Myosin phosphatase: Structure, regulation andfunction

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197

Myosin phosphatase: Structure, regulation and function

Masaaki Ito,

¹

Takeshi Nakano,

¹

Ferenc Erd di

²

and David J. Hartshorne

³

1First Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie, Japan; ²Department of Medical Chemistry, Medical and Health Science Center, University of Debrecen, Debrecen, H-4026, Hungary; ³Muscle Biology Group, University of Arizona, Tucson, AZ, USA

Abstract

Phosphorylation of myosin II plays an important role in many cell functions, including smooth muscle contraction. The level of myosin II phosphorylation is determined by activities of myosin light chain kinase and myosin phosphatase (MP). MP is composed of 3 subunits: a catalytic subunit of type 1 phosphatase, PP1c; a targeting subunit, termed myosin phosphatase target subunit, MYPT; and a smaller subunit, M20, of unknown function. Most of the properties of MP are due to MYPT and include binding of PP1c and substrate. Other interactions are discussed. A recent discovery is the existence of an MYPT family and members include, MYPT1, MYPT2, MBS85, MYPT3 and TIMAP. Characteristics of each are outlined. An important discovery was that the activity of MP could be regulated and both activation and inhibition were reported. Activation occurs in response to elevated cyclic nucleotide levels and various mechanisms are presented. Inhibition of MP is a major component of Ca²⁺-sensitization in smooth muscle and various molecular mechanisms are discussed. Two mechanisms are cited frequently:

(1) Phosphorylation of an inhibitory site on MYPT1, Thr696 (human isoform) and resulting inhibition of PP1c activity. Sev- eral kinases can phosphorylate Thr696, including Rho-kinase that serves an important role in smooth muscle function; and (2) Inhibition of MP by the protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17). Examples where these mechanisms are implicated in smooth muscle function are presented. The critical role of RhoA/Rho-kinase signaling in various systems is discussed, in particular those vascular smooth muscle disorders involving hypercontractility. (Mol Cell Biochem 259: 197–

209, 2004)

Key words: myosin phosphatase, type 1 phosphatase, MYPT1, Rho, Rho-kinase, CPI-17, smooth muscle, Ca²⁺ sensitization, cGMP-dependent protein kinase

Introduction

Phosphorylation of smooth muscle and non muscle myosin II is implicated in many physiological phenomena, including smooth muscle contraction, cell motility and cytokinesis [1]. A distinct phosphatase, termed myosin phosphatase (MP), is responsible for dephosphorylation of the phosphorylated myosin light chain and this is involved in relaxation of smooth muscle. The latter has attracted much recent attention since the inhibition of MP via a G-protein-coupled mechanism is a major contributor to agonist-induced Ca²⁺ sensitization in

many smooth muscles [2, 3]. The opposite effect, Ca²⁺ desensitization, implicating an activation of MP by cyclic nucleotides has also been observed [4, 5]. Thus, MP is a key regulatory component and by altering levels of myosin II phosphorylation can enhance or diminish the contractile/

motile response at a given Ca²⁺ concentration.

The initial characterization of MP from chicken gizzard was reported over a decade ago [6] and some of the earlier studies have been reviewed [7]. The aims of the present review will be to summarize earlier studies and to focus on more recent achievements within the last 5 years, or so.

Address for offprints: M. Ito, First Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie 514-8507, Japan (E-mail: naika1@clin.medic.mie-u.ac.jp)

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Structure of myosin phosphatase

Smooth muscle myosin phosphatase is a heterotrimer composed of a 38 kDa catalytic subunit of type 1 phosphatase δ isoform (PP1cδ), and two regulatory subunits, a 110 kDa target/regulatory subunit (myosin phosphatase target subunit 1;

MYPT1) and a 20 kDa small regulatory subunit (M20) [6, 7]. MYPT1 interacts with PP1cδ at the N-terminus and with M20 at the C-terminus, thereby forming the holoenzyme of MP (Fig. 1).

MYPT1

MYPT1 has been cloned from several species and many isoforms identified. The avian and mammalian isoforms contain about 1000 amino acids with an approximate mass of 110 kDa [7]. A characteristic structure of all MYPT1 molecules is the ankyrin repeat region at the N-terminus containing 7 ankyrin repeats. This region is one of the more conserved regions among MYPT isoforms. At the N-terminal margin of the ankyrin repeats is a PP1c-binding motif (K/R-I/V-X-F/W).

In some isoforms there are leucine zipper motifs (four hep- tad repeats) at the extreme C-terminus.

The MYPT gene seems to be a housekeeping gene [8] since MYPT1 is expressed in many tissues, although present at higher concentrations in smooth muscle [9, 10]. MYPT1 is encoded by a single gene on human chromosome 12q15-q21.2 [11]. The different MYPT1 isoforms arise from splicing varia- tions. One variation reflecting a cassette-type alternative splicing exon is located at the center of the molecule [12]. In chicken MYPT1, there are two isoforms representing the absence or presence of a central 123 nucleotide exon, termed M130 and M133, respectively [13]. In developing chicken gizzard there is a switching between these two isoforms at about the time of hatching. Increased expression of the smaller isoform was coincident with loss of Ca²⁺-sensitization (detected using GTPγS) thus suggesting a possible role for the central insert in Ca²⁺-sensitization [14]. In rat, the alternative exon found in chicken is constitutive and two immediately

downstream exons are alternative, resulting in five isoforms of the central insert [12]. The isoform containing the longest insert, originally reported as rat3 [15], is equivalent to human MYPT1 shown in Fig. 1.

