Éva Bakó

High complexity in the expression of the B′ subunit of protein phosphatase 2A0: Evidence for the existence of at least seven novel isoforms

Association of the catalytic subunit (C2) with a variety of regulatory subunits is believed to modulate the activity and specificity of protein phosphatase 2A (PP2A). In this study we report the cloning and expression of a new family of B-subunit, the B′, associated with the PP2A0 form. Polymerase chain reactions and cDNA library screening have identified at least seven cDNA isotypes, designated α, β1, β2, β3, β4, γ, and δ. The different β subtypes appear to be generated by alternative splicing. The deduced amino acid sequences of the α, β2, β3, β4 and γ isoforms predict molecular weights of 57,600, 56,500, 60,900, 52,500, and 68,000, respectively. The proteins are 60-80% identical and differ mostly at their termini. Two of the isoforms, B′β3 and B′γ, contain a bipartite nuclear localization signal in their COOH terminus. No homology was found with other B- or Brelated subunits. Northern analyses indicate a tissuespecific expression of the isoforms. Expression of B′α protein in Escherichia coli generated a polypeptide of ∼53 kDa, similar to the size of the B′ subunit present in the purified PP2A0. The recombinant protein was recognized by antibody raised against native B′ and interacted with the dimeric PP2A (A•C2) to generate a trimeric phosphatase. The deduced amino acid sequences of the B′ isoforms show significant homology to mammalian, fungal, and plant nucleotide sequences of unknown function present in the data bases. Notably, a high degree of homology (55-66%) was found with a yeast gene, RTS1, encoding a multicopy suppressor of a rox3 mutant. Our data indicate that at least seven B′ subunit isoforms may participate in the generation of a large number of PP2A0 holoenzymes that may be spatially and/or functionally targeted to different cellular processes.

Decreased protein phosphatase 2A activity in hippocampal long-term potentiation.

Abstract: Using autophosphorylated Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) as substrate, we now find that long‐term potentian (LTP) induction and maintenance are also associated with a significant decrease in calyculin A‐sensitive protein phosphatase (protein phosphatase 2A) activity, without changes in Mg2+‐dependent protein phosphatase (protein phosphatase 2C) activity. This decrease in protein phosphatase 2A activity was prevented when LTP induction was inhibited by treatment with calmidazolium or D‐2‐amino‐5‐phosphonopentanoic acid. In addition, the application of high‐frequency stimulation to 32P‐labeled hippocampal slices resulted in increases in the phosphorylation of a 55‐kDa protein immunoprecipitated with anti‐phosphatase 2A antibodies. Use of a specific antibody revealed that the 55‐kDa protein is the B′α subunit of protein phosphatase 2A. Following purification of brain protein phosphatase 2A, the B′α subunit was phosphorylated by CaM kinase II, an event that led to the reduction of protein phosphatase 2A activity. These results suggest that the decreased activity in protein phosphatase 2A following LTP induction contributes to the maintenance of constitutively active CaM kinase II and to the long‐lasting increase in phosphorylation of synaptic components implicated in LTP.

Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures

Biomechanical stimuli play important roles in the formation of articular cartilage during early foetal life, and optimal mechanical load is a crucial regulatory factor of adult chondrocyte metabolism and function. In this study, we undertook to analyse mechanotransduction pathways during in vitro chondrogenesis. Chondroprogenitor cells isolated from limb buds of 4-day-old chicken embryos were cultivated as high density cell cultures for 6 days. Mechanical stimulation was carried out by a self-designed bioreactor that exerted uniaxial intermittent cyclic load transmitted by the culture medium as hydrostatic pressure and fluid shear to differentiating cells. The loading scheme (0.05 Hz, 600 Pa; for 30 min) was applied on culturing days 2 and 3, when final commitment and differentiation of chondroprogenitor cells occurred in this model. The applied mechanical load significantly augmented cartilage matrix production and elevated mRNA expression of several cartilage matrix constituents, including collagen type II and aggrecan core protein, as well as matrix-producing hyaluronan synthases through enhanced expression, phosphorylation and nuclear signals of the main chondrogenic transcription factor Sox9. Along with increased cAMP levels, a significantly enhanced protein kinase A (PKA) activity was also detected and CREB, the archetypal downstream transcription factor of PKA signalling, exhibited elevated phosphorylation levels and stronger nuclear signals in response to mechanical stimuli. All the above effects were diminished by the PKA-inhibitor H89. Inhibition of the PKA-independent cAMP-mediators Epac1 and Epac2 with HJC0197 resulted in enhanced cartilage formation, which was additive to that of the mechanical stimulation, implying that the chondrogenesis-promoting effect of mechanical load was independent of Epac. At the same time, PP2A activity was reduced following mechanical load and treatments with the PP2A-inhibitor okadaic acid were able to mimic the effects of the intervention. Our results indicate that proper mechanical stimuli augment in vitro cartilage formation via promoting both differentiation and matrix production of chondrogenic cells, and the opposing regulation of the PKA/CREB-Sox9 and the PP2A signalling pathways is crucial in this phenomenon.

