The myristoylation of guanylate cyclase-activating protein-2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca²⁺

Guanylate cyclase activating protein‐2 (GCAP‐2) is a Ca²⁺‐binding protein of the neuronal calcium sensor (NCS) family. Ca²⁺‐free GCAP‐2 activates the retinal rod outer segment guanylate cyclases ROS‐GC1 and 2. Native GCAP‐2 is N‐terminally myristoylated. Detailed structural information on the Ca²⁺‐dependent conformational switch of GCAP‐2 is missing so far, as no atomic resolution structures of the Ca²⁺‐free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca²⁺‐myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP‐1, a Ca²⁺‐independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non‐myristoylated GCAP‐2 in their Ca²⁺‐bound and Ca²⁺‐free forms, respectively, to gain information on the nature of the Ca²⁺‐dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca²⁺, the stability of the myristoylated and non‐myristoylated forms was indistinguishable. Ca²⁺ exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP‐2. The stability data were corroborated by dye binding experiments performed to probe the solvent‐accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent‐exposed in Ca²⁺‐free GCAP‐2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca²⁺‐bound state.     

Influence of W-7, a calmodulin antagonist on phospholipid biosynthesis in Candida albicans

AIM: This study was undertaken to investigate the role of calmodulin in phospholipid biosynthesis in Candida albicans using W-7, a calmodulin antagonist. METHODS: Cells were grown as shake cultures in the absence and presence of W-7 at different concentrations. Changes in cell mass, phospholipid content and incorporation of labelled precursor into phospholipid and activities of respective enzymes have been studied. RESULTS: Decreased incorporation of labelled acetate into total lipids and phospholipids was observed in the presence of 40 microm of W-7 which was not as a consequence of altered growth of Candida in the presence of calmodulin antagonist. Further, a significant decrease in the levels of calmodulin and CaM dependent protein kinase activity was observed in cells grown with different concentrations of W-7. This was accompanied by decreased/increased activity of phosphatidic acid phosphatase and phospholipase A, respectively in W-7 grown cells as compared to controls. CONCLUSIONS: These findings suggest definite involvement of calmodulin in the regulation of phospholipid metabolism in Candida albicans.     

SERCA mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation

The sarco(endo)plasmic reticulum Ca²⁺‐ATPase (SERCA) couples ATP hydrolysis to transport of Ca²⁺. This directed energy transfer requires cross‐talk between the two Ca²⁺ sites and the phosphorylation site over 50 Å distance. We have addressed the mechano‐structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu³⁰⁹ contributes to Ca²⁺ coordination at site II, and a consensus has been that E309Q only binds Ca²⁺ at site I. The crystal structure of E309Q in the presence of Ca²⁺ and an ATP analogue, however, reveals two occupied Ca²⁺ sites of a non‐catalytic Ca₂E1 state. Ca²⁺ is bound with micromolar affinity by both Ca²⁺ sites in E309Q, but without cooperativity. The Ca²⁺‐bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A‐domain, requiring a shift of transmembrane segment M1 into an ‘up and kinked position’. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca²⁺ site II.     

Kinetics of Protein Phosphorylation in Microvessels Isolated from Rat Brain: Modulation by Second Messengers

The role of second messengers in the regulation of protein phosphorylation was studied in microvessels isolated from rat cerebral cortex. The phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the kinetics of 32P incorporation into specific protein substrates were evaluated by computer-aided x-ray film densitometry. With the use of this method, Ca2+-calmodulin (CAM)-, Ca2+/phospholipid (PK C)-, cyclic GMP (cGMP)-, and cyclic AMP (cAMP)-dependent protein kinases were detected. CAM-dependent protein kinase proved to be the major phosphorylating enzyme in the microvascular fraction of the rat cerebral cortex; the activity of cGMP-dependent protein kinase was much higher than that of the cAMP-dependent one. Autophosphorylation of both the alpha- and beta-subunits of CAM-dependent protein kinase and the proteolytic fragment of the PK C enzyme was also detected. The kinetics of phosphorylation of the individual polypeptides indicate the presence in the cerebral endothelium of phosphoprotein phosphatases. The phosphorylation of proteins in the cerebral capillaries was more or less reversible; the addition of second messengers initiated a very rapid increase in 32P incorporation, followed by a slow decrease. Because the intracellular signal transducers like Ca2+ and cyclic nucleotides are frequently regulated by different vasoactive substances in the endothelial cells, the modified phosphorylation evoked by these second messengers may be related in vivo to certain changes in the transport processes of the blood-brain barrier.     

Organ-specific distribution of the calcium sensor CaMKII in Locusta migratoria

The Ca(2+)/calmodulin-dependent kinase CaMKII is a key signaling component in Ca(2+)-dependent physiological processes. The expression and function of CaMKII in insect brain is well documented but less investigated for other tissues of insects. The present study demonstrates that in the locust Locusta migratoria CaMKII is widely expressed in various tissues. Relatively high expression levels of CaMKII were found in the brain, upper part of the digestive tract (pharynx, esophagus), and the flight and leg muscles. The different expression patterns of CaMKII in various tissues, as well as different molecular masses of CaMKII between 48 and 60 kDa indicate a tissue-specific expression of CaMKII variants. The expression was monitored with a polyclonal anti-(rat)CaMKII antibody. About 60% of total CaMKII activity in flight muscle cells is associated to the myofibril-rich, particulate fraction suggesting an important role of CaMKII in sarcomeric function.     

