Am J Physiol Lung Cell Mol Physiol 293: L843–L854, 2007.
First published August 10, 2007; doi:10.1152/ajplung.00120.2007.
Invited Review
Regulation of vascular endothelial cell barrier function and cytoskeleton
structure by protein phosphatases of the PPP family
Csilla Csortos,1,2 Irina Kolosova,1,3 and Alexander D. Verin1,4
1
Department of Medicine, Division of Biological Sciences, The University of Chicago, Chicago, Illinois; 2Department of
Medical Chemistry, Research Center for Molecular Medicine, University of Debrecen, Medical and Health Science Center,
Debrecen, Hungary; 3Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Case Western
Reserve University, Cleveland, Ohio; and 4Medical College of Georgia, Vascular Biology Center, Augusta, Georgia
endothelial barrier function; Ser/Thr protein phosphatases
(EC) monolayer serves as a
dynamic, semiselective barrier that regulates transport of fluid
and macromolecules between blood and the interstitium. A
variety of physical, inflammatory, and bioactive stimuli alter
the endothelial barrier, leading to formation of paracellular
gaps thereby increasing vessel permeability and compromising
organ function (37, 106). Appropriate functioning of the EC
barrier, therefore, necessitates proper shape/cytoskeletal structure of individual cells and integrity of the EC monolayer.
Integrity, however, requires uncompromised intercellular junctions and cell-matrix contacts. Vascular EC have gap, adherens, and tight junctions; the latter two play a role in paracellular permeability. These are communicating structures and
contain proteins for several signaling pathways (8, 31, 120).
The integral, transmembrane proteins (VE-cadherin, occludin,
claudins, PECAM, etc.) of junctions, at the cytoplasmic side,
are connected to cytoskeletal filaments (microfilaments, and
intermediate filaments) through linker intracellular proteins
(catenins, ␣-actinin, zonula occludens proteins, etc). The dynamic structures of the three major components of the EC
cytoskeleton, F-actin, microtubules (MT), and intermediate
filaments, are of critical importance in the balance of the
competing contractile and tethering forces determining the
actual shape of the cell. Multiple signal transduction pathways,
regulating EC contraction and barrier function, involve the
activity of several protein kinases [myosin light chain kinase
(MLCK), MAP kinases, protein kinase C, etc.] on junctional
THE VASCULAR ENDOTHELIAL CELL
Address for reprint requests and other correspondence: Alexander D. Verin,
Vascular Biology Center, CB-3210A, Medical College of Georgia, Augusta,
GA 30912-2500 (e-mail: [email protected]).
http://www.ajplung.org
and cytoskeletal/cytoskeleton-associated proteins (37, 106,
173). The activity of protein phosphatases is required to keep
balance with, or to reverse the effect of, protein kinases either
in resting cells or after stimuli. The activity of both the kinases
and the phosphatases is strictly controlled, many of them by
phosphorylation/dephosphorylation. Therefore, to better understand the regulation of EC barrier function, the molecular
and functional analyses of protein kinases and phosphatases in
EC are equally important. Numerous recent findings pertaining
to EC support their complexity both in structure and regulation.
Here we provide an overview on the present information
available about Ser/Thr-specific protein phosphatases involved
in EC barrier regulation.
CLASSIFICATION AND GENERAL PROPERTIES OF SER/THR
PROTEIN PHOSPHATASES
Protein phosphorylation/dephosphorylation is a well-recognized element in almost all signaling pathways of the cell. The
presence or absence of a phosphate group on the Ser, Thr, or
Tyr amino acid side chain of a protein effects its conformation
and may serve as an on and off signal in the regulation of its
physiological activity. As a primary effect, enzymes, for example, may become active or inactive; protein complexes may
form or loosen, etc. The covalent attachment of phosphate
groups is catalyzed by protein kinases, whereas protein phosphatases exhibit the opposite activity.
Protein phosphatases are sorted into three families according to
their capability to dephosphorylate amino acid residues. Ser/Thrspecific protein phosphatases dephosphorylate phosphoserine/
phosphothreonine residues. Tyr-specific protein phosphatases dephosphorylate phosphotyrosine residues alone. Dual specificity
1040-0605/07 $8.00 Copyright © 2007 the American Physiological Society
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Csortos C, Kolosova I, Verin AD. Regulation of vascular endothelial cell
barrier function and cytoskeleton structure by protein phosphatases of the PPP
family. Am J Physiol Lung Cell Mol Physiol 293: L843–L854, 2007. First published
August 10, 2007; doi:10.1152/ajplung.00120.2007.—Reversible phosphorylation
of cytoskeletal and cytoskeleton-associated proteins is a significant element of
endothelial barrier function regulation. Therefore, understanding the mechanisms
of phosphorylation/dephosphorylation of endothelial cell cytoskeletal proteins is
vital to the treatment of severe lung disorders such as high permeability pulmonary
edema. In vivo, there is a controlled balance between the activities of protein
kinases and phosphatases. Due to various external or internal signals, this balance
may be shifted. The actual balances at a given time alter the phosphorylation level
of certain proteins with appropriate physiological consequences. The latest information about the structure and regulation of different types of Ser/Thr protein
phosphatases participating in the regulation of endothelial cytoskeletal organization
and barrier function will be reviewed here.