The other splicing variant reflects the presence or absence of the C-terminal leucine zipper motifs. In chicken, skipping of a 31-nucleotide alternative exon generates a leucine zipper positive isoform, whereas inclusion of the exon shifts the reading frame, inducing a premature stop codon and resulting in the leucine zipper-negative MYPT1 [16]. This C-terminal splicing is developmentally regulated and tissue selective.

In aorta, the exon-excluded transcript (leucine zipper positive) is expressed predominantly. Other tissues, such as the portal vein, uterus and intestinal tracts, contain a combina- tion of the two transcripts. In chicken gizzard, the expression of MYPT1 is developmentally regulated, with switching of exon exclusion to inclusion about the time of hatching [16].

One of the important aspects of MYPT1 is that it is a platform for interaction with multiple ligands. Several proteins have been reported to bind to MYPT1. Obviously, the phosphorylated myosin-MYPT1 interaction is important. One binding site for myosin is within the C-terminal half of the ankyrin repeats [17, 18] and it is suggested that binding of the phosphorylated light chain with this region could reduce the K_m of PP1c for its substrate [19]. Phosphorylation of the ankyrin repeats by protein kinase C (PKC) was shown to attenuate binding of phosphorylated MLC to the N-terminal fragment of MYPT1, implying that this interaction may be modulated via phosphorylation [20]. An additional/alternative site for myosin binding, probably via its rod region, is proposed to be within the C-terminal part of MYPT1 and this may target MP to the myosin filaments [15]. An interesting suggestion was that phosphorylation of Thr850 (Thr853 in human MYPT1) by Rho-kinase reduced binding to myosin [21].

Other interactions with MYPT1 include: the binding of GTP-bound active RhoA (but not the inactive GDP-bound form) to the C-terminal region of MYPT1 [22]. Whether this interaction has any physiological role is not known, but it could represent an alternative (to the plasmalemma) docking

1 MYPT1 1030

PP1cδδδδ M20

35KVKF³⁸ (R/K-V/I-X-F/W motif)

Ankyrin repeats Leucine zipper

motif

Fig. 1. Subunit structure of smooth muscle myosin phosphatase.

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site for GTP·RhoA; the interaction of leucine zipper regions of cGMP-dependent protein kinase Iα (cGKIα) and MYPT1 [23]. This interaction may localize cGKIα to the contractile apparatus in smooth muscle and facilitate relaxation in response to increased cGMP levels; and the interaction of acidic phospholipids with the C-terminal region of MYPT1 [24]. This interaction inhibits PP1c activity and binding of phospholipids is reduced by phosphorylation of MYPT1 by cAMP-dependent protein kinase (PKA). Under certain conditions MYPT1 is localized at the plasmalemma and at cell-cell contacts [25, 26] and the affinity of MYPT1 for acidic phospholipids may be a factor in the membrane location. The various interactions of MYPT1 are summarized in Fig. 2.

Other MYPT isoforms

Within the newly-emerging MYPT family there are four other molecules with varying degrees of similarity to MYPT1, namely MYPT2, MBS85, MYPT3 and TIMAP (TGF-β-inhibited membrane associated protein).

MYPT2 was cloned from human brain library using a cDNA probe based on MYPT1 [27]. It contains 982 residues with a mass of 110 kDa. MBS85 was identified as a substrate for myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) and is composed of 782 residues and a mass of 85 kDa [28]. The genes for MYPT2 and MBS85 are found on human chromosomes 1q32.1 and 19q13.3-13.4, respectively.

MYPT3 was isolated by the yeast two hybrid screen of a mouse adipocyte cDNA library using PP1cα as bait. It con-

P P P

1

Thr696

MYPT1 1030

RhoA-binding domain

Phospholipid-binding domain Myosin-binding domain

M20-binding domain PP1cδ-binding domain

cGKIα-binding domain Phosphorylated

MLC-binding domain

Ser432/Thr435

P P

Thr853

P P

1 296

170 296

714

930

933 934

668

1030 1006

1030

Mitotic-specific phospohrylation sites Mitotic-specific phospohrylation sites

Inhibitory phosphorylation site

Myosin-binding regulatory phosphorylation site

849 1030

Fig. 2. Domain structure of human MYPT1 indicating phosphorylation sites and regions involved in various interactions.

tains 524 residues, a mass of 58 kDa and the gene is located on mouse chromosome 15 [29]. TIMAP was detected by representational difference analysis of the glomerular cell response to transforming growth factor-β1 (TGF-β1). This novel gene was mapped to human chromosome 20.q11.22.

[30].

For these four molecules the two more similar to MYPT1 are MYPT2 followed by MBS85 with overall identities (to MYPT1) of 61 and 39%, respectively. Within these MYPT isoforms there are three conserved regions: The N-terminal ankyrin repeats; a central sequence spanning the inhibitory phosphorylation site (see section ‘Inhibition of myosin phosphatase by MYPT1 phosphorylation’); and C-terminal leucine zipper motifs. Identities for these regions of MYPT2 and MBS85 compared to MYPT1 are: ankyrin repeats, 73 and 53%, respectively; central sequence, 91 and 86%, respectively; and the C-terminal motifs both 88%. Each MYPT isoform also contains a PP1c-binding motif at the N-terminal edge of the ankyrin repeats: KVKF in MYPT1, RVRF in MYPT2; and RTVRF in MBS85.

