Regulation of α-Transducin and α-Gustducin Expression by a High Protein Diet in the Pig Gastrointestinal Tract

BackgroundThe expression of taste receptors (TASRs) and their signalling molecules in the gastrointestinal (GI) epithelial cells, including enteroendocrine cells (EECs), suggests they participate in chemosensing mechanisms influencing GI physiology via the release of endocrine messengers. TASRs mediate gustatory signalling by interacting with different transducers, including α-gustducin (G_αgust) and α-transducin (G_αtran) G protein subunits. This study tested whether G_αtran and G_αgust immunoreactive (-IR) cells are affected by a short-term (3 days) and long-term (30 days) high protein (Hp) diet in the pig GI tract.ResultIn the stomach, G_αgust and G_αtran-IR cells contained serotonin (5-HT) and ghrelin (GHR), while in the small and large intestine, G_αgust and G_αtran-IR colocalized with 5-HT-, cholecystokinin (CCK)- and peptide YY (PYY)-IR. There was a significant increase in the density of G_αtran-IR cells in the pyloric mucosa in both short- and long-term Hp diet groups (Hp3 and Hp30) vs. the control group (Ctr) (P<0.05), while the increase of G_αgust-IR cells in the pyloric mucosa was significant in Hp30 group vs. Ctr and vs. Hp3 (P<0.05); these cells included G_αtran / 5HT-IR and G_αtran / GHR-IR cells (P<0.05 and P<0.001 vs. Ctr, respectively) as well as G_αgust /5-HT-IR or G_αgust / GHR-IR cells (P<0.05 and P<0.01 vs. Ctr, respectively). In the small intestine, we recorded a significant increase in G_αtran-IR cells in the duodenal crypts and a significant increase of G_αgust-IR cells in the jejunal crypts in Hp3 group compared to HP30 (P<0.05). With regard to the number of G_αtran-G_αgust IR cells colocalized with CCK or 5-HT, there was only a significant increase of G_αtran / CCK-IR cells in Hp3 group compared to Ctr (P = 0.01).ConclusionThis study showed an upregulation of selected subpopulations of G_αgust / G_αtran-IR cells in distinct regions of the pig GI tract by short- and long-term Hp diet lending support to TASR-mediated effects in metabolic homeostasis and satiety mechanisms.     

Expression of α4 Integrin mRNA and Protein and Fibronectin in the Early Chicken Embryo

α₄ integrins (α₄β₁ and α₄β₇) have been shown to mediate both cell-matrix adhesion to fibronectin and cell-cell adhesion to VCAM-1. These interactions have been suggested to contribute to hematopoiesis, lymphocyte homing, recruitment of inflammatory cells, neural crest cell migration and myogenesis. We report here the cloning of chicken α₄ cDNA and its use to define the patterns of expression of α₄ mRNA and protein in early chicken embryos (19–22 somite pairs), a stage at which neural crest cells can be examined at various points in their migration and somitic development and differentiation can also be observed at various stages. We observe widespread expression of both α₄ mRNA and protein, although the patterns of steady state expression do not conform precisely. Many neural crest cells contain significant levels of α₄ mRNA. Some neural crest cells express α₄ protein but its expression is transient and/or limited to a subset of these cells. α₄ is strongly expressed at both mRNA and protein levels by somitic cells and their derivatives in the sclerotome, dermatome and myotome and is also expressed in neural tube, otic placode, heart, gut endoderm and some other tissues. Comparison with the distributions of fibronectin shows that, although some α₄ expression occurs in locations consistent with a role in cell-matrix adhesion to fibronectin, α₄ is also expressed in other places where fibronectin is low or absent and a role for α₄ in cell-cell interactions appears more likely.     

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis

The Integrin β1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of theα5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hy-pertrophy.     

