Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases

Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.     

Maillard reaction-like lysine modification by a lipid peroxidation product: immunochemical detection of protein-bound 2-hydroxyheptanal in vivo

2-Hydroxyheptanal (2-HH) is one of the reactive aldehyde species generated during the peroxidation of n-6 polyunsaturated fatty acids, such as linoleic and arachidonic acids. Analogous to the Maillard reaction of reducing sugars, 2-HH readily reacts with lysine epsilon-amino groups. In the present study, to define the occurrence of the Maillard reaction-like lysine modification by 2-HH in vivo, we raised a monoclonal antibody directed to a trihydropyridinone (THPO) structure, 1-alkyl-4-butyl-5-pentyl-1,2,6-trihydropyridin-3-one, formed from 2-HH and lysine, and examined the presence of the antigenic structure in the human atherosclerotic aorta. Mice were immunized with the 2-HH-modified keyhole limpet hemocyanin (KLH) as the immunogen. Using a THPO-carrier protein conjugate, we screened the hybridomas and finally obtained a clone that produced the monoclonal antibody 3C8 (mAb3C8). The antibody strongly recognized bovine serum albumin (BSA) treated with 2-HH, but showed no cross-reactivity with BSAs modified with other related aldehydes. By using this antibody, it was revealed that the antigenic structure was indeed present in atherosclerotic lesions of the human aorta.     

Molecular modeling of manganese regulation of calmodulin-sensitive adenylyl cyclase from mammalian sperm

The soluble calmodulin-sensitive isoform of adenylyl cyclase isolated from equine sperm is unique because it requires Mn(2+) rather than Mg(2+) for activity. To gain insight into the molecular action of metals on sperm adenylyl cyclase, the kinetics of Mn(2+) and ATP effect was examined. A biphasic response to increases in ATP concentration was observed when metal was held constant. When [Mn(2+)] exceeded [ATP], however, greatly enhanced enzyme activity was observed. The kinetic profiles were consistent with allosteric activation of adenylyl cyclase by Mn(2+). Linear transformation of the data yielded an apparent K(m) for Mn-ATP of 5.8 mM and calculated V(max) of 12 nM cyclic AMP formed/min/mg. Data analysis using calculated equilibrium concentrations of free and complexed reactants provided similar estimates of these kinetic parameters.     

Selectivity of 4,5,6,7-tetrabromobenzimidazole as an ATP-competitive potent inhibitor of protein kinase CK2 from various sources

Like the previously reported 4,5,6,7-tetrabromobenzotriazole (TBBt), the structurally related 4,5,6,7-tetrabromobenzimidazole (TBBz) is a selective ATP-competitive inhibitor of protein kinase CK2 from such divergent sources as yeast, rat liver, Neurospora crassa and Candida tropicalis, with K(i) values in the range 0.5-1 microM. It is virtually inactive vs. PKA, PKC, and a very weak inhibitor of protein kinase CK1. The corresponding tetrachlorobenzimidazole (TCBz) is a much weaker inhibitor of CK2, like tetrachlorobenzotriazole (TCBt) relative to TBBt. Bearing in mind the similarity of the van der Waals radii of Br (1.95 A) and CH(3) (2.0 A), the corresponding much less hydrophobic 4,5,6,7-tetramethylbenzotriazole (TMeBt) was prepared and found to be a very weak inhibitor of CK2, as well as of CK1. An unexpected, and significant, difference between TBBt and TBBz are their inhibitory activities vs. the yeast protein kinase PK60S, which phosphorylates, both in vitro and in intact yeast cells, three of the five pp13 kDa ribosomal surface acidic proteins in yeast cells. TBBt was previously noted to be a more effective inhibitor of PK60S than of yeast CK2; by contrast, TBBz is a relatively feeble inhibitor of PK60S, hence more selective than TBBt vs. CK2 in yeast cells. TMeBt was virtually inactive vs PK60S. Like TBBt, TBBz is an additional lead compound for development of more potent inhibitors of CK2.     