Ca2+-induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle

The temporal relationship between Ca2+-induced contraction and phosphorylation of 20 kDa myosin light chain (MLC) during a step increase in Ca2+ was investigated using permeabilized phasic smooth muscle from rabbit portal vein and guinea-pig ileum at 25°C. We describe here a Ca2+-induced Ca2+ desensitization phenomenon in which a transient rise in MLC phosphorylation is followed by a transient rise in contractile force. During and after the peak contraction, the force to phosphorylation ratio remained constant. Further treatment with cytochalasin D, an actin fragmenting agent, did not affect the transient increase in phosphorylation, but blocked force development. Together, these results indicate that the transient phosphorylation causes the transient contraction and that neither inhomogeneous contractility nor reduced thin filament integrity effects the transient phosphorylation. Lastly, we show that known inhibitors to MLC kinase kinases and to a Ca2+-dependent protein phosphatase did not eliminate the desensitized contractile force. This study suggests that the Ca2+-induced Ca2+ desensitization phenomenon in phasic smooth muscle does not result from any of the known intrinsic mechanisms involved with other aspects of smooth muscle contractility.     

植物感受盐胁迫及相关钙信号的研究进展

盐胁迫对植物的生长和发育造成严重影响,其危害包括渗透胁迫、离子毒害等,严重损害了农业生产和粮食安全。在盐胁迫下,植物相关感受器接受刺激,使得Ca²⁺通过细胞膜以及细胞内钙库膜上打开的Ca²⁺通道进入细胞质基质,导致细胞质内Ca²⁺浓度升高,产生钙信号。钙离子作为重要的第二信使,在植物细胞内和细胞间传递信号,信号往下游传递,在不同生长和发育阶段引起植物一系列的生理响应来应对盐胁迫影响。钙信号主要通过钙调蛋白（CaM）、钙调素样蛋白（CML）、钙依赖性蛋白激酶（CDPK）、钙调磷酸酶B样蛋白（CBL）和CBL互作蛋白激酶（CIPK）感知并将特异的钙信号信息传递到下游;从而激活植物盐胁迫生理响应。本文主要综述植物如何感知盐胁迫刺激,以及钙信号产生与传导机制,并对该研究领域需解决的问题进行了展望。     

Bidirectional changes in synapsin I phosphorylation at MAP kinase-dependent sites by acute neuronal excitation in vivo

Synapsin I is a synaptic vesicle-associated protein which is phosphorylated at multiple sites by various kinases. It has been proposed to play a role in the regulation of neurotransmitter release and the organization of cytoskeletal architecture in the presynaptic terminal. To better understand the physiological regulation of its phosphorylation in vivo, we induced acute, reversible neuronal excitation by electroconvulsive treatment (ECT) in rats, and studied its effects on synapsin I phosphorylation at sites 3, 4/5 and 6 by immunoblot analyses of homogenates from hippocampus and parietal cortex using phospho-site-specific antibodies. A decrease in phosphorylation at all sites was observed soon after the electrical stimulation, followed by a large increase in phosphorylation at site 4/5 peaking at 5 min and a moderate increase in phosphorylation at site 6 peaking at 20 min. Systemic injection of SL327, a mitogen-activated protein kinase (MAPK) kinase inhibitor, prior to ECT, suppressed the increase in phospho-site 4/5 level, as well as that in MAPK activity, but not that in phospho-site 6 level. Thus, phosphorylation at site 4/5 of synapsin I has been shown to be regulated by MAPK in vivo.     

Ca2+-Dependent and Ca2+-Independent Protein Kinase C Changes in the Brains of Patients with Alzheimer's Disease

Abstract: We examined protein kinase C (PKC) activity in Ca²⁺-dependent PKC (Ca²⁺-dependent PKC activities) and Ca²⁺-independent PKC (Ca²⁺-independent PKC activities) assay conditions in brains from Alzheimer's disease (AD) patients and age-matched controls. In cytosolic and membranous fractions, Ca²⁺-dependent and Ca²⁺-independent PKC activities were significantly lower in AD brain than in control brain. In particular, reduction of Ca²⁺-independent PKC activity in the membranous fraction of AD brain was most enhanced when cardiolipin, the optimal stimulator of PKC-ε, was used in the assay; whereas Ca²⁺-independent PKC activity stimulated by phosphatidylinositol, the optimal stimulator of PKC-δ, was not significantly reduced in AD. Further studies on the protein levels of Ca²⁺-independent PKC-δ, PKC-ε, and PKC-ζ in AD brain revealed reduction of the PKC-ε level in both cytosolic and membranous fractions, although PKC-δ and PKC-ζ levels were not changed. These findings indicated that Ca²⁺-dependent and Ca²⁺-independent PKC are changed in AD, and that among Ca²⁺-independent PKC isozymes, the alteration of PKC-ε is a specific event in AD brain, suggesting its crucial role in AD pathophysiology.