MYPT2 has two splicing variants with differences at the C-terminal end [31]. The smaller MYPT2A (as originally cloned [27]; Fig. 3) is the product of alternatively spliced exon 24 and termination at the stop codon in exon 25. The larger MYPT2B (998 residues and mass of 112 kDa) is the product of exon 24-including mRNA and its stop codon is located in exon 24. Therefore, the C-terminal 28 residue sequence of MYPT2A is permuted to different sequences, although both isoforms contain the leucine zipper motifs. The same alternative splicing variants are observed in the heart-

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specific M20 subunit (hHS-M₂₁) which is a product of the MYPT2 gene (see section ‘M20’) [31]. The interaction of hHS-M₂₁ with MYPT2 appears weaker than the correspond- ing interaction of M20 and MYPT1 and thus the MYPT2- containing MP may be a heterodimer of MYPT2 and PP1cδ [31]. MYPT2 is expressed preferentially in heart, skeletal muscle and brain [27, 31] and in a specific complex with PP1cδ isoform may be the targeting subunit of MP in striated muscle [32].

MBS85 is ubiquitously expressed, although higher levels were detected in cardiac muscle [28]. As with MYPT1, the N-terminal region of MBS85 contains ankyrin repeats and the canonical PP1c-binding motif, suggesting binding to PP1c and phosphorylated MLC. A point of considerable interest is that the activity of the MBS85 holoenzyme was inhibited by phosphorylation of Thr560 in the inhibitory region by MRCKα.

MYPT3 is 30–40% identical with other members of the MYPT family and contains several features similar to MYPT1/

2, hence its name. It has 5 N-terminal/central ankyrin repeats and an N-terminal PP1c-binding motif (KHVLF). MYPT3 inhibited PP1cγ activity towards both phosphorylase a and phosphorylated gizzard myosin, in contrast to the MYPT1- PP1cδ complex that activates phosphatase activity with phosphorylated myosin. The regulatory phosphorylation site found in other MYPT family members is not present in MYPT3. However, other distinct signaling motifs are present in MYPT3 and not in MYPT1, including: an ATP/GTP binding motif (Walker A type); two SH3 sites; and a C-terminal

prenylation motif [29]. The latter suggests possible membrane association. In the mouse, MYPT3 was widely distributed and was high in heart, brain and kidney. Comparison of MYPT3 with genomic sequences revealed several homologous gene products and it was suggested that a distinct and previously unidentified subfamily of myosin targeting subunits exist in a variety of species.

In many respects the structure of TIMAP is similar to MYPT3 (44.7% identity). It consists of 567 residues (human isoform) mass 64 kDa and contains N-terminal nuclear localization consensus sequences, five ankyrin repeats, a PP1c- binding motif (KVSF) at the N-terminal margin of the ankyrin repeats and a CAAX box at the C-terminus (Fig. 3). Similar to MYPT3, it is suggested that TIMAP is prenylated and subject to plasamalemma localization. High expression of TIMAP was observed in endothelial and hematopoietic cells and mRNA distribution suggested expression also in CNS, adrenal, lung and spleen. The function of TIMAP is not defined but may involve interaction with PP1c. It is suggested that the transcriptional repression of TIMAP by TGF-β1 may be an important component of the apoptotic and/or capillary morphogenesis response of endothelial cells [30].

Specific roles for the above four isoforms are not established. It is assumed that MYPT2-PP1c is associated with dephosphorylation of striated muscle myosin, but alternative substrates are possible. For MBS85, MYPT3 and TIMAP very little is known and potential substrates associated with identified cell functions and cell localization(s) need to be established. Identification of regulatory mechanisms and their

Ankyrin repeats Leucine

zipper motif

110 kDa 57 188 216 316

Thr646

1 982

MYPT2

115 kDa 1

39 170 198 296

Thr696

P 1030

P MYPT1

P P

662 708 1007

613 658 959

50 198 226 324 1

P P

527 572

MBS85 782 ^{85 kDa}

100

Thr560

Phosphorylation inhibitory motif

759

528

567 1

1 1

TIMAP MYPT3

70

67 168

165 231

228 296

293

58 kDa

64 kDa CAAX

motif

Fig. 3. Human MYPT family.

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integration with known signaling pathways remains a major objective.

M20

M20 (also termed M₂₁ or M_18/21) is the small non-catalytic subunit of MP that binds to the C-terminal part of MYPT1 [7]. The smooth muscle M20 has been cloned only from chicken gizzard [33] in which there are two splicing varia- tions of 161 (M₁₈) and 186 (M₂₁) residues (masses approxi- mately 18 and 21 kDa, respectively) [34]. These isoforms differ in their C-termini and the larger isoform contains leucine zipper motifs. Expression of each isoform is tissue specific. M₁₈ is the major species in gizzard, both isoforms are equally expressed in stomach and M₂₁ appears to be the major isoform in some mammals, i.e. rat and ferret [34]. An interesting feature is that the last 120 residues of M₂₁ are re- markably similar to the last residues of MYPT2 (91% identity, 100% similarity) and this prompted the suggestion that M₂₁ may be produced from the gene encoding MYPT2 [32].