Invited Review
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EC BARRIER REGULATION AND SER/THR PHOSPHATASES
structure of the more than 50 potential R subunits share no
apparent similarities, except multiple, short interaction sites
with a conserved PP1c binding motif, (R/K)VxF (7, 28). It
seems that the isoforms of PP1c may associate with discrete
pools of R and possess diverse cellular functions. Different
R subunits may direct the PP1 holoenzyme to distinct
subcellular locations and enhance or suppress its activity
toward different substrates (7, 28).
PP2A also has a multimeric structure with a common core of
a dimer of the catalytic (PP2Ac, 36 kDa, 2 isoforms) (135) and
the structural A (PP2Aa, 65 kDa, 2 isoforms) (62) subunit
(Table 1). The latter one serves as a coordinator (57) to
assemble PP2Ac with one of the variable third subunits,
usually called the B subunit. The tertiary structure of PP2Aa
and its regions binding the PP2Ac and B subunits are well
characterized. As PP2Ac is thought to be constitutively associated with the PP2Aa, it implies that this association probably
does not represent a direct regulatory mechanism. Rather, the
activity of the heterotrimeric PP2A forms is controlled by
means of the B subunit termed B, B⬘, and B⬙. At least three
unrelated gene families encode the many isoforms and splice
variants of B subunits (79) (Table 1). The core dimer may also
associate with viral proteins (103, 132), and, in addition, with
a phosphotyrosyl phosphatase activator (PTPA) protein that
reversibly stimulates its variable phosphotyrosyl phosphatase
activity (25). The specific role and regulation of the individual
holoenzyme forms of PP2A are not yet clarified, although
PP2A regulates many cellular processes, and many of its
in vitro substrates were described as reviewed in Ref. 79.
PP2B, also called calcineurin, is a Ca2⫹-dependent enzyme
and it is the least varied in structure among the PPP family of
phosphatases. The enzyme consists of two tightly connected
subunits. Calcineurin A (58 – 64 kDa) is the catalytic subunit,
and calcineurin B (19 kDa) is a Ca2⫹-binding regulatory
subunit (87, 88) (Table 1). The latter subunit is highly conserved and encoded by a single gene, whereas the three
isoforms of calcineurin A are products of three different genes
(157). The NH2-terminal region of the A subunit is the cata-
Table 1. Subunit composition, isoforms, and nomenclature of common serine/threonine protein phosphatase (PPP) enzymes
Abbreviation Used in
the Present Paper
Protein phosphatase 1
Catalytic, C subunit
Regulatory subunits (selection)
Myosin phosphatase target subunit
Myosin binding subunit 85
Myosin phosphatase target subunit 3
TGF-␤-inhibited membrane-associated protein
Protein phosphatase 2A
Catalytic, C subunit
Regulatory, A subunit
Regulatory, B subunits
B subunit
B⬘ subunit
B⬙ subunit
Phosphotyrosyl phosphatase activator
Protein phosphatase 2B
Catalytic, A subunit
Regulatory, B subunit
PP1
PP1c
R
MYPT
MBS85
MYPT3
TIMAP
PP2A
PP2Ac
PP2Aa
PTPA
PP2B
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Alternative Name/
Abbreviation
Isoforms
Gene Symbol (HUGO)
␣, ␤, ␥1, 2
PPP1CA, PPP1CB, PPP1CC
MYPT1, MYPT2
PPP1R12A, PPP1R12B
PPP1R12C
PPP1R16A
PPP1R16B
␣, ␤
␣, ␤
PPP2CA, PPP2CB
PPP2R1A, PPP2R1B
PR52
B56
PR72, PR130
PR53
␣, ␤, ␥, ␦
␣, ␤, ␥, ␦, ε
␣, ␤
PPP2R2A-D
PPP2R5A-E
PPP2R3A-B
PPP2R4
Calcineurin A
Calcineurin B
␣, ␤, ␥
␣, ␤
PPP3CA, PPP3CB, PPP3CC
PPP3R1, PPP3R2
MBS
p85
PR65
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protein phosphatases dephosphorylate both phosphoserine/phosphothreonine and phosphotyrosine residues. The initial classification as type 1 (protein phosphatase 1, PP1) or type 2 (protein
phosphatase 2, PP2) of Ser/Thr-specific protein phosphatases was
based on their biochemical properties (72), such as their substrate specificity and their sensitivity toward heat-stable inhibitor proteins. Type 2 phosphatases were further assorted according to their metal ion dependency as PP2A, PP2B, and
PP2C. Cloning of Ser/Thr-specific protein phosphatases revealed that they are encoded by two different gene families,
termed PPP and PPM (6, 7, 27). PPP and PPM comprise both
classic and more recently identified, but less characterized,
members from different species (4, 7, 27). Tyrosine-specific
and dual-specificity protein phosphatases belong to a distinct
gene family, called PTP.