Phosphatase 2A is involved in endothelial cell microtubule remodeling and barrier regulation

We have recently shown that microtubule (MT) inhibitor, nocodazole (2–5 μM) significantly increases endothelial cells (EC) actomyosin contraction and permeability indicating the importance of MT in maintaining the EC barrier (Verin et al. [ 2001 ]: Cell Mol Physiol 281:L565–L574). Okadaic acid (OA, 2–5 nM), a powerful inhibitor of protein phosphatase 2A (PP2A), significantly potentiates the effect of submaximal concentrations of nocodazole (50–200 nM) on transendothelial electrical resistance (TER) suggesting the involvement of PP2A activity in the MT‐mediated EC barrier regulation. Immunofluorescent staining of EC revealed that in control cells PP2A distributes in a pattern similar to MT. Consistent with these results, we demonstrated that significant amounts of PP2A were present in MT‐enriched EC fractions indicating tight association of PP2A with MT in endothelium. Treatment of EC with OA leads to disappearance of MT‐like PP2A staining suggesting dissociation of PP2A from the MT network. Next, we examined the effect of PP2A inhibition on phosphorylation status of MT‐associated protein tau, which in its unphosphorylated form promotes MT assembly. OA caused significant increases in tau phosphorylation confirming that tau is a substrate for PP2A in endothelium. Immunofluorescent experiments demonstrated that the OA‐induced increases in tau phosphorylation strongly correlated with translocation of phospho‐tau to cell periphery and disassembly of peripheral MT. These results suggest the involvement of PP2A‐mediated tau dephosphorylation in alteration of EC MT structure and highlight the potential importance of PP2A in the regulation of EC the MT cytoskeleton and barrier function.

Functional analysis of the Neurospora crassa PZL-1 protein phosphatase by expression in budding and fission yeast

The gene pzl‐1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL‐1 protein, as well as its carboxyl‐terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL‐1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL‐1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL‐1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen‐activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL‐1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Δ strain in high potassium, but only partially complemented the sodium‐hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL‐1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH2‐terminal moieties. Copyright

Okadaic acid-induced inhibition of protein phosphatase 2A enhances chondrogenesis in chicken limb bud micromass cell cultures

The role of major cellular serine/threonine-specific protein phosphatases, protein phosphatase 1 and 2A, was investigated during chicken cartilage differentiation under in vitro conditions. Activity of protein phosphatase 2A decreased parallel to differentiation of chondrogenic cells, whereas activity of protein phosphatase 1 remained unchanged as assayed in the supernatants of the homogenised chicken limb bud micromass cell cultures. When okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A was applied in 20 nM concentration for 4 h during the second and third culturing days, it significantly increased the size of metachromatic cartilage areas measured in 6-day-old colonies. Following okadaic acid treatments, a significant inhibition in the activity of protein phosphatase 2A was found, while the activity of protein phosphatase 1 was unaffected as measured an days 2 and 3. TRITC-phalloidin labelling demonstrated that okadaic acid disorganised actin filaments and induced rounding of chondrogenic cells. This deterioration of actin filaments was reversible. Electron microscopy and biochemical analysis of colonies revealed that the ultrastructure and major components of cartilage matrix remained unchanged under the effect of okadaic acid. Okadaic acid-treatment applied to cultures containing predominantly differentiated chondrocytes (after day 4) did not influence the cartilage formation. 3H- thymidine and bromodeoxyuridine incorporation-assays demonstrated enhanced cell proliferation in the okadaic acid-treated colonies compared to that of the untreated ones. Our results indicate, for the first time, that pro- tein phosphatase 2A is involved in the regulation of chondrogenesis. Inhibition of protein phosphatase 2A with okadaic acid may result in increased chondrogenesis via modulation of proliferation and cytoskeletal or-ganisation, as well as via alteration of protein kinase A-signaling pathway of the chondrogenic cells.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Signalling Enhances Osteogenesis in UMR-106 Cell Line

