Subunit Of Protein Phosphatase 2B Research Articles

The β isoform of the catalytic subunit of protein phosphatase 2B restrains platelet function by suppressing outside‐in αIIbβ3 integrin signaling

Calcium-dependent signaling mechanisms play a critical role in platelet activation. Unlike calcium-activated protease and kinase, the contribution of calcium-activated protein serine/threonine phosphatase in platelet activation is poorly understood. To assess the role of catalytic subunit of protein phosphatase 2B (PP2B) or calcineurin in platelet function. Here, we showed that an increase in PP2B activity was associated with agonist-induced activation of human and murine platelets. Pharmacological inhibitors of the catalytic subunit of protein phosphatase 2B (PP2B-A) such as cyclosporine A or tacrolimus (FK506) potentiated aggregation of human platelets. Murine platelets lacking the β isoform of PP2B-A (PP2B-Aβ(-/-) ) displayed increased aggregation with low doses of agonist concentrations. Loss of PP2B-Aβ did not affect agonist-induced integrin αII b β3 inside-out signaling, but increased basal Src activation and outside-in αII b β3 signaling to p38 mitogen-activated protein kinase (MAPK), with a concomitant enhancement in platelet spreading on immobilized fibrinogen and greater fibrin clot retraction. Fibrinogen-induced increased p38 activation in PP2B-Aβ(-/-) platelets were blocked by Src inhibitor. Both PP2B-Aβ(-/-) platelets and PP2B-Aβ-depleted human embryonal kidney 293 αII b β3 cells displayed increased adhesion to immobilized fibrinogen. Filamin A, an actin crosslinking phosphoprotein that is known to associate with β3 , was dephosphorylated on Ser(2152) in fibrinogen-adhered wild-type but not in PP2B-Aβ(-/-) platelets. In a FeCl3 injury thrombosis model, PP2B-Aβ(-/-) mice showed decreased time to occlusion in the carotid artery. These observations indicate that PP2B-Aβ by suppressing outside-in αII b β3 integrin signaling limits platelet response to vascular injury.

Suppression of Murine Platelet Activation by the β Isoform of the Catalytic Subunit of Protein Phosphatase 2B

Abstract 190Signal transduction mediated by the kinases and phosphatases are critical for platelet activation at the site of vascular injury. Compared to the kinases, a role for phosphatases in platelet activation is less understood. Our previous studies have focused on the roles of serine/threonine protein phosphatase 1 (PP1) and 2A (PP2A) in regulating integrin αIIbβ3functions. However, platelets also express protein phosphatase 2B (PP2B) and its role in platelet function is unexplored. PP2B-Aα and PP2B-Aβ constitute two ubiquitous isoforms of the PP2B catalytic subunit. Due to the general concerns regarding the specificity of the PP2B inhibitors, we have utilized mice deficient in the β isoform of the catalytic subunit of PP2B (PP2B-Aβ) to explore the role of PP2B in platelet functions. Mice lacking PP2B-Aα are short lived and are not considered in this study. Loss of PP2B-Aβ did not cause any compensatory increase in the PP2B-Aα levels in platelets. Compared to the wild type (WT) platelets, PP2B-Aβ−/− platelets displayed increased aggregation in response to low doses of protease-activated receptor 4-activating peptide (PAR4-AP), ADP, collagen and collagen related peptide (CRP). Enhanced α granule secretion in response to the low doses of PAR4-AP and CRP was noticed in PP2B-Aβ−/− platelets, relative to the WT platelets. Functions regulated by the outside-in αIIbβ3 integrin signaling like adhesion to immobilized fibrinogen and fibrin clot retraction were enhanced in the PP2B-Aβ−/− platelets. These studies indicate that PP2B-Aβ negatively regulate platelet functions in vitro. Consistent with these observations, PP2B-Aβ−/− mice exhibited a shorter tail bleeding time compared to the WT mice. In a FeCl3 induced endothelial denudation injury model, PP2B-Aβ−/− mice showed decreased time to occlusion in the carotid artery, and reduced number of emboli compared to the WT mice. These studies indicate that PP2B-Aβ suppress multiple murine platelet functions that contribute to an occlusive thrombi. Unlike a positive thrombus promoting role for the PP1cγ that was noticed in our previous study, PP2B-Aβ suppressed murine platelet activation, suggesting that different subtypes of Ser/Thr phosphatases have distinct roles in murine platelet activation. Disclosures:No relevant conflicts of interest to declare.

