (c) the influence of mitochondria as modulators of cytoplasmic and nuclear Ca(2+) signals. Rhod2 and Fluo3 fluorescence determinations revealed composite Ca(2+) signals associated to the mitochondrial compartment in electrically stimulated (400 pulses, 45 Hz) skeletal myotubes. Similar Ca(2+) signals were detected when using a mitochondria-targeted pericam. The fast mitochondrial Ca(2+) rise induced by stimulation was inhibited by pre-incubation with ryanodine, whereas the phospholipase C inhibitor U73122 blocked the slow mitochondrial Ca(2+) signal, showing that mitochondria sense the two cytoplasmic Ca(2+) signal components. The fast but not the slow Ca(2+) transient enhanced mitochondrial ATP production. Inhibition of the mitochondrial Ca(2+) uniporter prevented the emergence of mitochondrial Ca(2+) transients and significantly increased the magnitude of slow cytoplasmic Ca(2+) signals after stimulation. Precluding mitochondrial Ca(2+) extrusion with the Na(+)/Ca(2+) exchanger inhibitor CGP37157 decreased mitochondrial potential, increased the magnitude of the slow cytoplasmic Ca(2+) signal and decreased the rate of Ca(2+) signal propagation from one nucleus to the next. Over expression of the mitochondrial fission protein Drp-1 decreased mitochondrial size and the slow Ca(2+) transient in mitochondria, but enhanced cytoplasmic and nuclear slow transients. The present results indicate that mitochondria play a central role in the regulation of Ca(2+) signals involved in gene expression in myotubes.     

Adrenergic stimulation of rat resistance arteries affects Ca(2+) sparks, Ca(2+) waves, and Ca(2+) oscillations

Confocal laser scanning microscopy and fluo 4 were used to visualize local and whole cell Ca(2+) transients within individual smooth muscle cells (SMC) of intact, pressurized rat mesenteric small arteries during activation of alpha1-adrenoceptors. A method was developed to record the Ca(2+) transients within individual SMC during the changes in arterial diameter. Three distinct types of "Ca(2+) signals" were influenced by adrenergic activation (agonist: phenylephrine). First, asynchronous Ca(2+) transients were elicited by low levels of adrenergic stimulation. These propagated from a point of origin and then filled the cell. Second, synchronous, spatially uniform Ca(2+) transients, not reported previously, occurred at higher levels of adrenergic stimulation and continued for long periods during oscillatory vasomotion. Finally, Ca(2+) sparks slowly decreased in frequency of occurrence during exposure to adrenergic agonists. Thus adrenergic activation causes a decrease in the frequency of Ca(2+) sparks and an increase in the frequency of asynchronous wavelike Ca(2+) transients, both of which should tend to decrease arterial diameter. Oscillatory vasomotion is associated with spatially uniform synchronous oscillations of cellular [Ca(2+)] and may have a different mechanism than the asynchronous, propagating Ca(2+) transients.     

Residence of adenylyl cyclase type 8 in caveolae is necessary but not sufficient for regulation by capacitative Ca(2+) entry.

Ca(2+)-sensitive adenylyl cyclases (ACs) depend on capacitative Ca(2+) entry (CCE) for their regulation. Residence of the endogenous Ca(2+)-inhibitable adenylyl cyclase of C6-2B glioma cells in cholesterol-enriched caveolae is essential for its regulation by CCE (Fagan, K. A., Smith, K. E., and Cooper, D. M. F. (2000) J. Biol. Chem. 275, 26530-26537). In the present study, we established that depletion of cellular cholesterol ablated the regulation by CCE of a Ca(2+)-stimulable adenylyl cyclase, AC8, heterologously expressed in HEK293 cells. We considered the possibility that a calmodulin-binding domain in the N terminus of AC8, which is not required for in vitro regulation by Ca(2+), might play a targeting role. Deletion and mutation of the N terminus did attenuate the enzyme's sensitivity to CCE without altering its in vitro responsiveness to Ca(2+)/calmodulin. Both N terminus-deleted AC8 and wild type AC8 were expressed at the plasma membrane, as shown by imaging analysis of green fluorescence protein-tagged constructs. However, not only wild type AC8 but also the CCE-insensitive mutants occurred in caveolar fractions of the plasma membranes, even though a Ca(2+)-insensitive adenylyl cyclase, AC7, was excluded from caveolae. Finally, the AC8 mutants were no more responsive to nonphysiological elevation of Ca(2+) than the wild type. We conclude that (i) not all adenylyl cyclases reside in caveolae, (ii) the calmodulin-binding domain in the N terminus of AC8 does not play a role in caveolar targeting, (iii) the N terminus does play a role in associating AC8 with factors that confer sensitivity to CCE, and (iv) residence of Ca(2+)-sensitive adenylyl cyclases in caveolae is essential but not sufficient for regulation by CCE.     

