Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.

Lobe specific Ca2+-calmodulin nano-domain in neuronal spines: a single molecule level analysis.

Calmodulin (CaM) is a ubiquitous Ca(2+) buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca(2+)-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca(2+)-CaM-dependent enzymes: Ca(2+)/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca(2+) and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca(2+) ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca(2+) and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca(2+) signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca(2+)-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca(2+) channels, and to the microscopic injection rate of Ca(2+) ions. We also demonstrate that Ca(2+) saturation takes place via two different pathways depending on the Ca(2+) injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca(2+) sensors that can differentially transduce Ca(2+) influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca(2+)-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.

Characterization of the human tissue transglutaminase gene promoter

Transglutaminases are a family of calcium-dependent enzymes, that catalyze the covalent cross-linking of proteins by forming $\varepsilon(\gamma$-glutamyl)lysine isopeptide bonds. In order to investigate the molecular mechanisms regulating the expression of the tissue transglutaminase gene and to determine its biological functions, the goal of this research has been to clone and characterize the human tissue transglutaminase promoter. Thirteen clones of the tissue transglutaminase gene were obtained from the screening of a human placental genomic DNA library. A 1.74 Kb fragment derived from DNA located immediately upstream of the translation start site was subcloned and sequenced. Sequence analysis of this DNA fragment revealed that it contains a TATA box (TATAA), a CAAT box (GGACAAT), and a series of potential transcription factor binding sites and hormone response elements. Four regions of significant homology, a GC-rich region, a TG-rich region, an AG-rich region, and HR1, were identified by aligning 1.8 Kb of DNA flanking the human, mouse, and guinea pig tissue transglutaminase genes.^ To measure promoter activity, we subcloned the 1.74 Kb fragment of the tissue transglutaminase gene into a luciferase reporter vector to generate transglutaminase promoter/luciferase reporter constructs. Transfection experiments showed that this DNA segment includes a functional promoter with high constitutive activity. Deletion analysis revealed that the SP1 sites or corresponding sequences contribute to this activity. We investigated the role of DNA methylation in regulating the activity of the promoter and found that in vitro methylation of tissue transglutaminase promoter/luciferase reporter constructs suppressed their basal activity. Methylation of the promoter is inversely correlated with the expression of the tissue transglutaminase gene in vivo. These results suggest that DNA methylation may be one of the mechanisms regulating the expression of the gene. The tumor suppressor gene product p53 was also shown to inhibit the activity of the promoter, suggesting that induction of the tissue transglutaminase gene is not involved in the p53-dependent programmed cell death pathway. Although retinoids regulate the expression of the tissue transglutaminase gene in vivo, retinoid-inducible activity can not be identified in 3.7 Kb of DNA 5$\sp\prime$ to the tissue transglutaminase gene.^ The structure of the 5$\sp\prime$ end of the tissue transglutaminase gene was mapped. Alignment analysis of the human tissue transglutaminase gene with other human transglutaminases showed that tissue transglutaminase is the simplest member of transglutaminase superfamily. Transglutaminase genes show a conserved core of exons and introns but diverse N-terminuses and promoters. These observations suggest that key regulatory sequences and promoter elements have been appended upstream of the core transglutaminase gene to generate the diversity of regulated expression and regulated activity characteristic of the transglutaminase gene family. ^

The dynamics of calmodulin signaling revealed using optical methods

The Ca2+-binding protein calmodulin (CaM) is a key transducer of Ca2+ oscillations by virtue of its ability to bind Ca 2+ selectively and then interact specifically with a large number of downstream enzymes and proteins. It remains unclear whether Ca2+ -dependent signaling alone can activate the full range of Ca 2+/CaM regulated processes or whether other regulatory schemes in the cell exist that allow specific targeting of CaM to subsets of Ca 2+/CaM binding sites or regions of the cell. Here we investigate the possibility that alterations of the availability of CaM may serve as a potential cellular mechanism for regulating the activation of CaM-dependent targets. By utilizing sensitive optical techniques with high spatial and temporal resolution, we examine the intracellular dynamics of CaM signaling at a resolution previously unattainable. After optimizing and characterizing both the optical methods and fluorescently labeled probes for intracellular measurements, the diffusion of CaM in the cytoplasm of HEK293 cells was analyzed. It was discovered that the diffusion characteristics of CaM are similar to that of a comparably sized inert molecule. Independent manipulation of experimental parameters, including increases in total concentrations of CaM and intracellular Ca2+ levels, did not change the diffusion of CaM in the cytoplasm. However, changes in diffusion were seen when the concentration of Ca2+/CaM-binding targets was increased in conjunction with elevated Ca2+. This indicates that CaM is not normally limiting for the activation of Ca 2+/CaM-dependent enzymes in HEK293 cells but reveals that the ratio of CaM to CaM-dependent targets is a potential mechanism for changing CaM availability. Next we considered whether cellular compartmentalization may act to regulate concentrations of available Ca2+/CaM in hippocampal neurons. We discovered changes in diffusion parameters of CaM under elevated Ca2+ conditions in the soma, neurite and nucleus which suggest that either the composition of cytoplasm is different in these compartments and/or they are composed of unique families of CaM-binding proteins. Finally, we return to the HEK293 cell and for the first time directly show the intracellular binding of CaM and CaMKII, an important target for CaM critical for neuronal function and plasticity. Furthermore, we analyzed the complex binding stoichiometry of this molecular interaction in the basal, activated and autophosphorylated states of CaMKII and determined the impact of this binding on CaM availability in the cell. Overall these results demonstrate that regulation of CaM availability is a viable cellular mechanism for regulating the output of CaM-dependent processes and that this process is tuned to the specific functional needs of a particular cell type and subcellular compartment. ^