Overproduction of an α-Amylase/Glucoamylase Fusion Protein in Aspergillus oryzae Using a High Expression Vector

The systemic immune response against orally administered antigens is suppressed (oral tolerance), and this has been postulated to avoid excess immunity against dietary constituents which are present in large amounts in the gastrointestinal tract. Taking into consideration that such orally administered protein antigens are subjected to enzymatic degradation in the gastrointestinal tract, we examined whether an enzymatic digest of milk proteins could induce oral tolerance. A tryptic digest of casein, containing mainly fragments smaller than 6000 Da, was fed to mice as a constituent of their diet. Mice fed with the casein-digest diet responded poorly to subsequent immunization with casein, indicating that oral tolerance to casein was induced in these animals. The results suggest the presence of immunosuppressive fragment(s) in the casein digest, which may be of use for preventing milk allergy.     

Dynamic Subunit Exchange and the Regulation of Microtubule Assembly by the Stress Response Protein Human αB Crystallin

Background

The small heat shock protein (sHSP), human αB crystallin, forms large, polydisperse complexes that modulate the tubulin-microtubule equilibrium using a dynamic mechanism that is poorly understood. The interactive sequences in αB crystallin for tubulin are surface exposed, and correspond to interactive sites for the formation of αB crystallin complexes.

Methodology/Principal Findings

There is sequence homology between tubulin and the interactive domains in the β8-strand of the core domain and the C-terminal extension of αB crystallin. This study investigated the hypothesis that the formation of tubulin and αB crystallin quaternary structures was regulated through common interactive domains that alter the dynamics of their assembly. Size exclusion chromatography (SEC), SDS-PAGE, microtubule assembly assays, aggregation assays, multiple sequence alignment, and molecular modeling characterized the dynamic response of tubulin assembly to increasing concentrations of αB crystallin. Low molar ratios of αB crystallin∶tubulin were favorable for microtubule assembly and high molar ratios of αB crystallin∶tubulin were unfavorable for microtubule assembly. Interactions between αB crystallin and unassembled tubulin were observed using SEC and SDS-PAGE.

Conclusions/Significance

Subunits of αB crystallin that exchange dynamically with the αB crystallin complex can interact with tubulin subunits to regulate the equilibrium between tubulin and microtubules.     

Protein electrostriction: a possibility of elastic deformation of the α-hemolysin channel by the applied field

While conformational flexibility of proteins is widely recognized as one of their functionally crucial features and enjoys proper attention for this reason, their elastic properties are rarely discussed. In ion channel studies, where the voltage-induced or ligand-induced conformational transitions, gating, are the leading topic of research, the elastic structural deformation by the applied electric field has never been addressed at all. Here we examine elasticity using a model channel of known crystal structure—Staphylococcus aureus -hemolysin. Working with single channels reconstituted into planar lipid bilayers, we first show that their ionic conductance is asymmetric with voltage even at the highest salt concentration used where the static charges in the channel interior are maximally shielded. Second, choosing 18-crown-6 as a molecular probe whose size is close to the size of the narrowest part of the -hemolysin pore, we analyze the blockage of the channel by the crown/K⁺ complex. Analysis of the blockage within the framework of the Woodhull model in its generalized form demonstrates that the model is able to correctly describe the crown effect only if the parameters of the model are considered to be voltage-dependent. Specifically, one has to include either a voltage-dependent barrier for crown release to the cis side of the channel or voltage-dependent interactions between the binding site and the crown. We suggest that the voltage sensitivity of both the ionic conductance of the channel seen at the highest salt concentration and its blockage by the crown reflects a field-induced deformation of the pore.     

Expression and activation of the oxytocin receptor in airway smooth muscle cells: Regulation by TNFα and IL-13

Background

During pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFα, cytokines known to be important in asthma.

Method

Expression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma.

Results

PCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFα) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFα had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma.

Conclusion

Taken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.     

UPR Activation and the Down–Regulation of α-Crystallin in Human High Myopia-Related Cataract Lens Epithelium

Purpose

To investigate the expression of αA- and αB-crystallin and the unfolded protein response in the lens epithelium of patients with high myopia-related cataracts.