Discrete mutations in the presequence of potato formate dehydrogenase inhibit the in vivo targeting of GFP fusions into mitochondria

Most mitochondrial proteins are encoded by the nucleus, translated in the cytosol, and imported. Mitochondrial precursors generally contain their targeting information in a cleavable N-terminal presequence, which is rich in hydroxylated and positively charged residues and can form amphiphilic alpha-helices. We report the in vivo targeting of green fluorescent protein (GFP) by the FDH presequence, as well as several truncated or mutated variants. Some of these mutations modify the amphiphilicity of the predicted alpha-helix. The removal of the first two residues abolishes import and some single amino acid mutations strongly inhibit import. Such strong effects on import had not been observed in similar studies on other plant mitochondrial presequences, suggesting that the FDH presequence is a particularly good model for functional studies.     

Teleost fish spermatozoa contain a cytosolic protein factor that induces calcium release in sea urchin egg homogenates and triggers calcium oscillations when injected into mouse oocytes

Established studies in a variety of organisms including amphibians, fish, ascidians, nemerteans, echinoderms, mammals, and even a species of flowering plant, clearly demonstrate that an increase in intracellular egg calcium is crucial to the process of egg activation at fertilization. In echinoderms, egg activation appears to involve an egg phospholipase C gamma (PLCgamma). However, numerous studies in mammalian species suggest that calcium is released from internal egg stores at fertilization by a sperm-derived cytosolic protein factor. Recent studies in the mouse have identified this sperm-derived factor as being a novel sperm-specific PLC isoform with distinctive properties (PLCzeta). Homologues of PLCzeta have since been isolated from human and cynomolgus monkey sperm. In addition, sperm factor activity has been detected in non-mammalian species such as chicken, Xenopus, and a flowering plant. Here we report evidence for the existence of a similar sperm-derived factor in a commercially important species of teleost fish, the Nile tilapia Oreochromis niloticus (L). Using an established bioassay for calcium release, the sea urchin egg homogenate, we demonstrate that protein extracts obtained from tilapia spermatozoa exhibit PLC activity similar to that seen in mammalian sperm extracts, and also induce calcium release when added directly to the homogenate. Further, tilapia sperm extracts induced calcium oscillations when injected into mouse oocytes.     

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.     

M-phase regulation of the recruitment of mRNAs onto polysomes using the CDK1/cyclin B inhibitor aminopurvalanol

Translation under the control of the universal cell cycle regulator CDK1/cyclin B was investigated during the first cell cycle in sea urchin embryos. The CDK1/cyclin B inhibitor aminopurvalanol arrested embryos at the G2/M transition. Polysomal mRNAs were purified from control and arrested embryos, and screened for specific mRNA recruitment or release at M-phase by subtractive hybridization. The polysomal repartition of clones issued from this screen was analyzed. Three specific mRNAs were selectively recruited onto polysomes at M-phase. Conversely, two other specific mRNAs were released from polysomes. The isolation of these translationally regulated mRNAs gives now important tools for insights into the regulation of protein synthesis by the cell cycle regulator CDK1-cyclin B.     

Purification of antifreeze proteins by adsorption to ice

Antifreeze proteins (AFPs) can protect organisms from freezing injury by adsorbing to ice and inhibiting its growth. We describe here a method where ice, grown on a cold finger, is used to selectively adsorb and purify these ice-binding proteins from a crude mixture. Type III recombinant AFP was enriched approximately 50-fold after one round of partitioning into ice and purified to homogeneity by a second round. This method can also be used to purify non-ice-binding proteins by linkage to AFP domains as demonstrated by the recovery of a 50 kDa maltose-binding protein-AFP fusion from a crude lysate of Escherichia coli.     

A hierarchy of lipid constructs for the sperm plasma membrane

We have presented a series of lipid constructs as models of the sperm plasma membrane. We also isolated the plasma membrane from rabbit sperm cells and characterized the lipid composition. The behavior of these various membrane systems was evaluated using a vesicle leakage assay, in which surfactant (nonoxynol-9, N-9; or benzalkonium chloride, BZK) exposure induced membrane permeabilization. These studies shed light on the relative importance and significance of particular components present in the lipid constructs. In particular, a highly unsaturated phospholipid component characterized by an ether-linkage to position 1 of the glycerol backbone (as opposed to the more conventional ester linkage) as well as the presence of sulfogalactosyl ceramide were found to have an effect on the surfactant-induced leakage response. The presence of cholesterol had the greatest influence on membrane behavior. The construct series also demonstrated the ability of the surfactants studied to discriminate between different membrane systems. We found that N-9 displayed little sensitivity to membrane composition while BZK showed specific behavior with the various membrane systems.