Csilla Csortos

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.

Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60 src

Inflammatory diseases of the lung are characterized by increases in vascular permeability and enhanced leukocyte infiltration, reflecting compromise of the endothelial cell (EC) barrier. We examined potential molecular mechanisms that underlie these alterations and assessed the effects of diperoxovanadate (DPV), a potent tyrosine kinase activator and phosphatase inhibitor, on EC contractile events. Confocal immunofluorescent microscopy confirmed dramatic increases in stress-fiber formation and colocalization of EC myosin light chain (MLC) kinase (MLCK) with the actin cytoskeleton, findings consistent with activation of the endothelial contractile apparatus. DPV produced significant time-dependent increases in MLC phosphorylation that were significantly attenuated but not abolished by EC MLCK inhibition with KT-5926. Pretreatment with the Rho GTPase-inhibitory C3 exotoxin completely abolished DPV-induced MLC phosphorylation, consistent with Rho-mediated MLC phosphatase inhibition and novel regulation of EC MLCK activity. Immunoprecipitation of EC MLCK after DPV challenge revealed dramatic time-dependent tyrosine phosphorylation of the kinase in association with increased MLCK activity and a stable association of MLCK with the p85 actin-binding protein cortactin and p60 src . Translocation of immunoreactive cortactin from the cytosol to the cytoskeleton was noted after DPV in concert with cortactin tyrosine phosphorylation. These studies indicate that DPV activates the endothelial contractile apparatus in a Rho GTPase-dependent fashion and suggests that p60 src -induced tyrosine phosphorylation of MLCK and cortactin may be important features of contractile complex assembly.Inflammatory diseases of the lung are characterized by increases in vascular permeability and enhanced leukocyte infiltration, reflecting compromise of the endothelial cell (EC) barrier. We examined potential molecular mechanisms that underlie these alterations and assessed the effects of diperoxovanadate (DPV), a potent tyrosine kinase activator and phosphatase inhibitor, on EC contractile events. Confocal immunofluorescent microscopy confirmed dramatic increases in stress-fiber formation and colocalization of EC myosin light chain (MLC) kinase (MLCK) with the actin cytoskeleton, findings consistent with activation of the endothelial contractile apparatus. DPV produced significant time-dependent increases in MLC phosphorylation that were significantly attenuated but not abolished by EC MLCK inhibition with KT-5926. Pretreatment with the Rho GTPase-inhibitory C3 exotoxin completely abolished DPV-induced MLC phosphorylation, consistent with Rho-mediated MLC phosphatase inhibition and novel regulation of EC MLCK activity. Immunoprecipitation of EC MLCK after DPV challenge revealed dramatic time-dependent tyrosine phosphorylation of the kinase in association with increased MLCK activity and a stable association of MLCK with the p85 actin-binding protein cortactin and p60(src). Translocation of immunoreactive cortactin from the cytosol to the cytoskeleton was noted after DPV in concert with cortactin tyrosine phosphorylation. These studies indicate that DPV activates the endothelial contractile apparatus in a Rho GTPase-dependent fashion and suggests that p60(src)-induced tyrosine phosphorylation of MLCK and cortactin may be important features of contractile complex assembly.

Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

The Ca2+/calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210–214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch (amino acids 436–505) that contains potentially important consensus sites for phosphorylation by p60Src kinase (Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256–267). We have now found that both recombinant EC MLCK splice variants exhibit comparable enzymatic activities but a 2-fold reduction ofV max, and a 2-fold increase inK 0.5 CaM when compared with the SM MLCK isoform, whereas K m was similar in the three isoforms. However, only EC MLCK-1 is readily phosphorylated by purified p60 Src in vitro, resulting in a 2- to 3-fold increase in EC MLCK-1 enzymatic activity (compared with EC MLCK-2 and SM MLCK). This increased activity of phospho-MLCK-1 was observed over a broad range of submaximal [Ca2+] levels with comparable EC50 [Ca2+] for both phosphorylated and unphosphorylated EC MLCK-1. The sites of tyrosine phosphorylation catalyzed by p60Src are Tyr464 and Tyr471 within the 69-residue stretch deleted in the MLCK-2 splice variant. These results demonstrate for the first time that p60Src-mediated tyrosine phosphorylation represents an important mechanism for splice variant-specific regulation of nonmuscle MLCK and vascular cell function.

Serine/threonine protein phosphatases in the control of cell function

Reversible protein phosphorylation is a fundamental mechanism by which many biological functions are regulated. Achievement of such control requires the coordinated action of the interconverting enzymes, the protein kinases and protein phosphatases. By comparison with protein kinases, a limited number of protein phosphatase catalytic subunits are present in the cell, which raises the question of how such a small number of dephosphorylating enzymes can counterbalance the action of the more numerous protein kinases. In mammalian cells, four major classes of Ser/Thr-specific phosphatase catalytic subunits have been identified, comprising two distinct gene families. The high degree of homology among members of the same family, PP1, PP2A and PP2B, and the high degree of evolutionary conservation between organisms as divergent as mammals and yeast, implies that these enzymes are involved in fundamental cell functions. Type 1 enzymes appear to acquire specificity by association with targeting regulatory subunits which direct the enzymes to specific cellular compartments, confer substrate specificity and control enzyme activity. In spite of the progress made in determining the structure of the PP2A subunits, very little is known about the control of this activity and about substrate selection. Recent studies have unravelled a significant number of regulatory subunits. The potential existence of five distinct B or B-related polypeptides, some of which are present in multiple isoforms, two A and two C subunit isoforms, raises the possibility that a combinatorial association could generate a large number of specific PP2A forms with different substrate specificity and/or cellular localization. Moreover, biochemical, biological and genetic studies all concur in suggesting that the regulatory subunits may play an important role in determining the properties of the Ser/Thr protein phosphatases and hence their physiological functions.