Mechanism of intracellular Ca(2+)-wave propagation elicited by mechanical stimulation in cultured endothelial CPAE cells

Intra- and intercellular Ca(2+)-signaling during mechanical stimulation in calf pulmonary artery endothelial cells (CPAE) was investigated with digital fluorescence microscopy. Mechanical stimulation of a CPAE cell in a Ca(2+)-containing solution revealed a rise of the free intracellular Ca(2+)-concentration ([Ca(2+)](i)) in the mechanically stimulated cell (MS) proceeding to the neighboring (NB) cells as an intercellular Ca(2+)-wave. Experiments in Ca(2+)-free solution, containing 2mM EGTA, demonstrated that a detectable [Ca(2+)](i)-transient in the MS cell is not always a requisite for intercellular communication (IC). The Ca(2+)-wave propagation was not affected by changes in membrane potential and was not mediated by voltage-dependent Ca(2+)-channels. Ca(2+)-influx through the Ni(2+)-sensitive Ca(2+)-pathway occurred in the MS as could be assessed by Mn(2+)-quenching experiments. The intra- and intercellular Ca(2+)-wave was triggered by the release of thapsigargin-sensitive intracellular Ca(2+)-stores. Phospholipase C (PLC) inhibition by U73122 reduced the Ca(2+)-amplitude of the MS cell and almost completely inhibited the IC, indicating that the Ca(2+)-release in the MS and NB cells is PLC/inositol 1,4,5-trisphosphate (IP(3)) mediated.     

The C2B domain of synaptotagmin is a Ca(2+)-sensing module essential for exocytosis

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca(2+) ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca(2+)-sensing module. Here, we report that Ca(2+) drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca(2)+ are mediated by a set of conserved acidic Ca(2)+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca(2)+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.     

Properties of intra- and intercellular Ca(2+)-wave propagation elicited by mechanical stimulation in cultured RPE cells

Membrane deformation induced by a mechanical stimulus increases the [Ca2+]i in cultured retinal pigment epithelial (RPE) cells, and in many other cell types. In this study, confocal microscopy and Ca(2+)-measurements using the fluorescent dye fluo-3 were used to measure the spatiotemporal characteristics of the Ca(2+)-wave propagation during a mechanical stimulation in Long Evans (LE) RPE cells or dystrophic Royal College of Surgeons (RCS) RPE cells. Ca2+ signals were recorded in the mechanically stimulated cell and in the neighboring cells. A regenerative Ca(2+)-wave with a decreasing rate of propagation was found in the stimulated cells. The rate of propagation was significantly slower in RCS-RPE cells compared to LE-RPE cells. Incubation with thapsigargin significantly lowered the propagation rate in both LE- and RCS-RPE cells. The amplitude of the [Ca2+]i-rise in the nucleus and cytoplasm was differentially modulated by protein kinase C in RCS-RPE cells, but not in LE-RPE cells. It is concluded that RCS-RPE cells have intracellular Ca(2+)-regulating properties which are different from those of LE-RPE cells.     