PP1, PP2A, as well as PP2B, belong to the PPP family
(Table 1), whereas PP2C belongs to the PPM family. These
four enzymes account for the majority of the Ser/Thr phosphatase activity in vivo. The catalytic subunits of mammalian PP1,
PP2A, and PP2B share a highly conserved homologous catalytic domain (40 – 60% identity). Interestingly, although PP2C,
a monomer enzyme with two domains, does not have resemblance in sequence, its three-dimensional structure is remarkably similar to that of the PPP family members (30).
The high versatility of PPP activities is due to multisubunit
structure of its members (Table 1). One or two subunits from
a diverse array of phosphatase regulatory or targeting (R)
proteins associate with the catalytic subunit and create various
holoenzyme forms. The R subunits, i.e., the holoenzyme composition, have high impact on the substrate specificity, localization, and regulation of Ser/Thr-specific phosphatases. Posttranslational modifications (phosphorylation, methylation) of
the catalytic and regulatory subunits are also possible, although
their exact role in the regulation is not yet completely understood (26, 35, 170).
PP1 holoenzymes are mostly dimers of one of the four
isoforms, ␣-, ␤-, ␥1-, or ␥2-, of the catalytic subunit (PP1c,
⬃330 aa) (29, 118), and an R subunit (Table 1). The primary
Invited Review
EC BARRIER REGULATION AND SER/THR PHOSPHATASES
PP1: MYOSIN PHOSPHATASE AND MORE?
Myosin phosphorylation is critical in the regulation of endothelial barrier function. Phosphorylation of the myosin regulatory light chain (MLC) at Ser19 and Thr18 by Ca2⫹/
calmodulin-dependent MLCK results in actin-myosin interaction, stress fiber formation, and cell contraction in nonmuscle
and smooth muscle (SM) cells. ATP, Ca2⫹, calmodulin, and
MLCK were shown as required elements for EC retraction (9,
167, 168), indicating that the phosphorylation level of MLC
has a central, although not exclusive, role in the control of EC
contraction-relaxation and intercellular gap formation. Indeed,
it was shown that inflammatory agonists, thrombin and histamine, produce a rapid increase in MLC phosphorylation and
actomyosin interaction and increased EC permeability (51, 53,
126, 147). Transforming growth factor (TGF)-␤1 also induces
MLC phosphorylation and increases EC permeability; however, the effects are deferred by 1–2 h, suggesting a less direct
effect of the agonist, supposedly involving changes in gene
expression (55). The critical roles of EC MLCK activity and
phosphorylation level of MLC are recognized in the endothelial barrier function based on the above-mentioned and further
experimental data. MLC phosphorylation and permeability
increases were detected after direct introduction of activated
MLCK (143). MLCK inhibitors significantly attenuate the
effects of thrombin and histamine on EC permeability and actin
stress-fiber formation (51, 69, 147). EC MLCK is expressed as
a high-molecular-mass protein (214 kDa) from a single gene on
chromosome 3 in humans; the same gene also encodes the
smaller SM MLCK. Detailed characterization of EC MLCK
revealed both its similarities and unique properties compared
with its counterpart in SM (10, 52, 93, 153, 154).
PP1 activity in EC. Studies with the combined usage of
semiselective PPP inhibitors, such as okadaic acid and calyculin A, and thrombin treatment followed by cell fractionation
AJP-Lung Cell Mol Physiol • VOL
indicated that a substantial portion of PP1 activity is associated
with the myosin filaments and suggested the predominant role
of PP1 activity in MLC dephosphorylation. Calyculin A
(0.1–10 nM), but not okadaic acid (1–100 nM), produced
significant dose-dependent enhancement of both MLC phosphorylation and EC permeability in bovine pulmonary artery
EC (BPAEC); moreover, the majority of the phosphatase
activity in the myosin-enriched fraction of BPAEC was attributed to PP1 (155). Thrombin treatment evoked barrier disruption
and partial translocation of the PP1 catalytic subunit from the
myosin-enriched fraction and inhibited myosin phosphatase activity (125, 151, 155). The mechanism responsible for thrombininduced PP1 translocation is unclear; one possible explanation
is the dissociation of the catalytic subunit from its targeting
subunit. This mechanism was also proposed for the regulation
of glycogen-associated PP1 (127). Further results also point
out the significance of the interaction between PP1c and its
regulatory subunits in the determination of subcellular localization (40, 166). Experiments with rat pulmonary microvascular EC led to a similar conclusion about the pivotal role of
PP1 activity in barrier function. A substantial increase was
detected in the MLC phosphorylation level after PPP inhibition; however, the authors imply that the inhibition of PPP
activities results in loss of barrier function by a mechanism
independent from MLC phosphorylation (34). These results
suggest a critical, but not exclusive, role for MLC phosphorylation and indicate a complex task for PP1 activity in EC.
Further studies with PPP inhibitors suggest a possible wider
substrate spectrum of PP1 among cytoskeleton-associated/regulatory proteins, but the particular regulatory subunits, i.e., the
actual holoenzyme forms, are not clear (83, 146).
The catalytic subunit. All four isoforms of the PP1 catalytic
subunit are present in human and bovine endothelium, as
shown by Northern blot analysis; however, only the ␤-isoform
is stably associated with the actomyosin complex (152). This
suggests that, like in smooth muscle cells, PP1c␤ is responsible
for the dephosphorylation of MLC.