A similar situation exists between telokin and the smooth muscle myosin light chain kinase gene. However, it is sur- prising that the M20 subunit is not detected in those tissues expressing MYPT2, i.e. brain and striated muscle [32].

The human heart-specific M₂₁ has also been cloned [31].

Again, there are two splicing variants whose sequences match the C-terminal sequences of two MYPT2 isoforms, A and B.

As these M20 isoforms are expressed only in heart it is suggested that the N-terminal part of smooth muscle M20 is encoded within an intron of the MYPT2 gene and that the C- terminal residues are translated from the following MYPT2 exon [31, 32]. Cloning of the human smooth muscle M20 is required to test this hypothesis.

The function of M20 in smooth muscle is not established, although it was suggested that the heart-specific M₂₁ subunits increased sensitivity to Ca²⁺ in permeabilized renal artery (and cardiac myocytes) [31]. Binding of M20 to MYPT1 does not affect phosphatase activity [7]. At high molar excess it was proposed that M20 could bind to the myosin dimer [15]. Also the suggestion was made that M20 may bind to microtubules and influence microtubule dynamics [35].

PP1c

In mammals the catalytic subunits of type 1 phosphatase (PP1c) are products of three genes, i.e. α, γ and δ. The α and γ isoforms each have two splicing variants, thus generating five possible PP1c isoforms. Of these isoforms it is thought that MYPT1 and MBS85 bind specifically to PP1cδ. The differences between the PP1c isoforms reside in the N- and

C-terminal sequences [36]. However, the role of differing sequences is not appreciated with respect to PP1c function and interactions. It is possible that the sequences unique to each isoform are involved in interactions with specific target/regulatory proteins.

Subunit interactions

The heterotrimer characteristic of smooth muscle MP is con- structed by binding of PP1cδ to the N-terminal region of MYPT1 and binding of M20 to its C-terminal region. The binding sites for PP1c involve the 38 N-terminal residues and the ankyrin repeats and possibly the acidic region C-terminal to the ankyrin repeats [18, 19, 37, 38]. Within these multiple binding sites there appears to be a hierarchy of interactions.

From studies using surface plasmon resonance it appeared that the PP1c-binding motif (³⁵KVKF³⁸) is the crucial site [38]. Following the prerequisite interaction at this site subsequent sites are utilized. These include: interaction with residues 1–22; binding to the ankyrin repeats (residues 40–

296); and interaction within the sequence of residues 304–

511. The binding of PP1c to residues 1–296 is relatively strong and this fragment of MYPT1 has been used to affinity-purify PP1c [19]. Interaction of PP1cδ with MYPT1 alters the substrate specificity of the catalytic subunit and, in general, the various interactions of PP1c and MYPT1 are re- flected by alterations in MP activity. The overall effect for the MP holoenzyme compared to the isolated PP1c is en- hanced activity toward phosphorylated myosin and inhibition of activity with phosphorylase a [15]. Activation of PP1c activity by MYPT1 or, various fragments is usually 10–15 fold. The shortest fragment of MYPT1 capable of PP1c activation is the N-terminal sequence, 1–38. However, full activation is only achieved with the 1–296 fragment, i.e.

combining the ankyrin repeats [19, 38]. Also, it was suggested that the acidic region (residues 324–372) was involved in PP1c activation [18]. For the 1–296 fragment it was proposed that residues 1–38 were involved in activation of PP1c (ef- fect on k_cat) and that the ankyrin repeats were involved in substrate binding (K_m) [19]. The latter is consistent with binding of phosphorylated MLC to the ankyrin repeats [18].

M20 binds to the C-terminal part of MYPT1. The leucine zipper motifs, found in some isoforms of MYPT1 and M20, are not required for interaction. Within MYPT1 the binding site for M20 lies in the C-terminal 72 residues of the leucine- zipper-minus MYPT1 isoform (chicken M133 isoform) and within M20 the site for MYPT1 is located on the N-terminal 120 residues [15]. Recently, it was shown that the region of M20 including residues 77–116 is an α-helix and can form a homodimer or, a heterodimer via coiled-coil interaction with MYPT1 [39].

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Regulation of myosin phosphatase activity

Inhibition of MP via a G-protein coupled mechanism is thought to be responsible for Ca²⁺-sensitization of the contractile response in smooth muscle [2, 3]. Inhibition of MP is more extensively documented and several putative mechanisms have been proposed [7]. These include: phosphorylation of MYPT1; dissociation of the MP holoenzyme; a change in targeting function or localization; and inhibition by inhibitory proteins (e.g. CPI-17). Activation of MP has also been proposed, but molecular mechanisms are not defined. In general, activation of MP is thought to be linked to an increase in cyclic nucleotides and would promote Ca²⁺-desensitization.

In the following section various aspects of MP regulation will be presented.

Inhibition of myosin phosphatase by MYPT1 phosphorylation

This idea originated from results with α-toxin permeabilized rabbit portal vein in which incubation with ATPγS induced Ca²⁺-sensitization of force, an increase in MLC phosphorylation and the concomitant thiophosphorylation of MYPT1. MP activity also was inhibited, under these conditions [40]. Sub- sequently, it was found that phosphorylation of MYPT1 at Thr695 by an endogenous kinase inhibited activity of the gizzard MP holoenzyme [41].