Role of protein phosphatase 2A in the regulation of endothelial cell cytoskeleton structure

Our recently published data suggested the involvement of protein phosphatase 2A (PP2A) in endothelial cell (EC) barrier regulation (Tar et al. [2004] J Cell Biochem 92:534–546). In order to further elucidate the role of PP2A in the regulation of EC cytoskeleton and permeability, PP2A catalytic (PP2Ac) and A regulatory (PP2Aa) subunits were cloned and human pulmonary arterial EC (HPAEC) were transfected with PP2A mammalian expression constructs or infected with PP2A recombinant adenoviruses. Immunostaining of PP2Ac or of PP2Aa + c overexpressing HPAEC indicated actin cytoskeleton rearrangement. PP2A overexpression hindered or at least dramatically reduced thrombin‐ or nocodazole‐induced F‐actin stress fiber formation and microtubule (MT) dissolution. Accordingly, it also attenuated thrombin‐ or nocodazole‐induced decrease in transendothelial electrical resistance indicative of barrier protection. Inhibition of PP2A by okadaic acid abolished its effect on agonist‐induced changes in EC cytoskeleton; this indicates a critical role of PP2A activity in EC cytoskeletal maintenance. The overexpression of PP2A significantly attenuated thrombin‐ or nocodazole‐induced phosphorylation of HSP27 and tau, two cytoskeletal proteins, which potentially could be involved in agonist‐induced cytoskeletal rearrangement and in the increase of permeability. PP2A‐mediated dephosphorylation of HSP27 and tau correlated with PP2A‐induced preservation of EC cytoskeleton and barrier maintenance. Collectively, our observations clearly demonstrate the crucial role of PP2A in EC barrier protection. J. Cell. Biochem. 98: 931–953, 2006.

Separation of rabbit liver latent and spontaneously active phosphorylase phosphatases by chromatography on heparin- Sepharose

Latent and spontaneously active forms of phosphorylase phosphatase were separated by heparin-Sepharose chromatography of rabbit liver extract. The latent enzyme had an absolute polycation (histone H1, polybrene) requirement for the activity assayed with phosphorylase a and phosphorylase kinase substrates. Ethanol treatment resulted in the activation of both phosphatases by dissociating of 150-180 kDa holoenzymes to 33-38 kDa catalytic subunits as judged by gel filtration. The latent and spontaneously active phosphatases were differentiated according to their abilities to dephosphorylate the alpha and the beta subunits of phosphorylase kinase and sensitivities to inhibition by inhibitor-2 or heparin, and were classified as type-2A and type-1 phosphatases, respectively.

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.

TIMAP is a positive regulator of pulmonary endothelial barrier function

TGF-beta-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.

Cytoskeletal mechanisms regulating vascular endothelial barrier function in response to acute lung injury

Endothelial cells (EC) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. In acute lung injury (ALI) the EC barrier is weakened leading to increased vascular permeability. It is widely accepted that EC barrier integrity is critically dependent upon intact cytoskeletal structure and cell junctions. Edemagenic agonists, like thrombin or endotoxin lipopolysaccharide (LPS), induced cytoskeletal rearrangement, and EC contractile responses leading to disruption of intercellular contacts and EC permeability increase. The highly clinically-relevant cytoskeletal mechanisms of EC barrier dysfunction are currently under intense investigation and will be described and discussed in the current review.

Characterization of the protein phosphatase 1 catalytic subunit in endothelium: involvement in contractile responses.

We have previously demonstrated the direct involvement of a type 1 Ser/Thr phosphatase (PPase 1) in endothelial cell (EC) barrier regulation [Am. J. Physiol. 269:L99–L108, 1995]. To further extend this observation, we microinjected either the Ser/Thr PPase inhibitor, calyculin, or the PPase 1 inhibitory protein, I‐2 into bovine pulmonary artery EC and demonstrated both an increase in F‐actin stress fibers and a shift from a regular polygonal shape to a spindle shape with gaps apparent at the cell borders. Northern blot analysis with specific cDNA probes revealed the presence of three major PPase 1 catalytic subunit (CS1) isoforms (α, δ, and γ) in human and bovine EC. To characterize the myosin‐associated EC CS1 isoform, myosin‐enriched bovine EC fraction was screened with anti‐CS1α and anti‐CS1δ antibodies The anti‐CS1δ antiserum, but not anti‐CS1α antiserum cross reacts with the CS1 isoform present in myosin‐enriched fraction and CS1δ was found in stable association with EC myosin/myosin light chain kinase (MLCK) complex in MLCK immunoprecipitates under nondenaturing conditions. Consistent with these data, overexpression of CS1δ‐GFP construct in bovine endothelium followed by immunoprecipitation of CS1 with anti‐GFP antibody revealed the stable association of CS1δ with actomyosin complex. Finally, screening of a human EC oligo(dT)‐primed cDNA library with a probe encoding a rat CS1δ cDNA segment yielding several positive clones that encoded the entire CS1δ open reading frame and partially noncoding regions. Sequence analysis determined a high homology (≈99%) with human CS1δ derived from a teratocarcinoma cell line. Together, these data suggest that CS1δ is the major of PPase 1 isoform specifically associated with EC actomyosin complex and which participates in EC barrier regulation. J. Cell. Biochem. 79:113–125, 2000.