The holoenzyme form of myosin phosphatase. SM MLC
phosphatase, termed myosin phosphatase (MP), is composed of
PP1c␤ and two regulatory subunits, namely, a larger targeting/
regulatory subunit (MP target subunit 1, MYPT1; ⬃110 kDa)
and a smaller regulatory subunit (M20; 20 kDa) (2, 75). The
activity of the holoenzyme is increased by a factor of 10 toward
phosphorylated myosin compared with the PP1c monomer
(80). Human MYPT1 and its spliced variants are encoded by
one single gene on human chromosome 12q15– q21.2 (136).
The domain structure, phosphorylation sites, and regions involved in various interactions of SM MYPT1 are well explored
and understood. The most characteristic features of MYPT1
structure are the short PP1c binding motif (KVKF), which is
close to the NH2 terminus and is followed by the seven ankyrin
repeats and a regulatory/inhibitory phosphorylation site,
Thr696 (75) (Fig. 1). While MYPT1 is expressed in SM and in
most of the nonmuscle cells, its isoform, MYPT2, is expressed
predominantly in skeletal and cardiac muscles and was also
found in the brain (45). The specific role(s) of the small MP
subunit (M20), however, has not yet been established.
Western immunoblot and immunoprecipitation experiments
with cell extracts and fractions from porcine and bovine EC
verified that the endothelial counterpart of SM MYPT1 has a
similar size, ⬃115–130 kDa (65, 156, 158). However, an
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lytic domain, whereas its COOH-terminal region contains
binding sites for the B subunit, calmodulin, and an autoinhibitory domain as well. In the absence of Ca2⫹/calmodulin, the
autoinhibitory domain inhibits the enzyme by binding to the
active site cleft. Ca2⫹ stimulation of PP2B requires association of
Ca2⫹ to both the B subunit and to calmodulin. Calcineurin is
involved in the regulation of several cellular events in mammals,
such as apoptosis, gene regulation, T cell activation, etc. (116).
The study of individual PPP type activities is not easy,
because the in vivo and in vitro substrate specificities of protein
phosphatases are not the same; usually the latter one is broader.
Therefore, several specific inhibitory toxins are utilized in
in vitro assays to differentiate and identify the family members.
For example, okadaic acid and calyculin A are both potent
inhibitors of PP1 and PP2A at nanomolar concentrations. PP2B
is much less sensitive, and PP2C is not affected. Okadaic acid
inhibition of PP2A is stronger than that of PP1, Ki ⫽ 0.2 nM
and Ki ⫽ 3 nM, respectively (128). Many of the naturally
occurring toxins are cell permeable and are utilized in in vivo
studies.
Western blot analysis and cloning work indicates that EC
expresses all four major types of Ser/Thr phosphatases; moreover, a growing body of evidence points to their involvement
in the regulation of endothelial barrier function (34, 100, 121,
139, 151, 152, 155).
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EC BARRIER REGULATION AND SER/THR PHOSPHATASES
Fig. 1. Structural features of myosin phosphatase
subunit 1 (MYPT1) and MYPT1-related proteins.
aa, amino acid.
AJP-Lung Cell Mol Physiol • VOL
protein, which in unphosphorylated (active) form directly
binds actin and bundles actin filaments. As a consequence,
adducin can modulate the lattice structure of the cytoskeleton
and the exposure of transmembrane proteins. Similar to ezrin,
the phosphorylation state of adducin is regulated by activity
balance between ROCK and MP (84). The discovery that ERM
and adducin can bind MYPT1 expanded our understanding of
the MP function from strictly myosin-targeting enzyme to a
broader involvement of MP in control of actomyosin cytoskeleton.
Further MP targeting/regulatory proteins. Several MYPT1/
2-related proteins, MYPT3, TIMAP, and MBS85, were identified and partially characterized recently (Fig. 1).
MYPT3 is a novel PP1c-interacting protein cloned from
3T3-L1 adipocyte cDNA library (130). It shares some structural features with MYPT1/2, namely NH2-terminal ankyrin
repeats and a preceding PP1c binding motif; on the other hand,
its size is considerably smaller, 58 kDa, and it has a COOHterminal prenylation motif suggesting possible membrane association. It was shown that MYPT3 binds PP1c␣ and inhibits
PP1c␥ activity toward phospho-MLC in vitro (130). The regulatory phosphorylation site identified in MYPT1 is not present
in MYPT3 (Fig. 1). However, a more recent paper reports that
MYPT3 can be a substrate for protein kinase A (PKA) and that
the phosphorylation of MYPT3 resulted in PP1c activation
toward phospho-MLC (172). This is in agreement with previous data showing that PKA attenuates agonist-induced EC
barrier dysfunction (15, 99, 111). Furthermore, it was also
found that activation of PKA may improve vascular endothelial
dysfunction (124). Still, the exact physiological role and regulation of MYPT3 is not clear.