There is considerable support for the participation of the monomeric G protein, RhoA in Ca²⁺-sensitization of several smooth muscles [7] and indeed the first identified kinase that achieved phosphorylation of MYPT1 and MP inhibition was Rho-kinase [22]. The latter is a member of the myotonic dystrophy family of protein kinases and its Ser/Thr kinase activity is activated by GTP·RhoA [42]. There are two major phosphorylation sites for Rho-kinase on avian MYPT1, Thr695 and Thr850 (equivalent sites for the human MYPT1 isoform are Thr696 and Thr853; see Fig. 2). Thr695/696 is the inhibitory phosphorylation site [43].

Several other kinases also can phosphorylate the inhibitory site on MYPT1. A ZIP-like kinase (more recently termed MYPT1 kinase) thought to be a kinase endogenous to the MP holoenzyme, was effective in this regard [44]. Also, a constitutively-active fragment of ZIP kinase elicited contraction of β-escin permeabilized rabbit ileal smooth muscle at low Ca²⁺ concentrations and increased phosphorylation of the inhibitory site in MYPT1 [45]. Integrin-linked kinase (ILK) also may be one of the kinases endogenous to the MP holoenzyme. Native ILK is effective in inhibiting MP activity via phosphorylation of the inhibitory site on MYPT1 [46]. In platelet cytoskeleton ILK was also identified as the major

kinase that phosphorylates and inhibits myosin phosphatase activity [47]. Phosphatase inhibition was correlated with the extent of the phosphorylation at Thr695. In addition, these kinases (Rho-kinase, MYPT1 kinase and ILK) can also directly phosphorylate MLC at Ser19 and Thr18 (the sites phosphorylated by MLCK) and have been implicated in Ca²⁺-independent contraction of smooth muscle [48–52].

Two other kinases regulated by members of the Rho family of monomeric G proteins are capable of phosphorylating the inhibitory site of MYPT1. These are myotonic dystrophy protein kinase (DMPK) [53] and p21-activated protein kinase (PAK) [54]. DMPK is activated by Rac-1 and PAK by Rac and/or Cdc 42. In addition, the Ser/Thr kinase Raf-1 (a component of the ERK1/ERK2 MAPK pathway) can associate with MP [55]. Stimulation of Raf-1 (by Ras GTP) phosphorylates MYPT1 at the inhibitory site, under physiological conditions, leading to MP inhibition and cytoskeletal reorganization. Overexpression of a constitutively-active fragment of DMPK in lens cells also led to reorganization of the actin cytoskeleton and apoptotic-like blebbing of the plasmalemma [56].

The molecular basis for inhibition of MP by phosphorylation of the inhibitory site of MYPT1 is not understood. PP1c binds to the N-terminal half of the molecule. In vitro studies showed that phosphorylation of MP by Rho-kinase caused phosphatase inhibition with several substrates [43], notably phosphorylase a. The inhibition was due mainly to a decrease in V_max. However, from these studies the theory that inhibition reflects dissociation of MP can be discounted since the dissociated PP1c would have increased activity with phos- phorylase a. It is possible that the phosphorylated Thr696 interacts with the PP1c associated with MYPT1, either at the active site or, by inducing conformational changes in PP1c.

For MBS85 it was shown that phosphorylation of Thr560 was essential for interaction with PP1c and the phosphorylated peptide (representing the conserved region of 50 residues flanking the phosphorylation site) inhibited PP1cδ activity [28]. Also, the phosphorylated C-terminal fragment of MYPT1, but not the dephosphorylated form, inhibited PP1c activity [46]. A tentative model for inhibition of PP1c activity as a consequence of MYPT1 phosphorylation therefore is that Thr696 interacts either with the active site of PP1c or, disrupts PP1c-substrate interaction. For intramolecular inhibition this would require that the MYPT1 molecule folds to facilitate the Thr696-PP1c interaction. There is no evidence for or against intermolecular inhibition, an example of which could be an antiparallel dimer of MYPT1. That MYPT1 may form a dimer has been suggested previously [18, 39]. All models at this stage are speculative and further data are required to generate a more realistic mechanism for inhibition.

The physiological significance of MYPT1 phosphorylation at the inhibitory site has been addressed in both smooth muscle fibers and various cell types. In fibroblasts it was

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shown using a site- and phosphorylation-state specific anti- body for P-Thr695, that stimulation with lysophosphatidic acid increased phosphorylation at Thr695 and that this phosphorylation was sensitive to Y-27632 (a Rho-kinase inhibitor) [43]. RhoA or Rho-kinase also increased phosphorylation at the inhibitory site in platelets stimulated by thromboxane A₂ analogues [57] and in cultured vascular smooth muscle cells [58, 59]. However, recent reports have pointed out that phosphorylation of MYPT1 may not correspond to Ca²⁺- sensitization during the contractile cycle in smooth muscle [60, 61]. In permeabilized portal vein the level of Thr695 phosphorylation was not changed in response to agonist or GTPγS, but the levels of CPI-17 phosphorylation at Thr38 (see section on CPI-17) and phosphorylation of MYPT1 at Thr850 showed a correlation with force and MLC phosphorylation [60]. In intact and permeabilized rabbit vas deferens, where CPI-17 expression is low, a similar pattern for phosphorylation of Thr695 and Thr850 was observed [60]. This was reported also in rabbit femoral artery [61]. In contrast, a positive correlation between Thr695 phosphorylation and tension was observed in rabbit aorta stimulated by prostaglandin F_2α [62]. The reasons for this controversy are not clear.