Methods and Materials

The central portion of the human anterior lens capsule together with the adhering epithelial cells, approximately 5 mm in diameter, were harvested and processed within two hours after cataract surgery from high myopia-related (spherical equivalent ≥-10.00 diopters) and age-related cataract patients or from high myopia but non-cataractous patients (tissue were collected from ocular trauma patients with high myopia and lens trauma). Anterior lens samples from fresh cadaver normal human eyes were used as normal control (collected within 6 hours from death). Real-time PCR was performed to detect the mRNA levels of α-crystallins as well as unfolded protein response (UPR)-related GRP78, spliced-XBP1, ATF4 and ATF6. Western blot analysis was used to determine the protein level of α-crystallin, GRP78, p-IRE1α, p-eIF2α and ATF6.

Results

In the lens epithelium of the high myopia-related cataract group and the age related cataract group, the mRNA and soluble protein expression of αA- and αB-crystallin were both decreased; additionally, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased relative to the normal control.

Conclusion

These results suggest the significant loss of soluble α-crystallin and the activation of the UPR in the lens epithelium of patients with high myopia-related cataract, which may be associated with the cataractogenesis of high myopia-related cataract.     

Regulation of CCAAT/Enhancer-binding Protein Homologous Protein (CHOP) Expression by Interleukin-1β in Pancreatic β Cells

Apoptosis contributes to immune-mediated pancreatic β cell destruction in type I diabetes. Exposure of β cells to interleukin-1β (IL-1β) causes endoplasmic reticulum stress and activates proapoptotic networks. Here, we show that nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways regulate the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), which mediates endoplasmic reticulum stress-induced apoptosis. Both CHOP mRNA and protein increase in β cells treated with IL-1β. In addition, prolonged exposure to high glucose further increases IL-1β-triggered CHOP expression. IL-1β also causes increased expression of C/EBP-β and a reduction of MafA, NFATc2, and Pdx-1 expression in β cells. Inhibition of the NF-κB and MAPK signaling pathways differentially attenuates CHOP expression. Knocking down CHOP by RNA interference protects β cells from IL-1β-induced apoptosis. These studies provide direct mechanistic links between cytokine-induced signaling pathways and CHOP-mediated apoptosis of β cells.     

Cloning-Independent Expression and Analysis of ω-Transaminases by Use of a Cell-Free Protein Synthesis System