Another potential regulator of PP1c is TIMAP (for TGF-␤1inhibited membrane-associated protein), a 64-kDa protein expressed at high levels in endothelial cells. It is highly homologous to MYPT3 and shares its structural features, i.e., PP1c
binding motif, ankyrin repeats, and prenylation motif (Fig. 1)
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additional 70-kDa immunoreactive protein band was also detected in BPAEC with antibodies specific for the entire SM
MYPT1 protein or its NH2- or COOH-terminal fragments,
suggesting the presence of further MYPT1-related proteins in
EC (156). Screening the porcine aortic EC cDNA library
yielded two partial NH2-terminal fragments closely related to
SM MYPT1 (65). Our cloning work also revealed two MYPT1
variants in BPAEC (Kolosova, Verin, unpublished observations) and the same two, and two more potential, variants in
human pulmonary artery EC (HPAEC) (Csortos, Verin, unpublished observations). The two variants differ by the presence or
absence of a central 168-bp/56-aa insert and correspond to
MYPT1 splice variants 1 and 4, which were identified in rat
aorta earlier (33), and to human MYPT1 and variant 2, described by Xia et al. (169) (Fig. 1). Although this region is not
yet recognized as having any special feature, still, it is questionable whether the two alternate forms of MYPT1 have
different localizations and physiological functions in SM
and EC.
Other MP substrates. It is highly possible that MLC is not
the only in vivo substrate for MP; for example, association of
MYPT1 and moesin was shown in Madin-Darby canine kidney
cells, and the same work suggests that MP and Rho-activated
kinase (ROCK) regulate the phosphorylation state of moesin
(47). Moesin is a member of the so-called ERM family of three
closely related proteins: ezrin, radixin, and moesin. Their
NH2-terminal regions contain binding sites for membrane adhesion molecules, whereas their COOH-terminal part may bind
actin; thus they mediate binding of actin filaments with membrane proteins (144). Their conformations may change upon
phosphorylation (by ROCK or PKC) of a conserved Thr side
chain close to their COOH terminus (101, 104). The conformation of nonphosphorylated ERM is folded; the binding sites
are masked; they become unmasked after phosphorylation.
Another MYPT-associated protein was identified as adducin
(84). Adducin is a ubiquitously expressed membrane-skeletal
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EC BARRIER REGULATION AND SER/THR PHOSPHATASES
dissociation of the MP holoenzyme, change in localization, and
the effect of inhibitory proteins (61, 75). Phosphorylation of
Thr696, the inhibitory site of human MYPT1, can be catalyzed
by several kinases including ROCK (43, 81, 85). ROCK,
therefore, may increase MLC phosphorylation by two mechanisms: directly via phosphorylation of MLC at Ser19 and
Thr18 and indirectly via phosphorylation of MYPT1 at Thr696
and Thr853, which leads to MP inactivation and dissociation
from myosin (46).
Substantial work demonstrates the involvement of the vasoactive agent-induced Rho pathway in increased endothelial
permeability (163). For instance, inactivation of RhoA in
cultured endothelial monolayers with C3 toxin caused a decline
in MLC phosphorylation and attenuated the hyperpermeability
evoked by either thrombin, TNF-␣, or diperoxovanadate (23,
39, 54, 162). The permeability enhancing activity of RhoA
appears to be mainly mediated through ROCK, which phosphorylates and inactivates MP in EC (22, 39, 148, 149). Our
recent works have provided further evidence that thrombininduced EC barrier dysfunction in BPAEC and HPAEC involves membrane translocation and direct activation of small
GTPase Rho and ROCK. Translocation of the upstream Rho
activator, guanosine nucleotide exchange factor, p115-RhoGEF, to the membrane was also detected, demonstrating its
involvement in Rho regulation in EC. Furthermore, ROCK
induced phosphorylation of MYPT1 at the inhibitory Thr696
site and at Thr853, which resulted in MP inactivation, accumulation of diphospho-MLC, and cell contraction (11, 16)
(Fig. 2). An interesting and novel observation of our laboratory
demonstrates that MT disruption correlates with increased lung
permeability as well as with decreased MP activity and increased MLC phosphorylation, suggesting the possibility for
cross talk between specific signaling pathways of the MT and
microfilament network (17, 150) (Fig. 2). The critical role of
MT dynamics in thrombin- or TGF-␤1-induced lung vascular
EC barrier dysfunction and the G protein-dependent mechanisms of MT alterations were also shown (12, 14).
Fig. 2. Possible involvements of PPPs in endothelial cell (EC) barrier regulation. MLCK,
myosin light chain kinase.
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(21). Yeast and bacterial two-hybrid screening revealed several
potential protein partners for TIMAP (1, 83). For instance,
TIMAP interacts with the 37/67-kDa laminin receptor (LAMR1).
It was suggested that TIMAP targets PP1c to LAMR1, and
LAMR1 is a TIMAP-dependent PP1c substrate (83). Although
protein-protein interaction between TIMAP and PP1c was shown
by immunoprecipitation, its role in regulating PP1c activity is not
yet clarified. TIMAP mRNA synthesis is strongly downregulated
by TGF-␤1 (21); it is possible to assume that TIMAP may be an
important component of endothelial response to TGF-␤1, including apoptosis, capillary morphogenesis, and barrier dysfunction.