They may lie partly in the types of cells/tissues employed or, methods of stimulation, but clearly further studies are necessary to clarify the situation.

Another confusing aspect is the diversity of kinases capable of phosphorylating Thr695 and inhibiting MP activity.

Since RhoA has been shown to be important in the Ca²⁺- sensitization pathway it is reasonable to expect a downstream role for Rho-kinase. For the other kinases, that are components of different signaling pathways, their roles with respect to the contractile cycle of smooth muscle are less obvious.

However, for the cell studies it is possible that a variety of stimuli could signal to different pathways that converge at phosphorylation of Thr695 of MYPT1. For example, these diverse pathways could alter aspects of cytoskeletal structure or function. The integration of these pathways and an under- standing of their roles in cell function is an important objective.

Regulation by subunit dissociation and targeting function In permeabilized smooth muscle maintained at constant Ca²⁺

levels stimulation with arachidonic acid increased force and MLC phosphorylation and decreased phosphatase activity [63]. The complex of MYPT1-PP1cδ has higher activity towards phosphorylated myosin than isolated PP1cδ, thus dissociation of the MP holoenzyme would decrease myosin phosphatase activity. It was suggested that arachidonic acid operated via this mechanism and induced dissociation of the MP holoenzyme [63]. In addition, it was shown that the arachidonic acid effect was sensitive to Y-27632, thus implicat-

ing Rho-kinase in the arachidonic acid-induced Ca²⁺-sensitization [64, 65]. Purified Rho-kinase is activated by arachidonic acid independent of RhoA [66] and an alternative explanation for the arachidonic acid effect on smooth muscle fibers is via direct activation of Rho-kinase.

A change in targeting function may also regulate MP activity. Thr850, one of the major phosphorylation sites on MYPT1 for Rho-kinase [43], is located within the putative myosin-binding region at the C-terminal end of the molecule [15]. Phosphorylation of Thr850 was suggested to decrease binding to myosin [21]. Since the level of phosphorylated Thr850 was thought to correlate with Ca²⁺-sensitization (as discussed above) this mechanism should be considered as a candidate for the Rho-kinase induced inhibition of MP.

Recently, the effect of agonist stimulation on the localization of PP1cδ and MYPT1 was investigated using confocal microscopy [67]. Stimulation of isolated smooth muscle cells from ferret portal vein with prostaglandin F_2α induced translocation of MP from the cytosol to the plsamalemma and subsequent dissociation of subunits. MYPT1 remained at the membrane and PP1c returned to the cytosol. The translocation was inhibited by Y-27632. In these experiments phosphorylation at Thr695 preceded translocation of the holoenzyme to the plasmalemma, but phosphorylation appeared to be required for translocation. (Thus, there is no evidence for dissociation of MP by direct phosphorylation of MYPT1). At the membrane the phosphorylated MP dissociated, possibly as a result of lipid interactions with MYPT1. The released PP1c would have reduced activity toward phosphorylated myosin and this is consistent with the prolonged phase of elevated phosphorylated MLC associated with prostaglandin F_2α stimulation. On the other hand stimulation with phenyle- phrine does not induce translocation and here the profile of MLC phosphorylation is more phasic.

CPI-17

CPI-17 is a phosphorylation-dependent inhibitory protein for MP [68]. CPI-17 is composed of 147 residues with a mass of 17 kDa [69]. It is expressed in smooth muscle and brain [69].

The amino acid sequences of CPI-17 are well conserved in mammalians (more than 80%), especially in the N-terminal half (residues 1–67) encoded by exon 1 [70]. In humans a splicing variant of CPI-17, which lacks 27 residues (sequence 68–94) encoded by exon 2, also is expressed in aorta [70].

In contrast to other PP1 inhibitors such as inhibitor-1, CPI- 17 can inhibit the activity of the MP holoenzyme as well as that of isolated PP1c. Phosphorylation of CPI-17 at Thr38 en- hances its inhibitory potency about 1000 fold and the kinase responsible is primarily PKC, especially the α and δ isoforms [68, 71]. The central domain of CPI-17 (residues 35–120) is required for recognition of MP and Tyr41 is necessary to

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protect against dephosphorylation of Thr38 by MP [72].

Addition of thiophosphorylated CPI-17 or CPI-17 plus PKCα markedly induced potentiation of both contraction and MLC phosphorylation in Triton X-100 demembranated smooth muscle at constant [Ca²⁺] [73, 74]. In vitro several kinases can phosphorylate CPI-17 at Thr38, including: Rho-kinase [75];

protein kinase N (PKN) [76] (both Rho-kinase and PKN are targets of RhoA); MYPT1-kinase [77]; ILK [78]; PAK [54];

and cAMP-dependent protein kinase (PKA) [79]. It is interesting that these kinases, except PKN and PKA, also can phosphorylate MYPT1 at the inhibitory site (at least under in vitro conditions).

In intact and permeabilized smooth muscle fibers, CPI-17 was phosphorylated at Thr38 following agonist stimulation (histamine) and phosphorylation was reduced by inhibitors of both PKC and Rho-kinase. These observations raised the possibility that CPI-17 could be a downstream target for both Rho-kinase and PKC [80]. Phosphorylation of CPI-17 correlates well with force and MLC phosphorylation on agonist stimulation in various smooth muscle preparations, indicating a potential role for CPI-17 in Ca²⁺-sensitization [60, 61].