Herewith we report the expression and screening of microbial enzymes without involving cloning procedures. Computationally predicted putative ω-transaminase (ω-TA) genes were PCR amplified from the bacterial colonies and expressed in a cell-free protein synthesis system for subsequent analysis of their enzymatic activity and substrate specificity. Through the cell-free expression analysis of the putative ω-TA genes, a number of enzyme-substrate pairs were identified in a matter of hours. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function to accelerate the discovery of novel enzymes.Recent advances in genome sequencing technology have accumulated enormous amounts of sequence information (12). Although protein function encoded in nucleotide sequences can be annotated using computational alignment tools, in many cases, significant similarity to proteins with known function is hard to establish (5, 18). To understand the biological function of these unknown proteins, as well as to validate the computer-annotated results, efficient methods that enable rapid translation of genetic information into protein function are in high demand. The availability of high-throughput method for protein generation is also essential for accelerating the discovery and evolution of biocatalysts (3, 4, 6, 14, 22, 23) used in industry. While gene cloning and cultivation of transformed cells have long been used as standard methods for production of recombinant proteins, the vast amount of sequence information from various genome sequencing projects is now demanding a throughput of protein expression that exceeds that of the present in vivo expression techniques.Compared to cell-based gene expression, cell-free protein synthesis offers substantial advantages in speed and flexibility for the simultaneous expression of multiple proteins (7, 9, 13, 16, 19, 21). As a part of our efforts to extend the application of cell-free protein synthesis into the field of enzyme technology, we report in this paper an integrated methodology for fast expression screening of enzymes using ω-transaminases (ω-TAs) as a model target. Transaminases are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze reversible transfer of amine groups to keto acids, producing diverse proteogenic or nonproteogenic amino acids (1).In this work, ω-TA genes from microbial colonies were amplified by PCR and directly expressed in a cell-free protein synthesis system. Expressed enzymes were then screened for their activity toward different amine donors by colorimetric measurement of the changes in the concentration of pyruvate, which was used as a common amine acceptor. As a result, analysis of the substrate specificities of the enzymes encoded by 11 ω-TA genes toward 16 amine-donating compounds were completed within a matter of hours, identifying a number of enzyme-substrate matches.We started by examining whether sufficient amount of proteins could be generated for enzymatic analysis of ω-TAs when the PCR products amplified from the bacterial colonies were used as the template for cell-free synthesis reactions. The efficiency of protein synthesis was compared for reactions programmed with a plasmid-cloned ω-TA gene from Vibrio fluvialis (Vfω-TA) (pIVEX2.3d ω-TA Vf) and reactions programmed with the same gene prepared by two-step PCR from a bacterial colony (Vibrio fluvialis JS17 [20]), as depicted in Fig. ​Fig.1.1. The ω-TA genes examined in this study are listed in Table S1 in the supplemental material along with their bacterial sources.Open in a separate windowFIG. 1.Experimental scheme for cloning-independent cell-free expression screening of ω-transaminases. The expression templates for cell-free synthesis were prepared through two-step amplification of the target open reading frame (ORF) from bacterial genomes. PCR products were translated into corresponding enzymes in microtiter plates as described in the text. Upon completion of the synthesis reaction, the reaction mixture was sequentially supplied with the assay mixture and chromogenic compound to determine the residual pyruvate concentration after the amine transfer reaction. Abbreviations: For and Rev Primer, forward and reverse primers, respectively; 5′-UTR, 5′ UTR; T7P, T7 promoter; RBS, ribosome binding site; T7T, T7 terminator; ω-TAp, putative ω-transaminase; ω-TA Rs, ω-transaminase from Rhodobacter sphaeroides; ω-TA At, ω-transaminase from Agrobacterium tumefaciens.The templates for cell-free synthesis of ω-TA were prepared by colony PCR and subsequent second-round PCR using the MEGA primers flanking the T7 promoter, ribosome binding site, polyhistidine tag, and the T7 terminator. All of the PCRs were carried out using LA Taq DNA polymerase (Takara Bio Inc., Otsu, Japan). PCR products were directly used as the template for protein synthesis without purification. The standard cell-free reaction mixture consisted of the following components in a final volume of 50 μl: 57 mM HEPES-KOH (pH 7.5); 1.2 mM ATP; 0.85 mM (each) CTP, GTP, and UTP; 1.7 mM dithiothreitol; 0.17 mg/ml Escherichia coli total tRNA mixture (from strain MRE600); 90 mM potassium glutamate; 80 mM ammonium acetate; 12 mM magnesium acetate; 34 μg/ml l-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid); 1.5 mM (each) 20 amino acids; 2% polyethylene glycol 8000 (PEG 8000); 67 mM creatine phosphate; 