Requirement of pyk2 for the activation of the MAP kinase cascade induced by Ca(2+) (but not by PKC or G protein) in PC12 cells

The role of the Ca(2+)-activated tyrosine kinase, Pyk2, in the pleiotropic coupling of nerve cell stimulation to the MAP kinase cascade still remains undefined. Using a panel of PC12 clones, one of which was defective in Pyk2, we demonstrate (1) that the MAP kinase response induced by a [Ca(2+)](i) rise (following application of the Ca(2+) ionophore, ionomycin) is inappreciable in the defective clone and is re-established after Pyk2 transfection; and (2) that the responses to both protein kinase C and P(2y2) receptor activation occur normally even in the defective cells. We conclude that Pyk2 is the key mediator in the pathway activated by Ca(2+) but has minor roles with the other types of stimulation.     

L-Type Ca(2+) channel charge movement and intracellular Ca(2+) in skeletal muscle fibers from aging mice

In this work we tested the hypothesis that skeletal muscle fibers from aging mice exhibit a significant decline in myoplasmic Ca(2+) concentration resulting from a reduction in L-type Ca(2+) channel (dihydropyridine receptor, DHPR) charge movement. Skeletal muscle fibers from the flexor digitorum brevis (FDB) muscle were obtained from 5-7-, 14-18-, or 21-24-month-old FVB mice and voltage-clamped in the whole-cell configuration of the patch-clamp technique according to described procedures (Wang, Z.-M., M. L. Messi, and O. Delbono. 1999. Biophys. J. 77:2709-2716). Total charge movement or the DHPR charge movement was measured simultaneously with intracellular Ca(2+) concentration. The maximum charge movement (Q(max)) recorded (mean +/- SEM, in nC microF(-1)) was 53 +/- 3.2 (n = 47), 51 +/- 3.2 (n = 35) (non-significant, ns), and 33 +/- 1.9 (n = 32) (p < 0.01), for the three age groups, respectively. Q(max) corresponding to the DHPR was 43 +/- 3.3, 38 +/- 4.1 (ns), and 25 +/- 3.4 (p < 0.01) for the three age groups, respectively. The peak intracellular [Ca(2+)] recorded at 40 mV (in microM) was 15.7 +/- 0. 12, 16.7 +/- 0.18 (ns), and 8.2 +/- 0.07 (p < 0.01) for the three age groups, respectively. No significant changes in the voltage distribution or steepness of the Q-V or [Ca(2+)]-V relationship were found. These data support the concept that the reduction in the peak intracellular [Ca(2+)] results from a larger number of ryanodine receptors uncoupled to DHPRs in skeletal muscle fibers from aging mammals.     

Intracellular Ca(2+) oscillations induced by over-expressed Ca(V)3.1 T-type Ca(2+) channels in NG108-15 cells

T-type Ca²⁺ channel family includes three subunits Ca_V3.1, Ca_V3.2 and Ca_V3.3 and have been shown to control burst firing and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Here, we investigated whether Ca_V3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca_V3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca_V3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca²⁺]_i oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around −61 mV. [Ca²⁺]_i oscillations were critically dependent on Ca²⁺ influx through Ca_V3.1 channels and did not involve Ca²⁺ release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca_V3.1 channels. The trigger of the oscillations was the Ca_V3.1 window current. This current induced continuous [Ca²⁺]_i increase at −60 mV that depolarized the cells and triggered MPOs. Shifting the Ca_V3.1 window current potential range by increasing the external Ca²⁺ concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I_h) was not required to induce MPOs, but when expressed together with Ca_V3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca_V3.1 window current is critical in triggering intrinsic electrical and [Ca²⁺]_i oscillations.     

Neuronal galvanotropism is independent of external Ca(2+) entry or internal Ca(2+) gradients