In addition, the dephosphorylation of CPI-17 showed a tem- poral association with activation of MP during NO-induced relaxation [81], the apparent activation being due to removal of inhibition by the phosphorylated CPI-17. In vitro, it is sug- gested that type 2A, 2B and 2C protein phosphatases can dephosphorylate CPI-17, but not type 1 [82]. The expression of CPI-17 varies for different smooth muscles. It is higher in vascular tissue and lower in visceral muscle [83]. CPI-17 may play only a minor role in Ca²⁺-sensitization in those tissues expressing low levels of CPI-17, e.g. vas deferens [60]. In brain, it was suggested that PKC-mediated phosphorylation of CPI-17 may be involved in cerebellar long-term synaptic depression [84].

Activation of myosin phosphatase

cAMP, cGMP and the catalytic subunits of each respective kinase can relax permeabilized smooth muscle at constant [Ca²⁺]. The role of cyclic nucleotides in this Ca²⁺-desensitization process (review [85]) is thought to reflect an increase in MP activity [4, 5]. Direct phosphorylation of MYPT1 by either PKA [46] or cGK [86] had no effect on MP activity.

These observations imply that activation of MP via cyclic nucleotide-dependent protein kinases is indirect and could involve other components of MP. Several mechanisms have been suggested.

As discussed previously, MYPT1 interacts via leucine zip- pers with cGKIα [23] and only those smooth muscles expressing the leucine zipper positive isoform of MYPT1 show Ca²⁺-desensitization via cGMP-mediated activation of MP [16]. Uncoupling the cGKIα-MYPT1 interaction prevented

cGMP-dependent MLC dephosphorylation and these data support a role for this interaction in regulation of vascular smooth muscle tone [23]. Although in this scenario an obvious deficiency is the identity of the target protein for cGKIα.

Inhibition of RhoA activity as a result of cyclic nucleotide signaling is another potential mechanism. Both PKA and cGK phosphorylate RhoA at Ser188 and thereby inhibit RhoA activation [87-89]. In vivo and in vitro studies indicate that Ser188 phosphorylation modifies interaction of RhoA and RhoGDI and reduces the active GTP-bound form of RhoA [90]. Also, a reduced level of GTP-RhoA may result from modification of the upstream trimeric G protein, G_α12/13 [91].

Phosphorylation by PKA of a conformation-sensitive switch region in G_α13 increases interaction with the G-protein-coupled receptor(s) and this results in a decreased level of RhoA activation [92]. It is assumed that the effects on RhoA decrease Rho-kinase activity and prevent inhibition of MP (by phosphorylation of MYPT1 or CPI-17). However, this would not result in net activation of MP (above the non-phosphorylated control level) but merely reduce inhibition.

Telokin is an independently-expressed protein that is de- rived from the smooth muscle MLCK gene and contains the C-terminal domain of MLCK [93]. It is expressed at relatively high levels in phasic smooth muscles. Telokin has been proposed as another candidate implicated in cyclic nucleotide- induced Ca²⁺ desensitization [85]. It induces a dose-dependent relaxation and MLC dephosphorylation in permeabilized smooth muscle (ileum) and the phosphorylation of telokin by PKA or cGK increases its effect on relaxation.

An activation of MP activity could also theoretically be achieved by an increased affinity of MYPT1 to its substrate, phosphorylated myosin. Such was suggested to occur during cell division [94]. A mitosis-specific phosphorylation of MYPT1 increased affinity for phosphorylated myosin and increased MP activity (supported by in vivo and in vitro ob- servations). The functional phosphorylation site(s) were identified as Ser430 and/or Ser427 (in human MYPT1 these sites correspond to Thr435 and Ser432, respectively. See Fig. 2).

The kinase involved is not identified. The sequence around the phosphorylation sites, 430 and 427, is also conserved in MYPT2. It is suggested that activation of MP during mitosis would enhance dephosphorylation of MLC and lead to dis- assembly of stress fibers during prophase.

Roles of myosin phosphatase in physiological and pathological conditions

The phosphorylation of myosin II is important for a broad range of cellular functions, including: smooth muscle and cell

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contraction; cell motility; cell attachment and cytoskeletal structure; cytokinesis; platelet activation; and effects on various ion channels. For many of these processes the ‘usual’

substrate for MP on MLC is Ser19, but it will also dephosphorylated Thr18 (a second MLCK site) and Ser9 (a PKC site) [95]. Other substrates include moesin [96] and adducin [97]. It is likely that other substrates remain to be identified and it is possible that additional cellular functions will be linked to MP and the target function of MYPT1.

Recently it was reported that in Drosophila MP and Rho- kinase function antagonistically and that the MYPT homo- logue is essential for many developmental processes such as cell sheet movement during dorsal closure, morphogenesis during eye development and ring canal growth during oog- enesis [98, 99]. Similarly in C. elegance embryonic elon- gation driven by cell shape change requires a Rho-kinase (LET-502) and is opposed by the activity of the MP regulatory subunit (MEL-11) [100, 101].

