11 research outputs found
Protein tyrosine kinase regulates the number of renal secretory K channels
No full textThe apical small conductance (SK) channel plays a key role in K secretion in the cortical collecting duct (CCD). A high-K intake stimulates renal K secretion and involves a significant increase in the number of SK channels in the apical membrane of the CCD. We used the patch-clamp technique to examine the role of protein tyrosine kinase (PTK) in regulating the activity of SK channels in the CCD. The application of 100 μM genistein stimulated SK channels in 11 of 12 patches in CCDs from rats on a K-deficient diet, and the mean increase in NPo, a product of channel number ( N) and open probability ( Po), was 2.5. In contrast, inhibition of PTK had no effect in tubules from animals on a high-K diet in all 10 experiments. Western blot analysis further shows that the level of cSrc, a nonreceptor type of PTK, is 261% higher in the kidneys from rats on a K-deficient diet than those on a high-K diet. However, the effect of cSrc was not the result of direct inhibition of channel itself, because addition of exogenous cSrc had no effect on SK channels in inside-out patches. In cell-attached patches, application of herbimycin A increased channel activity in 14 of 16 patches, and the mean increase in NPowas 2.4 in tubules from rats on a K-deficient diet. In contrast, herbimycin A had no effect on channel activity in any of 15 tubules from rats on a high-K diet. Furthermore, herbimycin A pretreatment increased NPoper patch from the control value (0.4) to 2.25 in CCDs from rats on a K-deficient diet, whereas herbimycin failed to increase channel activity ( NPo: control, 3.10; herbimycin A, 3.25) in the CCDs from animals on a high-K diet. We conclude that PTK is involved in regulating the number of apical SK channels in the kidney.</jats:p
Resveratrol Inhibits Aggregation of Platelets from High-risk Cardiac Patients with Aspirin Resistance
No full textBiomolecular condensates in cell biology and virology: Phase-separated membraneless organelles (MLOs)
No full textMembraneless organelles (MLOs) in the cytoplasm and nucleus in the form of 2D and 3D phase-separated biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions. These functions include cell signaling, immune synapse function, nuclear transcription, RNA splicing and processing, mRNA storage and translation, virus replication and maturation, antiviral mechanisms, DNA sensing, synaptic transmission, protein turnover and mitosis. Components comprising MLOs often associate with low affinity; thus cell integrity can be critical to the maintenance of the full complement of respective MLO components. Phase-separated condensates are typically metastable (shape-changing) and can undergo dramatic, rapid and reversible assembly and disassembly in response to cell signaling events, cell stress, during mitosis, and after changes in cytoplasmic crowding (as observed with condensates of the human myxovirus resistance protein MxA). Increasing evidence suggests that neuron-specific aberrations in phase-separation properties of RNA-binding proteins (e.g. FUS and TDP-43) and others (such as the microtubule-binding protein tau) contribute to the development of degenerative neurological diseases (e.g. amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer\u27s disease). Thus, studies of liquid-like phase separation (LLPS) and the formation, structure and function of MLOs are of considerable importance in understanding basic cell biology and the pathogenesis of human diseases
Role of the NH<sub>2</sub>terminus of the cloned renal K<sup>+</sup>channel, ROMK1, in arachidonic acid-mediated inhibition
No full textWe have previously demonstrated that the ROMK channel maintains the property of arachidonic acid (AA) sensitivity observed originally in the native ATP-sensitive K+channel of the rat cortical collecting duct (16). We used the patch-clamp technique to extend these studies to other NH2-terminal splice variants of the ROMK channel family, ROMK2 and ROMK3, expressed in Xenopus oocytes to determine the mechanism by which AA inhibits channel activity. Although the conductance, channel open probability, and open/closed times of the three homologs were determined to be similar, addition of 5–10 μM AA caused only a moderate inhibition of ROMK2 (15 ± 8%) and ROMK3 (13 ± 9%) activity, indicating that differences in the NH2termini of ROMK channels strongly influence the AA action. We consequently examined the effect of AA on a ROMK1 variant, R1ND37, in which the NH2terminal amino acids 2–37 were deleted, and on a mutant ROMK1, R1S4A, in which the serine-4 residue was mutated to alanine. Like ROMK2 and ROMK3, AA had a diminished effect on these variants. Addition of 1 nM exogenous protein kinase C (PKC) inhibited ROMK1 but not the mutant, R1S4A. However, the effect of AA is not a result of stimulation of a membrane bound PKC, since PKC inhibitors, calphostin C and chelerythrine, failed to abolish the AA-induced inhibition. In contrast, application of 5 μM staurosporine, a nonspecific protein kinase inhibitor at high concentration, abolished the effect of AA. We conclude that phosphorylation of serine-4 residue in the NH2terminus plays a key role in determination of AA effect on ROMK channels.</jats:p