A third putative regulatory subunit, called myosin binding
subunit 85, MBS85 (or p85), was cloned from human genomic
library (138). Similar to other members of MYPT family, it
contains PP1c binding motif and ankyrin repeats, and it also
contains an inhibitory phosphorylation site equivalent to
Thr696 in human MYPT1 (Fig. 1). Using Northern blot and
RT-PCR methods, we found that mRNA homologous to
MBS85 is present both in human and bovine pulmonary
arterial EC, although it apparently differs from MBS85 at the
5⬘-region. We employed different RACE-PCR approaches using HPAEC and BPAEC mRNA, and human heart mRNA
where higher expression level was detected (138), but failed to
identify the very 5⬘-piece of the coding region (Tar K, Kolosova I, Verin AD, unpublished observations). Our previous
Western analysis using anti-MYPT1 antibodies, however,
showed the presence of a ⬃70-kDa protein in EC, which might
be the endothelial homolog of MBS85 (156).
Regulation of MP via Rho pathway. Studies with SM provided evidence that the level of MLC phosphorylation can be
modulated by changes in Ca2⫹ concentration, as MLCK requires Ca2⫹/calmodulin for its activity, or without a change
in the Ca2⫹ concentration, a process called Ca2⫹ sensitization.
The latter process includes the inhibition of MP activity, i.e.,
the balance between MLCK and MP is shifted toward phosphorylation (131). Although the exact mechanism for MP
inhibition has not yet been completely clarified, several mechanisms were suggested, including phosphorylation of MYPT1,
Invited Review
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EC BARRIER REGULATION AND SER/THR PHOSPHATASES
PP2A AND CYTOSKELETON
Much less is known about the involvement of PP2A activity
in the regulation of cytoskeletal structure compared with PP1,
although many data suggest its critical role. Inhibition of PP2A
in the nonmetastatic Lewis lung carcinoma cells, for example,
caused a rapid change in morphology and an increase in their
in vitro invasiveness (77). Translocation and activation of
PP2A during mast cell secretion were reported; furthermore,
both secretion and cytoskeletal reorganization were affected by
PP2A inhibition (66, 98). Gene disruption studies in fission
yeast indicated that PP2A plays a critical role in cell morphogenesis, probably through the regulation of the cytoskeletal
network and cell wall synthesis (107). There are also data
demonstrating a possible role of PP2A in the regulation of
cytoskeletal protein complex formation (107, 113). Cytoskeletal targets of PP2A, and its participation in cell contraction/
relaxation, are not well characterized, although recent data
support its involvement in the regulation of several cytoskeletal
and cytoskeleton-associated protein targets. For instance,
in vitro experiments suggest that PP2A is responsible for the
dephosphorylation of CPI-17 in SM (137). A recent paper
reports that okadaic acid-induced phosphorylation and translocation of MYPT1 is dependent on PP2A, and to varying
extents, on ROCK in HepG2 cells (97). These results suggest
that PP2A might be involved in the regulation of MP.
Some actin-binding proteins, like caldesmon, cofilin, the
small heat shock protein HSP27, and the MT-associated proAJP-Lung Cell Mol Physiol • VOL
tein tau, are further cytoskeletal targets of PP2A (3, 20, 110,
171). A growing body of evidence indicates the association
between PP2A and microtubules and shows the substantial role
of PP2A in MT stability (64, 133, 134). In vitro experiments
demonstrate that only PP2A was able to dephosphorylate
␤III-tubulin and inhibit MT assembly (82), and PP2A was
found to be the major phosphatase dephosphorylating tau in
human brain extract (96). Several data suggest that only the B,
not the B⬘ subunit containing PP2A heterotrimer form, is able
to bind to MT (64, 113). Indeed, B subunit binding is critical
for cell viability under conditions of MT damage in yeast (42).
The participation of PP2A activity in maintaining EC cytoskeletal organization has been suggested rarely until recently
(50, 89). Colocalization of PP2A with MT was shown in
unstimulated EC, but exposure to carcinoma-derived angiogenic products resulted in a more diffuse distribution of PP2A
and a loss of filamentous tubulin (161). Similarly, we found a
substantial amount of PP2A, along with HSP27 and tau, two
phosphorylatable regulators of the cytoskeleton, in the MTenriched fraction of pulmonary artery EC (139). Small HSPs
are proposed to control actin filament dynamics and to stabilize
microfilaments after dissociating from large to small aggregates and after phosphorylation. Following exposure to stress,
HSP27, as a terminal substrate of p38 MAPK-pathway, becomes phosphorylated and promotes F-actin formation (58, 68,
92) (Fig. 2). However, we detected the majority of HSP27 in a
MT-enriched fraction of pulmonary artery EC (139), and
interaction between tubulin/MT and HSP27 in other cell types
has also been reported (63), raising the question of whether
HSP27 is a link between MT and microfilaments in agonistevoked responses. On the other hand, tau is directly related to
MT; its unphosphorylated form supports MT assembly and
inhibits the rate of depolymerization (36). Phosphorylation of
tau by several kinases decreases its ability to bind MT and to
promote MT assembly (60, 95). Tau is predominantly found in
neuronal cells but has been reported in several nonneuronal
cells as well (73, 140, 142). Both HSP27 and tau become
phosphorylated after thrombin treatment or MT disruption (13,
14). We studied both endogenous and overexpressed PP2A in
HPAEC using pharmacological effectors, thrombin and nocodazole, and specific phosphatase inhibitors, calyculin A and
okadaic acid (139, 140). Our data suggest that HSP27 and tau
are potential substrates of PP2A activity with an impact on EC
barrier function. PP2A overexpression significantly attenuated
thrombin- or nocodazole-induced phosphorylation of HSP27
and tau, suggesting a barrier-protective role for this phosphatase (Fig. 2). PP2A-mediated dephosphorylation of these two
cytoskeletal proteins correlates with the PP2A-induced preservation of EC cytoskeleton and barrier maintenance (140).