Presence of the pituitary adenylate cyclase-activating polypeptide (PACAP) signalling has been proved in various peripheral tissues. PACAP can activate protein kinase A (PKA) signalling via binding to pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1), vasoactive intestinal polypeptide receptor (VPAC) 1 or VPAC2 receptor. Since little is known about the role of this regulatory mechanism in bone formation, we aimed to investigate the effect of PACAP on osteogenesis of UMR-106 cells. PACAP 1-38 as an agonist and PACAP 6-38 as an antagonist of PAC1 were added to the culture medium. Surprisingly, both substances enhanced protein expressions of collagen type I, osterix and alkaline phosphatase, along with higher cell proliferation rate and an augmented mineralisation. Although expression of PKA was elevated, no alterations were detected in the expression, phosphorylation and nuclear presence of CREB, but increased nuclear appearance of Runx2, the key transcription factor of osteoblast differentiation, was shown. Both PACAPs increased the expressions of bone morphogenetic proteins (BMPs) 2, 4, 6, 7 and Smad1 proteins, as well as that of Sonic hedgehog, PATCH1 and Gli1. Data of our experiments indicate that activation of PACAP pathway enhances bone formation of UMR-106 cells and PKA, BMP and Hedgehog signalling pathways became activated. We also found that PACAP 6-38 did not act as an antagonist of PACAP signalling in UMR-106 cells.

Calcineurin regulates endothelial barrier function by interaction with and dephosphorylation of myosin phosphatase

AIMS Calcineurin (CN) influences myosin phosphorylation and alters endothelial barrier function; however, the molecular mechanism is still obscure. Here we examine whether CN controls myosin phosphorylation via mediating the phosphorylation state of Thr696 in myosin phosphatase (MP) target subunit 1 (MYPT1), the phosphorylation site inhibitory to the catalytic activity of MP. METHODS AND RESULTS Exposure of bovine or human pulmonary artery endothelial cells (BPAECs or HPAECs) to the CN inhibitor cyclosporin A (CsA) induces a rise in intracellular Ca(2+) and increases the phosphorylation level of cofilin(Ser3) and MYPT1(Thr696) in a Ca(2+)-and Rho-kinase-dependent manner. An active catalytic fragment of CN overexpressed in tsA201 cells decreases endogenous MYPT-phospho-Thr696 (MYPT1(pThr696)) levels. Purified CN dephosphorylates (32)P-labelled MYPT1, suggesting direct action of CN on this substrate. Interaction of MYPT1 with CN is revealed by MYPT1 pull-down experiments and colocalization in both BPAECs and HPAECs as well as by surface plasmon resonance (SPR)-based binding studies. Stabilization of the MYPT1-CN complex occurs via the MYPT1(300PLIEST305) sequence similar to the CN substrate-docking PxIxIT-motif. Thrombin induces a transient increase of MYPT1(pThr696) in BPAECs, whereas its combination with CsA results in maintained phosphorylation levels of both MYPT1(pThr696) and myosin. These phosphorylation events might correlate with changes in endothelial permeability since CsA slows down the recovery from the thrombin-induced decrease of the transendothelial electrical resistance of the BPAEC monolayer. CONCLUSION CN may improve endothelial barrier function via inducing dephosphorylation of cofilin(pSer3) and by interaction with MYPT1 and activating MP through MYPT1(pThr696) dephosphorylation, thereby affecting actin polymerization and decreasing myosin phosphorylation.

ISOLATION AND CHARACTERIZATION OF THE CATALYTIC SUBUNIT OF PROTEIN PHOSPHATASE 2A FROM NEUROSPORA CRASSA

The catalytic subunit of protein phosphatase 2A (PP2Ac) was purified from Neurospora crassa extract by (NH4)2SO4-ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/g with a 2% yield. The apparent M(r) of PP2Ac was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2Ac was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the alpha-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2Ac is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2Ac did not cross-react with N. crassa PP2Ac, indicating sequence differences outside the catalytic core of the enzyme.

Optimalized transient transfection of chondrogenic primary cell cultures

We aimed to find a transfection method which provides high efficiency with minimal cytotoxic and/or apoptotic effects for gene transfer into multilayer primary chondrogenic cell cultures. The pEGFP-C1 plasmid was introduced into the cell culture and the efficiency of transformation quantified by GFP fluorescence; the resulting nucleofection was effective but resulted in severe apoptosis. Two liposomal reagents designed to allow transfection into adherent cells did not deliver the plasmids sufficiently and cartilage formation did not occur. In addition, a third liposomal compound, recommended for transfection into either adherent or suspension cell cultures, lead to acceptable transfection efficiency but no cartilage formation. When an amphiphilic reagent was used however, there was acceptable transfection efficiency as well as cartilage formation. The viability of the cells which were transfected using the amphiphilic reagent remained unaffected but proliferation was severely diminished, particularly in the presence of GFP. In addition, the amount of cartilage decreased when GFP was expressed, despite unchanged levels of mRNAs of sox9 and aggrecan core protein, factors reflecting on the efficiency of chondrogenesis. Overexpression of both the constitutively active delta and gamma isoforms of catalytic subunit of calcineurin, a protein phosphatase described as a positive regulator of chondrogenesis, decreased protein level of Sox9 and subsequent cartilage formation. In conclusion, we found that amphiphilic reagent applied prior to the adhesion of cells provides a useful means to transfer plasmids to primary differentiating chondrogenic cells.