Threonine phosphorylation of integrin β3 in calyculin A-treated platelets is selectively sensitive to 5′-iodotubercidin
No full textAbstractExposure of platelets to toxins (calyculin A or okadaic acid) that inhibit protein serine/threonine phosphatases types 1 and 2A, at concentrations that block aggregatory and secretory responses, results in the phosphorylation of several platelet proteins including integrin β3. Since protein phosphorylation represents a balance between kinase and phosphatase activities, this increase in phosphorylation reflects either the removal of phosphatases that oppose constitutively active kinases known to reside in the platelet (e.g., casein kinase 2) or the activation of endogenous kinases. In this study, we demonstrate that the addition of calyculin A promotes the activation of several endogenous platelet protein kinases, including p42/44mapk, p38mapk, Akt/PKB, and LKB1. Using a pharmacologic approach, we assessed whether inhibition of these and other enzymes block phosphorylation of β3. Inhibitors of p38mapk, casein kinase, AMP kinase, protein kinase C, and calcium–calmodulin-dependent kinases did not block phosphorylation of β3 on thr753. In contrast, 5′-iodotubercidin, at 50 μM, blocks β3 phosphorylation without affecting the efficacy of calyculin A to inhibit platelet aggregation and spreading. These data dissociate threonine phosphorylation of β3 molecules and inhibition of platelet responses by protein phosphatase inhibitors
Nuclear Localization of Type II cAMP-Dependent Protein Kinase during Limb Cartilage Differentiation Is Associated with a Novel Developmentally Regulated A-Kinase Anchoring Protein
No full textAbstractDifferentiation of chicken limb cartilage is accompanied by a rise in intracellular cyclic AMP, an inducer of cartilage-specific gene expression. A basic ∼35-kDa protein, designated p35, is the major nuclear substrate for cAMP-dependent protein kinase (PKA) during this process. Here we show that whereas both precartilage and cartilage nuclei contain p35, only precartilage nuclei contain PKA. The phosphorylation of p35 in isolated cell fractions was used as an index of changes in the cellular comparmentalization of components of PKA during chondrogenesis. Both the catalytic subunit and type II regulatory subunit (RII) of PKA were present in the precartilage nuclear fraction, but were undetectable or present in only trace amounts in the cartilage nuclear fraction. Furthermore, a novel ∼150-kDa A-kinase anchoring protein (AKAP), which binds to RII, was detected in the nuclear matrix of precartilage nuclei but, like RII, was virtually absent in the nuclei of fully differentiated cartilage cells. In limb mesenchymal cells undergoing chondrogenesis in culture a corresponding set of changes occurred: cartilage differentiation was accompanied by a marked reduction in the amounts of both nuclear RII and nAKAP150. These observations indicate that type II PKA holoenzyme is imported into the mesenchymal cell nucleus prior to chondrogenesis, an event that appears to depend on the the activity of the developmentally regulated nAKAP150
A regulatory network of two galectins mediates the earliest steps of avian limb skeletal morphogenesis
No full textAbstract
Background
The skeletal elements of vertebrate embryonic limbs are prefigured by rod- and spot-like condensations of precartilage mesenchymal cells. The formation of these condensations depends on cell-matrix and cell-cell interactions, but how they are initiated and patterned is as yet unresolved.
Results
Here we provide evidence that galectins, β-galactoside-binding lectins with β-sandwich folding, play fundamental roles in these processes. We show that among the five chicken galectin (CG) genes, two, CG-1A, and CG-8, are markedly elevated in expression at prospective sites of condensation in vitro and in vivo, with their protein products appearing earlier in development than any previously described marker. The two molecules enhance one another's gene expression but have opposite effects on condensation formation and cartilage development in vivo and in vitro: CG-1A, a non-covalent homodimer, promotes this process, while the tandem-repeat-type CG-8 antagonizes it. Correspondingly, knockdown of CG-1A inhibits the formation of skeletal elements while knockdown of CG-8 enhances it. The apparent paradox of mutual activation at the gene expression level coupled with antagonistic roles in skeletogenesis is resolved by analysis of the direct effect of the proteins on precartilage cells. Specifically, CG-1A causes their aggregation, whereas CG-8, which has no adhesive function of its own, blocks this effect. The developmental appearance and regulation of the unknown cell surface moieties ("ligands") to which CG-1A and CG-8 bind were indicative of specific cognate- and cross-regulatory interactions.
Conclusion
Our findings indicate that CG-1A and CG-8 constitute a multiscale network that is a major mediator, earlier-acting than any previously described, of the formation and patterning of precartilage mesenchymal condensations in the developing limb. This network functions autonomously of limb bud signaling centers or other limb bud positional cues.
</jats:sec
Cyclic arginine-glycine-aspartic acid peptide inhibits macrophage infiltration of the kidney and carotid artery lesions in apo-E-deficient mice
No full text