Most likely, tau and HSP27 are not the sole substrates of
PP2A; rather, the high versatility of PP2A holoenzyme forms
raises the possibility of a more complex role of PP2A in EC
cytoskeleton regulation at different levels. For example, PAK1,
one of the p21-activated kinases (PAKs), which are a group of
recognized p38 activators (174), was found in a protein complex with PP2A isolated from rat brain (160), suggesting that
PP2A may regulate the p38 MAP kinase pathway at different
levels. Another interesting, but unexplored, issue pertains to
intermediate filaments (IF). The organization of the IF network
depends on its phosphorylation state. Selective inhibition of
PP2A, but not PP1, led to hyperphosphorylation and concom293 • OCTOBER 2007 •
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MP activity can also be regulated through a PKC potentiated
inhibitory protein of 17 kDa, called CPI-17 (41). It may affect
isolated PP1c or the holoenzyme form of MP without dissociating its subunits (122). Recent phospho-pivot modeling predicts specific interactions of PP1c with CPI-17 (105). The MP
inhibitory effect of CPI-17 is dramatically increased by its
phosphorylation at Thr38. Even though in vitro, numerous
kinases are capable of phosphorylating CPI-17, including
ROCK, MYPT1-kinase, etc. (75), PKC is thought to be primarily responsible for the in vivo phosphorylation of CPI-17
(41). PKC activation in vascular smooth muscle by phorbol
ester was shown to induce MP inhibition, which led to an
increase both in MLC phosphorylation and in contraction
(102). In addition, contraction of permeabilized SM was induced by phospho-CPI-17 (86, 94). We studied endogenous
and overexpressed CPI-17 in microvascular and macrovascular
EC, respectively. Both the inflammatory agonist histamine,
and, to a much smaller extent, thrombin, were able to evoke
CPI-17 phosphorylation (Fig. 2). Utilizing various approaches,
we found that the contribution of PKC is more significant than
that of ROCK in agonist-induced CPI-17 phosphorylation (90).
These data suggest that MP inhibition/MLC phosphorylation
can be regulated via Rho-ROCK or/and PKC-CPI-17 pathways
depending on the agonist.
The observation that MP activity is inhibited during agonistinduced EC barrier dysfunction suggests a barrier-maintaining/
protective role for MP. Increased release of ATP from EC
during acute inflammation (19) and ATP-induced EC barrier
enhancement (59, 78) were reported. We found, in agreement
with the assumed barrier-protective role of MP, that the mechanism of ATP-induced barrier enhancement involves MP activity (91).
Invited Review
EC BARRIER REGULATION AND SER/THR PHOSPHATASES
PP2B AND EC BARRIER FUNCTION
PP2B is thought to be involved in the maintenance of a
prolonged state of contraction of molluscan smooth muscle
(24), and PP2B isolated from scallop smooth muscle dephosphorylated regulatory MLC prepared from the same source
(74). In nonmuscle cells, tight association was found between
the cytoskeleton and PP2B/calcineurin (44, 109). Human endothelium expresses all three isoforms of calcineurin A, the
catalytic subunit of PP2B (Bako E, Verin AD, unpublished
observations). We also showed PP2B associating with a detergent-insoluble actin-enriched cellular fraction of pulmonary
artery EC (151). Thrombin treatment considerably increased
the activity of PP2B; the activation correlated with the phosphorylation of the PP2B catalytic subunit. Furthermore, pharmacological inhibition of PP2B attenuated thrombin-induced
cytoskeletal protein dephosphorylation and increased thrombin-induced albumin clearance (151). These results suggest
that PP2B may also be involved in the regulation of EC barrier
function. Recent results of Lum et al. (100) confirmed our
observations, as their data provided further evidence that PP2B
activity is a significant participant in EC barrier regulation in
response to thrombin. PP2B inhibition potentiated thrombininduced increase in PKC activity and PKC␣ but not PKC␤
phosphorylation in pulmonary microvascular EC. PP2B inhibition also extended the thrombin-induced barrier dysfunction;
however, downregulation of PKC by phorbol ester treatment
rescued the inhibition of recovery, indicating that PP2B may
play a physiologically important role in returning EC barrier
function to normal through the regulation of PKC (100). In
addition, our data demonstrated that PKA inhibition increased
costaining of actin with both PP2B and MLCK, which correlated with reorganization of focal adhesions, and increased
F-actin content and organization into stress fibers, suggesting a
link between PP2B, MLCK, and PKA activities (111). A
AJP-Lung Cell Mol Physiol • VOL
further question is whether there is a direct or indirect connection between PP2B and other enzymes involved in the regulation of EC cytoskeleton. This requires future studies.