3.2 μg/ml creatine kinase; 10 μM l-[U-¹⁴C]leucine (11.3 GBq/mmol); 0.5 μg/ml PCR-amplified DNA; and 14 μl of the S12 extract (11). Cell-free synthesized proteins were quantified by measuring trichloroacetic acid (TCA)-insoluble radioactivity (10), and the size and relative solubility of the synthesized protein were determined by Western blot analysis on a 12% Tricine-SDS-polyacrylamide gel (17). Mouse anti-histidine-tagged IgG antibody (Merck KGaA, Darmstadt, Germany) and rabbit anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Sigma, St. Louis, MO) were used as the primary and secondary antibodies, respectively. The PCR products served as translation substrates appropriate for producing as much protein as the corresponding plasmid-cloned gene when expressed in the reaction mixture (see Fig. S1 in the supplemental material).We next proceeded to amplify 11 ω-TA genes, including 9 putative ones from the colonies of bacterial origins (see Table S2 in the supplemental material for the list of primers used in this study), and express them in the reaction mixture prepared in two microtiter plates. Each of the 11 target genes was added to the plates by column on the plate (columns 2 through 12). Column 1 was used for negative-control reactions without any template DNAs (Fig. ​(Fig.1).1). After the PCR-amplified template DNAs were added to the plates, the plates were sealed with a plastic film to prevent evaporation and incubated at 37°C. From the measurements of TCA-insoluble radioactivity, it was estimated that 301 (±13) to 501 (±9) μg/ml of the encoded enzymes were produced after 90 min of incubation (see Fig. S2A in the supplemental material). Although most of the cell-free synthesized ω-TAs was highly insoluble in the initial experiments conducted under standard reaction conditions, the relative amounts of the soluble enzymes were markedly improved by using the GroEL/ES-enriched S12 extract (8) (Fig. S2B).Instead of the conventional high-performance liquid chromatography (HPLC) methods which have limited throughput for handling many reaction samples from different enzyme-substrate combinations, in this study, we used a simple colorimetric method for combinatorial analysis of the cell-free synthesized ω-TAs with different amine-donating substrates. Using Vfω-TA as a model ω-TA, it was first examined whether the progress of amine transfer reaction can be assayed quantitatively by colorimetric measurement of the residual pyruvate concentration. On the basis of our previous finding that Vfω-TA takes amine donors containing aryl groups as effective substrates (20), cell-free synthesized Vfω-TA was incubated with α-methylbenzylamine or benzylamine in the presence of pyruvate, and the residual pyruvate concentration in the assay mixture was determined. In brief, the amine transfer reaction was initiated by adding 50 μl of assay mixture (50 mM Tris-HCl buffer [pH 7.2], 10 mM sodium pyruvate, 10 mM [each] amine donors, and 20 μM pyridoxal-5′-phosphate) to the completed cell-free synthesis reaction mixtures (50 μl) in a 96-well plate. After 3 h of incubation at 37°C, the assay mixture was diluted with an equal volume of distilled water. When 100 μl of the diluted solution was transferred to a fresh plate containing 50 μl of 0.5 mM 2,4-dinitrophenylhydrazine (DNP), a yellow precipitate of pyruvate-dinitrophenylhydrazone (PA-DNPH) derivative was formed instantly. The absorbance at 450 nm was measured in a microplate reader (Bio-Rad, Hercules, CA) and compared with a standard curve to determine the amount of residual pyruvate in each well. Although the sensitivity of the residual pyruvate assay was as low as 0.01 mM, considering the error range, it was determined that a sensitivity of 0.1 mM should be used instead. When the optical density at 450 nm (OD₄₅₀) was measured after the addition of DNP and referred to a standard curve, the conversion yield based on the amount of residual pyruvate concentration showed good correlation with the results from standard HPLC assay (Table ​(Table11 and Fig. ​Fig.2A,2A, insets) where acetophenone or benzaldehyde generated from the corresponding substrates was separated in the Discovery HS F5 (5-μm particle size; 150- by 4.6-mm inner diameter [i.d.]; Supelco, Bellefonte, PA) column and measured at 254 nm. In addition, the relative amount of pyruvate was also able to be compared visually by adding 100 μl of 4 N NaOH solution, which turned the color of PA-DNPH to dark red (2, 15).Open in a separate windowFIG. 2.(A) Reactivity of 16 amine donors toward Vibrio fluvialis ω-TA. Reduced amounts of pyruvate concentration after the amine transfer reactions are plotted. Substrate number abbreviations: S01, α-methylbenzylamine; S02, α-ethylbenzylamine; S03, benzylamine; S04, 3-phenyl-1-propylamine; S05, phenylbutylamine; S06, 1-aminoindan; S07, ethylamine; S08, propylamine; S09, butylamine; S10, amylamine; S11, isopropylamine; S12, sec-butylamine; S13, β-alanine; S14, 3-amino-n-butyric acid; S15, phenylalanine; S16, 3-amino-3-phenylpropionic acid. The insets show HPLC traces of acetophenone and benzaldehyde after termination of transamination reaction by use of Vibrio fluvialis ω-TA against α-methylbenzylamine and benzylamine. mAU, milliabsorbance units. (B) Photo image of the assay plate after the addition of DNP and NaOH. ω-TA Vf, ω-TA from Vibrio fluvialis.

