A novel mechanism of G protein-dependent phosphorylation of vasodilator-stimulated phosphoprotein

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) alpha-thrombin induced phosphorylation of VASP. Specific inhibition of Galpha13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Galpha13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-kappaB signaling pathway. alpha-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Galpha13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Galpha13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IkappaB and pretreatment with the proteasome inhibitor MG-132 abolished Galpha13- and alpha-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Galpha13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IkappaB, release of PKA catalytic subunit from the complex with IkappaB and NF-kappaB, and subsequent phosphorylation of VASP.     

Reaction of ferric cytochrome P450cam with peracids: kinetic characterization of intermediates on the reaction pathway

Reactions of substrate-free ferric cytochrome P450cam with peracids to generate Fe=O intermediates have previously been investigated with contradictory results. Using stopped-flow spectrophotometry, the reaction with m-chloroperoxybenzoic acid demonstrated an Fe(IV)=O + porphyrin pi-cation radical (Cpd I) (Egawa, T., Shimada, H., and Ishimura, Y. (1994) Biochem. Biophys. Res. Commun. 201, 1464-1469). By contrast, with peracetic acid, Fe(IV)=O plus a tyrosyl radical were observed by freeze-quench Mossbauer and EPR spectroscopy (Schunemann, V., Jung, C., Trautwein, A. X., Mandon, D., and Weiss, R. (2000) FEBS Lett. 479, 149-154). Our detailed kinetic studies have resolved these contradictory results. At pH >7, a significant fraction of Cpd I is formed transiently, whereas at low pH only a species with a Soret band at 406 nm, presumably Fe(IV)=O + tyrosyl radical, is observed. Evidence for formation of an acylperoxo complex en route to Cpd I was obtained. Because of rapid heme destruction, steps subsequent to formation of the highly oxidized forms could not be fully characterized. Heme destruction was avoided by including peroxidase substrates (e.g. guaiacol), which were oxidized to characteristic peroxidase products as the Fe(III)-P450 was regenerated. Addition of ascorbate to either of the high valent species also reforms the Fe(III) state with only a small loss of heme absorbance. These results indicate that typical peroxidase chemistry occurs with P450cam and offer an explanation for the contrasting results reported earlier. The delineation of improved conditions (pH, temperature, choice of peracid) for generating highly oxidized species with P450cam should be valuable for their further characterization.     

LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.     

Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.     

Natural microbial diversity in superficial sediments of Milazzo Harbor (Sicily) and community successions during microcosm enrichment with various hydrocarbons

Hydrocarbon-contaminated superficial sediments collected from the Harbor of Milazzo (Tirrenean Sea, northern Sicily), a zone strongly affected by anthropogenic activities, were examined for in situ biodegradative capacities. A culture-independent molecular phylogenetic approach was used to study the influence of hydrocarbon and nutrient addition on the activity and diversity of the indigenous microbiota during a microcosm evaluation. The autochthonous microbial community in non-polluted sediments was represented by eubacterial phylotypes grouped within Proteobacteria, CFB and Firmicutes. The archaeal domain was represented by members of Marine Group I of Crenarchaeota. The majority of recovered sequences was affiliated with heterotrophic genera Clostridium and Vibrio, typical members of eutrophic coastal environments. Amendments of hydrocarbons and mineral nutrients to microcosms dramatically changed the initial diversity of the microbial community. Only bacterial phylotypes affiliated with Proteobacteria and CFB division were detected. The decrease in diversity observed in several microcosms could be explained by the strong selection for microorganisms belonging to group of marine hydrocarbonoclastic gamma-Proteobacteria, namely Alcanivorax, Cycloclasticus, Marinobacter, Marinobacterium/Neptunomonas and Thalassolituus. This study demonstrated that nutrient amendment to hydrocarbon-contaminated superficial sediments enhanced the indigenous microbial biodegradation activity and that highly specialized marine hydrocarbonoclastic bacteria, representing a minor fraction in the natural microbial community, play an important role in the biodegradation of petroleum hydrocarbons accidentally entering the coastal environment.     

Premicellar complexes of sphingomyelinase mediate enzyme exchange for the stationary phase turnover

During the steady state reaction progress in the scooting mode with highly processive turnover, Bacillus cereus sphingomyelinase (SMase) remains tightly bound to sphingomyelin (SM) vesicles (Yu et al., Biochim. Biophys. Acta 1583, 121-131, 2002). In this paper, we analyze the kinetics of SMase-catalyzed hydrolysis of SM dispersed in diheptanoylphosphatidyl-choline (DC7PC) micelles. Results show that the resulting decrease in the turnover processivity induces the stationary phase in the reaction progress. The exchange of the bound enzyme (E*) between the vesicle during such reaction progress is mediated via the premicellar complexes (E(i)#) of SMase with DC7PC. Biophysical studies indicate that in E(i)# monodisperse DC7PC is bound to the interface binding surface (i-face) of SMase that is also involved in its binding to micelles or vesicles. In the presence of magnesium, required for the catalytic turnover, three different complexes of SMase with monodisperse DC7PC (E(i)# with i=1, 2, 3) are sequentially formed with Hill coefficients of 3, 4 and 8, respectively. As a result, during the stationary phase reaction progress, the initial rate is linear for an extended period and all the substrate in the reaction mixture is hydrolyzed at the end of the reaction progress. At low mole fraction (X) of total added SM, exchange is rapid and the processive turnover is limited by the steps of the interfacial turnover cycle without becoming microscopically limited by local substrate depletion or enzyme exchange. At high X, less DC7PC will be monodisperse, E(i)# does not form and the turnover becomes limited by slow enzyme exchange. Transferred NOESY enhancement results show that monomeric DC7PC in solution is in a rapid exchange with that bound to E(i)# at a rate comparable to that in micelles. Significance of the exchange and equilibrium properties of the E(i)# complexes for the interpretation of the stationary phase reaction progress is discussed.     