INTERCELLULAR JUNCTION PROTEINS AND PPPS
Tight junctions (TJ) and adherens junctions (AJ) are formed
by several transmembrane adhesion proteins and their intracellular partners. For a detailed review of EC cell-cell junctions,
see Refs. 8 and 31. The organization of AJ and TJ depends on
protein-protein interactions, which in turn are determined in
many cases by the phosphorylation state of proteins. It seems
that both Ser/Thr and Tyr phosphorylation, in many cases of
the same protein, play a regulatory role in TJ and AJ structure
and eventually influence paracellular permeability. For example, serine phosphorylation of cadherin, the major transmembrane component of AJ, leads to specific interactions with one
of its intracellular partners, ␤-catenin; on the other hand,
tyrosine phosphorylation of ␤-catenin reduces its affinity to
bind to cadherin (67, 115). There are further recent data
showing that tyrosine phosphorylation of VE-cadherin prevents binding of ␤-catenin and p120, another member of the
catenin family, and inhibits EC barrier function (112). Furthermore, serine phosphorylation and proteosomal degradation of
␤-catenin are modulated by platelet EC adhesion molecule-1
(PECAM-1), another transmembrane protein in AJ (18). Similarly, tyrosine and serine/threonine phosphorylation of TJ
components is a critical factor in TJ assembly and endothelial
permeability. Localization of the transmembrane protein occludin changes with its phosphorylation state; its highly phosphorylated form was detected to be selectively concentrated at
TJ (117). VEGF increased the phosphorylation level of occludin and its intercellular partner, zona occludens-1 (ZO-1) (5).
However, the amino acid side chains involved are not yet
identified.
Much less is known about the dephosphorylation of AJ and
TJ proteins. The inflammatory barrier-disruptive agents histamine and VEGF both induce a decrease in Ser/Thr phosphorylation of p120 (114, 164), and that must imply protein
phosphatase activity. Okadaic acid and calyculin A, inhibitors
of PP1 and PP2A, induce complete disruption of cell-cell
contacts and increase the Ser/Thr phosphorylation level of
␤-catenin in epidermal cells (123). Another work showed
cellular redistribution of ZO-1 after okadaic acid treatment in
epithelial cells (129). However, the applied concentrations of
PPP inhibitors in these experiments are not suitable to differentiate between PP1 and PP2A activities. A specific study of
PP2A in epithelial cells indicated that it negatively regulates TJ
assembly (108). Expression of PP2Ac prevented TJ assembly;
on the other hand, okadaic acid inhibition of PP2A supported
the phosphorylation and recruitment of ZO-1, occludin, and
claudin-1 to the TJ during junctional biogenesis (108).
Overall, the above data clearly indicate the crucial involvement of PPP activities in AJ/TJ-mediated cellular permeability.
CONCLUSION
During the last decade, it has become clear that EC barrier
function is finely regulated via the coordinated complex actions
of protein kinases and phosphatases on cytoskeletal/cytoskeleton-associated and intercellular junction proteins. The phosphorylation level of these proteins is crucial in determining the
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itant disassembly of vimentin, characterized by a collapse into
bundles around the nucleus in mammalian fibroblasts (145).
Impaired dephosphorylation of vimentin and increased phosphorylation of tau were described in mutant PP2A transgenic
mice (119). Vimentin contains many sites that can be phosphorylated by a variety of kinases with variable impact on IF
structure at different physiological conditions (38, 71, 76). It
will be a difficult task to identify the physiological phosphatase
enzyme(s) of these sites and to learn about their regulation.
Endothelial nitric oxide synthase (eNOS) is considered to be
a major vasoprotective molecule; its function is fundamentally
modulated by phosphorylation at multiple sites (49, 165).
Several agonists, like vascular endothelial growth factor
(VEGF), sphingosine-1-phosphate, or hydrogen peroxide, may
produce Akt activation and phosphorylation of Ser1179 (bovine)/1177 (human) of eNOS, a site with a central role in
eNOS regulation (32, 48, 70, 141). Dephosphorylation of
serine 1179 and one more site (Thr497) by PP2A was reported,
suggesting a key role for PP2A in the control of nitric oxidedependent signaling pathways in vascular EC (56). Moreover,
recent work revealed a new pathway for eNOS regulation
including PP2A, namely, proteasome inhibition did not modulate eNOS phosphorylation and activity by affecting its turnover, but it caused ubiquitination and translocation of PP2A
from cytosol to membrane (159).
L849
Invited Review
L850
EC BARRIER REGULATION AND SER/THR PHOSPHATASES
11.
12.
13.
14.
15.
16.
17.
18.
ACKNOWLEDGMENTS
We express special thanks to Dr. P. Singleton and E. Broman for careful
reading of the manuscript and G. Terbova for assistance in manuscript
preparation.
19.
GRANTS
20.
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-58064, HL-67307, HL-80675, and HL-083327 (A. D. Verin) and
by the Hungarian Scientific Research Fund T043133 (C. Csortos).
21.
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