TABLE 1.

Activity comparison of Vibrio fluvialis ω-TA by HPLC and colorimetric method

Regulation of Human and Pig Renal Na+,K+-ATPase Activity by Tyrosine Phosphorylation of Their α1-Subunits

Modulation of the physiologically influential Na⁺,K⁺-ATPase is a complex process involving a wide variety of factors. To determine the possible effects of the protein tyrosine phosphatase (PTP) inhibitors dephostatin and Et-3,4-dephostatin on human and pig, renal cells and enzymatic extracts, we treated our samples (15 min–24 h) with those PTP inhibitors (0–100 μM). PTP inhibitors were found to possess a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in both human and pig samples. The inhibition was similarly demonstrated on all cellular, microsomal fraction and purified Na⁺,K⁺-ATPase levels. Despite rigorous activity recovery attempts, the PTP inhibitors’ effects were sustained on Na⁺,K⁺-ATPase activity. Western blotting experiments revealed the expression of both α₁- and β₁-subunits in both human and pig tissues. α₁-Subunits possessed higher tyrosine phosphorylation levels with higher concentrations of PTP inhibitors. Meanwhile, serine/threonine residues of both α₁- and β₁-subunits demonstrated diminished phosphorylation levels upon dephostatin treatment. Accordingly, we provide evidence that Na⁺,K⁺-ATPase can be regulated through tyrosine phosphorylation of primarily their α₁-subunits, using PTP inhibitors.     

Direct Binding and Regulation of RhoA Protein by Cyclic GMP-dependent Protein Kinase Iα

Vascular smooth muscle cell (VSMC) tone is regulated by the state of myosin light chain (MLC) phosphorylation, which is in turn regulated by the balance between MLC kinase and MLC phosphatase (MLCP) activities. RhoA activates Rho kinase, which phosphorylates the regulatory subunit of MLC phosphatase, thereby inhibiting MLC phosphatase activity and increasing contraction and vascular tone. Nitric oxide is an important mediator of VSMC relaxation and vasodilation, which acts by increasing cyclic GMP (cGMP) levels in VSMC, thereby activating cGMP-dependent protein kinase Iα (PKGIα). PKGI is known to phosphorylate Rho kinase, preventing Rho-mediated inhibition of MLC phosphatase, promoting vasorelaxation, although the molecular mechanisms that mediate this are unclear. Here we identify RhoA as a target of activated PKGIα and show further that PKGIα binds directly to RhoA, inhibiting its activation and translocation. In protein pulldown and immunoprecipitation experiments, binding of RhoA and PKGIα was demonstrated via a direct interaction between the amino terminus of RhoA (residues 1–44), containing the switch I domain of RhoA, and the amino terminus of PKGIα (residues 1–59), which includes a leucine zipper heptad repeat motif. Affinity assays using cGMP-immobilized agarose showed that only activated PKGIα binds RhoA, and a leucine zipper mutant PKGIα was unable to bind RhoA even if activated. Furthermore, a catalytically inactive mutant of PKGIα bound RhoA but did not prevent RhoA activation and translocation. Collectively, these results support that RhoA is a PKGIα target and that direct binding of activated PKGIα to RhoA is central to cGMP-mediated inhibition of the VSMC Rho kinase contractile pathway.