The mechanism by which growing neurites sense and respond to small applied electrical fields is not known, but there is some evidence that the entry of Ca(2+) from the external medium, with the subsequent formation of intracellular Ca(2+) gradients, is important in this process. We have employed two approaches to test this idea. Xenopus spinal neurites were exposed to electrical fields in a culture medium in which Ca(2+) was chelated to very low levels compared to the normal extracellular concentration of 2 mM. In other experiments, loading the neurites with the calcium buffer, 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), disrupted the putative internal Ca(2+) gradients, and the effects on the electrical response were determined. Fields of 100 mV/mm were applied for 12 h, and no difference was detected in the cathodal turning response between the treated neurites and the untreated controls. Using the Differential Growth Index (DGI), an asymmetry index, to quantitate the turning response, we recorded DGIs of -0.64, -0.65, and -0.62 for control cells, cells in Ca(2+)-free medium, and cells preloaded with BAPTA, respectively. Furthermore, we detected an increase in neurite length for those neurons cultured in Ca(2+)-free medium; they were 1.5-1.7 times as long as neurites from neurons cultured in normal Ca(2+) medium. Likewise, we found that BAPTA-loaded neurites were longer than control neurites. Our data indicate that neuronal galvanotropism is independent of the entry of external Ca(2+) or of internal Ca(2+) gradients. Both cell-permeant agonistic and antagonistic analogs of cyclic 3',5'-adenosine monophosphate (cAMP) increased the response to applied electrical fields.     

UmuC function is not essential for the production of all targeted lacI mutations induced by ultraviolet light

Up to a quarter or more of the normal yield of lacI- mutations could be induced by ultraviolet light in a uvrA6 umuC122:: Tn5 strain if they were detected by plating on 5-bromo-4-chloro-3-indolyl-beta-D-galactoside medium, where all surviving cells can form colonies. Using phenyl beta-D-galactoside selection, which curtails post-irradiation growth, only low yields of mutations were induced. Nucleotide sequence analysis of 134 spontaneous and 145 ultraviolet light-induced mutations shows that broadly similar kinds of mutations were induced in the umuC mutant and its uvrA6 umuC+ counterpart. In particular, these data offer no reason for believing that most of the mutations induced in the umuC mutant were other than normal, targeted events. We conclude that UmuC function, rather than being essential, facilitates recovery and specifically, following the model of Bridges & Woodgate, that it facilitates the prompt resumption of chain elongation.     

Plasma membrane Ca(2+)-pump ATPase is not a substrate for cGMP-dependent protein kinase.

A plasma membrane Ca(2+)-pump ATPase preparation purified from porcine aorta was incubated with cGMP-dependent protein kinase (G-kinase) under the conditions under which dose-dependent stimulation of the enzyme by G-kinase was observed. Several proteins were phosphorylated, but two isoforms of plasma membrane Ca(2+)-pump ATPase with molecular masses of 135- and 145-kDa were not phosphorylated. The protein that was phosphorylated by G-kinase and identified in our previous study as the 135-kDa isoform of Ca(2+)-pump ATPase, on the basis of its almost identical mobility on SDS-PAGE, was found to be another protein with a molecular mass of 138 kDa. Fractionation of the enzyme preparation after incubation with G-kinase by a newly developed calmodulin affinity chromatographic method resulted in the separation of all the G-kinase substrates from the two isoforms of plasma membrane Ca(2+)-pump ATPase. These results suggest that the direct phosphorylation of the Ca(2+)-pump ATPase does not occur in association with the stimulation of the plasma membrane Ca(2+)-pump ATPase by G-kinase.     

Bak but not Bax is essential for Bcl-xS-induced apoptosis

Bcl-x(S), a proapoptotic member of the Bcl-2 protein family, is localized in the mitochondria and induces apoptosis in a caspase- and BH3-dependent manner by a mechanism involving cytochrome c release. The way in which Bcl-x(S) induces caspase activation and cytochrome c release, as well as the relationship between Bcl-x(S) and other proapoptotic members of the Bcl-2 family, is not known. Here we used embryonic fibroblasts derived from mice deficient in the multidomain proapoptotic members of the Bcl-2 family (Bax and Bak) and the apoptotic components of the apoptosome (Apaf-1 and caspase-9) to unravel the cascade of events by which Bcl-x(S) promotes apoptosis. Our results show that Bak but not Bax is essential for Bcl-x(S)-induced apoptosis. Bcl-x(S) induced activation of Bak, which in turn promoted apoptosis by apoptosome-dependent and -independent pathways. These findings provide the first evidence that a proapoptotic Bcl-2 family protein induces apoptosis exclusively via Bak.