Mutation of the putative hydrogen-bond donor to P700 of photosystem I

The primary electron donor of photosystem I (PS1), called P(700), is a heterodimer of chlorophyll (Chl) a and a'. The crystal structure of photosystem I reveals that the chlorophyll a' (P(A)) could be hydrogen-bonded to the protein via a threonine residue, while the chlorophyll a (P(B)) does not have such a hydrogen bond. To investigate the influence of this hydrogen bond on P(700), PsaA-Thr739 was converted to alanine to remove the H-bond to the 13(1)-keto group of the chlorophyll a' in Chlamydomonas reinhardtii. The PsaA-T739A mutant was capable of assembling active PS1. Furthermore the mutant PS1 contained approximately one chlorophyll a' molecule per reaction center, indicating that P(700) was still a Chl a/a' heterodimer in the mutant. However, the mutation induced several band shifts in the visible P(700)(+) - P(700) absorbance difference spectrum. Redox titration of P(700) revealed a 60 mV decrease in the P(700)/P(700)(+) midpoint potential of the mutant, consistent with loss of a H-bond. Fourier transform infrared (FTIR) spectroscopy indicates that the ground state of P(700) is somewhat modified by mutation of ThrA739 to alanine. Comparison of FTIR difference band shifts upon P(700)(+) formation in WT and mutant PS1 suggests that the mutation modifies the charge distribution over the pigments in the P(700)(+) state, with approximately 14-18% of the positive charge on P(B) in WT being relocated onto P(A) in the mutant. (1)H-electron-nuclear double resonance (ENDOR) analysis of the P(700)(+) cation radical was also consistent with a slight redistribution of spin from the P(B) chlorophyll to P(A), as well as some redistribution of spin within the P(B) chlorophyll. High-field electron paramagnetic resonance (EPR) spectroscopy at 330-GHz was used to resolve the g-tensor of P(700)(+), but no significant differences from wild-type were observed, except for a slight decrease of anisotropy. The mutation did, however, provoke changes in the zero-field splitting parameters of the triplet state of P(700) ((3)P(700)), as determined by EPR. Interestingly, the mutation-induced change in asymmetry of P(700) did not cause an observable change in the directionality of electron transfer within PS1.     

Myosin light chain kinase (210 kDa) is a potential cytoskeleton integrator through its unique N-terminal domain

Recently discovered 210-kDa myosin light chain kinase (MLCK-210) is identical to 108-130 kDa MLCK, the principal regulator of the myosin II molecular motor, except for the presence of a unique amino terminal extension. Our in vitro experiments and transfected cell studies demonstrate that the N-terminal half of MLCK-210 unique tail domain has novel microfilament and microtubule binding activity. Consistent with this activity, the MLCK-210 domain codistributes with microfilaments and microtubules in cultured cells and with soluble tubulin in nocodazole-treated cells. This domain is capable of aggregating tubulin dimers in vitro, causing bundling and branching of microtubules induced by taxol. The N-terminal actin-binding region of MLCK-210 has lower affinity to actin (K(d) = 7.4 microM) than its central D(F/V)RXXL repeat-based actin-binding site and does not protect stress fibers from disassembly triggered by MLCK inhibition in transfected cells. Obtained results suggest that while being resident on microfilaments, MLCK-210 may interact with other cytoskeletal components through its N-terminal domain. Based on available evidence, we propose a model in which MLCK-210 could organize cell motility by simultaneous control of cytoskeleton architecture and actomyosin activation through the novel protein scaffold function of the unique tail domain and the classical MLCK catalytic function of the kinase domain.     

New approach for specific determination of antibiotics by use of luminescent Escherichia coli and immune serum

This paper describes a possible application of luminescent Escherichia coli activated by blood serum for high-sensitivity and high-specificity assays of antibiotics in solutions. Antibiotics inhibited luminescence of a genetically engineered E. coli strain; the system sensitivity to some antibiotics grew notably after the cells had been preactivated by blood serum. The highest level of sensitivity (2.8 +/- 0.6 ng/ml) of luminescent cells was obtained for aminoglycoside antibiotics (gentamicin and streptomycin). It is feasible to create the specific biosensor for antibiotics on the basis of bioluminescent E. coli strains by applying sera containing antibodies against the antibiotic under assay. The presence of antibodies specific for gentamicin in serum affects inhibition of luminescent cells by gentamicin but not inhibition by other antibiotics.