SUBCELLULAR LOCALIZATION, PURIFICATION AND PROPERTIES OF A CDP-DIACYLGLYCEROL - DEPENDENT PHOSPHATIDYLSERINE SYNTHASE IN BACILLUS LICHENIFORMIS

A CDP-diacylglycerol dependent phosphatidylserine synthase was detected in three species of gram-positive bacilli, viz. Bacillus licheniformis, Bacillus subtilis and Bacillus megaterium; the enzyme in B. licheniformis was studied in detail. The subcellular distribution experiments in cell-free extracts of B. licheniformis using differential centrifugation, sucrose gradient centrifugation and detergent solubilization showed the phosphatidylserine synthase to be tightly associated with the membrane. The enzyme was shown to have an absolute requirement for divalent metal ion for activity with a strong preference for manganese. The enzyme activity was completely dependent upon the addition of CDP-diacylglycerol to the assay system; the role of the liponucleotide was rigorously shown to be that of phosphatidyl donor and not just a detergent-like stimulator. This enzyme was then solubilized from B. licheniformis membranes and purified to near homogeneity. The purification procedure consisted of CDP-diacylglycerol-Sepharose affinity chromatography followed by substrate elution from blue-dextran Sepharose. The purified preparation showed a single band with an apparent minimum molecular weight of 53,000 when subjected to SDS polyacrylamide gel electrophoresis. The preparation was free of any phosphatidylglycerophosphate synthase, CDP-diacylglycerol hydrolase and phosphatidylserine hydrolase activities. The utilization of substrates and formation of products occurred with the expected stoichiometry. Radioisotopic exchange patterns between related substrate and product pairs suggest a sequential BiBi reaction as opposed to the ping-pong mechanism exhibited by the well studied phosphatidylserine synthase of Escherichia coli. Proteolytic digestion of the enzyme yielded a smaller active form of the enzyme (41,000 daltons) which appears to be less prone to aggregation.^ This has been the first detailed study in a well-defined bacillus species of the enzyme catalyzing the CDP-diacylglycerol-dependent formation of phosphatidylserine; this reaction is the first committed step in the biosynthetic pathway to the major membrane component, phosphatidylethanolamine. Further study of this enzyme may lead to understanding of new mechanisms of phosphatidyl transfer and novel modes of control of phospholipid biosynthetic enzymes. ^

Novel mechanisms for PKC family enzymes to regulate PI3K signaling pathway

Phosphatidylinositol 3-kinase (PI3K) generates membrane phospholipids that serve as second messengers to recruit signaling proteins to plasma membrane consequently regulating cell growth and survival. PI3K is a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit. Association of the p85 with other signal proteins is critical for induced PI3K activation. Activated PI3K, in turn, leads to signal flows through a variety of PI3K effectors including PDK1, AKT, GSK3, BAD, p70 S6K and NFκB. The PI3K pathway is under regulation by multiple signal proteins representing cross-talk between different signaling cascades. In this study, we have evaluated the role of protein kinase C family kinases on signaling through PI3K at multiple levels. Firstly, we observed that the action of PKC specific inhibitors like Ro-31-8220 and GF109203X was associated with an increased AKT phosphorylation and activity, suggesting that PKC kinases might play a negative role in the regulation of PI3K pathway. Then, we demonstrated the stimulation of AKT by PKC inhibition was dependent on functional PI3K enzyme and able to be transmitted to the AKT effector p70 S6K. Furthermore, we showed an inducible physical association between the PKCζ isotype and AKT, which was accompanied by an attenuated AKT activity. However, a kinase-dead form of PKC failed to affect AKT. In the second part of our research we revealed the ability of a different PKC family member, PKCδ to bind to the p85 subunit of PI3K in response to oxidative stress, a process requiring the activity of src tyrosine kinases. The interaction was demonstrated to be a direct and specific contact between the carboxyl terminal SH2 domain of p85 and tyrosine phosphorylated PKCδ. Several different types of agonists were capable to induce this association including tyrosine kinases and phorbol esters with PKCδ tyrosine phosphorylation being integral components. Finally, the PKCδ-PI3K complex was related to a reduction in the AKT phosphorylation induced by src. A kinase-deficient mutant of PKCδ was equally able to inhibit AKT signal as the wild type, indicative of a process independent of PKCδ catalytic activity. Altogether, our data illustrate different PKC isoforms regulating PI3K pathway at multiple levels, suggesting a mechanism to control signal flows through PI3K for normal cell activities. Although further investigation is required for full understanding of the regulatory mechanism, we propose that complex formation of signal proteins in PI3K pathway and specific PKC isoforms plays important role in their functional linkage. ^