Critical role of divalent cations and Na+ efflux in Arabidopsis thaliana salt tolerance

AbstractDetrimental effects of salinity on plants are known to be partially alleviated by external Ca2+. Previous work demonstrated that the Arabidopsis SOS3 locus encodes a Ca2+‐binding protein with similarities to CnB, the regulatory subunit of protein phosphatase 2B (calcineurin). In this study, we further characterized the role of SOS3 in salt tolerance. We found that reduced root elongation of sos3 mutants in the presence of high concentrations of either NaCl or LiCl is specifically rescued by Ca2+ and not Mg2+, whereas root growth is rescued by both Ca2+ and Mg2+ in the presence of high concentrations of KCl. Phenocopies of sos3 mutants were obtained in wild‐type plants by the application of calmodulin and calcineurin inhibitors. These data provide further evidence that SOS3 is a calcineurin‐like protein and that calmodulin plays an important role in the signalling pathways involved in plant salt tolerance. The origin of the elevated Na : K ratio in sos3 mutants was investigated by comparing Na+ efflux and influx in both mutant and wild type. No difference in Na+ influx was recorded between wild type and sos3; however, sos3 plants showed a markedly lower Na+ efflux, a property that would contribute to the salt‐oversensitive phenotype of sos3 plants.

A Novel Ca2+-binding Protein, p22, Is Required for Constitutive Membrane Traffic

We have identified a novel protein, p22, required for "constitutive" exocytic membrane traffic. p22 belongs to the EF-hand superfamily of Ca2+-binding proteins and shows extensive similarity to the regulatory subunit of protein phosphatase 2B, calcineurin B. p22 is a cytosolic N-myristoylated protein that undergoes conformational changes upon binding of Ca2+. Antibodies against a p22 peptide block the targeting/fusion of transcytotic vesicles with the apical plasma membrane, but recombinant wild-type p22 overcomes that inhibition. Nonmyristoylated p22, or p22 incapable of undergoing Ca2+-induced conformational changes, cannot reverse the antibody-mediated inhibition. The data suggest that p22 may act by transducing cellular Ca2+ signals to downstream effectors. p22 is ubiquitously expressed, and we propose that its function is required for membrane trafficking events common to many cells.

Identification of a third alternatively spliced cDNA encoding the catalytic subunit of protein phosphatase 2Bβ

Complementary DNA coding for a catalytic subunit of protein phosphatase 2B was isolated from a human teratocarcinoma library. It encodes a third isoform of protein phosphatase 2Bβ, and differs from the cDNA for the second isoform by a deletion of 30 base pairs in the coding region. The deletion results in the loss of ten amino acids between the putative calmodulin site and a postulated autoinhibitory domain. An identical deletion occurs in one of the two alternatively spliced isoforms of PP2Bα.

19] Identification of N-terminal myristyl blocking groups in proteins

This chapter illustrates the identification of N-terminal myristyl blocking groups in proteins. The catalytic subunit of cyclic-AMP-dependent protein kinase (C subunit) and the regulatory subunit of protein phosphatase 2B contain myristic acid covalently attached to their amino-terminus. The discovery of myristic acid at the N-terminus of a protein phosphatase and a protein kinase suggests that this unusual blocking group may be involved in the interaction of converter enzymes with their protein substrates. The myristyl group could also play a role in maintaining the subunit-subunit interactions between the regulatory (B) and catalytic (A) subunits of calcineurin and between the regulatory and catalytic subunits of cyclic-AMP-dependent protein kinase. The myristyl group may be possibly involved in the translocation of the C subunit from the cytoplasm to the nucleus, across the nuclear membrane. Fast atom bombardment (FAB) mass spectrometry is considered the most useful mass spectrometric technique for identification of myristic acid in blocked peptides. Involatile and thermally labile compounds can be studied by this method and very small amounts of material can be used.