It is clear that MP regulates many smooth muscle-associated functions such as vascular tone, blood pressure control, gastrointestinal motility, airway resistance, erectile function and uterine contraction. These functions and their related pathological conditions have been investigated mainly with respect to the RhoA/Rho-kinase pathway. A compelling rea- son for this approach is the availability of relatively specific inhibitors for Rho-kinase, namely Y-27632 [102] and fasudil (HA-1077) [103].

Several lines of evidence suggest that activation of the Rho-kinase pathway and possible inhibition of MP are involved in vascular smooth muscle hypercontractility as manifested by hypertension, coronary spasm and cerebral vasospasm. In various rat model of hypertension, Y-27632 dramatically corrected hypertension but had little effect on normotensive rats, thus indicating an augmentation of the RhoA/Rho-kinase signaling pathway in hypertension [102].

This selective effect may reflect either higher expression of RhoA [104] or increased levels of activated GTP·RhoA in the hypertensive state. Seko et al. [59] found that in several rat models of hypertension the level of ‘activated’ RhoA, i.e.

GTP·RhoA was higher than in control tissues. Also it was shown that the expression of RhoA, Rho-kinase (both isoforms) MYPT1, CPI-17 and MLCK were not significantly different in aortas from various types of hypertensive and control rats. In coronary vasospasm, Rho-kinase may induce a regional hypercontraction. In porcine models, the spastic site showed an increase in Rho-kinase message associated with MYPT1 phosphorylation that could be inhibited by Y- 27632 [105]. Intracoronary infusion of fasudil markedly at- tenuated the coronary constriction induced by acetylcholine in patients with vasospastic angina [106]. Also, in cerebral vasospasm-induced subarachnoid hemorrhage the activation of Rho-kinase and phosphorylation of MLC and MYPT1 occurred concomitantly during vasospasm. Thus indicating

that inhibition of MP via a RhoA/Rho-kinase pathway is an important contributor to cerebral vasospasm [107].

Rho-kinase also has been implicated in vascular remodel- ing including neointimal formation and growth following balloon injury [108, 109]. The invasive activity of MM1 hepatoma cells was blocked by Y-27632 [110] and thus Rho- kinase may be involved in processes related to tumor development, invasion and metastasis. Other disorders in which Rho-kinase may be involved include: bronchial asthma; endothelial dysfunction; and myocardial hypertrophy. The potential roles of RhoA/Rho-kinase signaling in various disease states have been reviewed recently [111]. From these multiple lines of evidence, it is reasonable to assume that modula- tion of MP by Rho-kinase is implicated in several disorders.

The components of the RhoA/Rho-kinase signaling pathway offer novel therapeutic targets for treatment of these disorders.

Conclusion

Various aspects of myosin phosphatase (MP) have been discussed. It is clear that the majority of the characteristics of MP are determined by the targeting subunit, MYPT. These include binding of the catalytic subunit, PP1cδ, and interaction with the substrate, phosphorylated myosin. In addition, MYPT provides a platform for several other interactions, although in general the in vivo roles for these are not estab- lished. MYPT may also have a regulatory function and phosphorylation of Thr696 (in the human MYPT1 isoform) is thought to inhibit MP activity. An important component of Ca²⁺-sensitization in smooth muscle is RhoA and its downstream partner, Rho-kinase, can phosphorylate Thr696 on MYPT1 and inhibit MP activity. The importance of RhoA in smooth muscle function also is illustrated in several examples of smooth muscle hypercontractility in which inhibition of Rho-kinase frequently is beneficial. One of the problems associated with the MYPT1 phosphorylation theory is that several kinases, associated with different signal transduction pathways, can also inhibit MP activity. How these kinases and implicated signaling pathways can be integrated into identified cell functions is a major challenge. However, it should be stressed that myosin phosphorylation is involved in many aspects of cell function, not just contraction of smooth muscle and the multiple signaling routes could reflect this diversity and the varied signals may converge at a common focus, namely phosphorylation of MYPT1. Another frequently-cited mechanism for inhibition of MP is via the phosphorylation- dependent inhibitor protein, CPI-17. Unlike other ‘classical’

protein inhibitors of type 1 phosphatase, CPI-17 is effective with the holoenzyme, not just the isolated catalytic subunit.

Although originally described as a PKC potentiated inhibitor it is now apparent that several kinases, including Rho-

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kinase can phosphorylate CPI-17 and increase its inhibitory potency. Thus, a similar situation exists for phosphorylation of both MYPT1 and CPI-17 in that several signaling pathways may be implicated.

A relatively recent development is the discovery of a putative MYPT family. Its members include MYPT1, MYPT2, MBS85, MYPT3 and TIMAP. In addition, data base searches indicate several other candidates. MYPT1 and MYPT2 are different gene products but similar proteins. It is assumed that each is involved in dephosphorylation of myosin II, MYPT1 in smooth muscle and non-muscle cells and MYPT2 in striated muscle. MYPT2 also is expressed in brain, together with MYPT1, and the requirement for both isoforms is not apparent. Thus, particularly for MYPT2, the possibility of different substrates cannot be overlooked. With respect to the other family members the pertinent substrates are not identified and an exciting aspect of future research will be to identify cell functions associated with these MYPT-like molecules.

Acknowledgements

Authors are supported by Grants-in-Aid for Scientific Re- search for the Ministry of Education, Culture, Sports, Sci- ence and Technology, Japan (to MI and TN), by grant OTKA T043296 (to FE), and by grants from the National Institutes of Health (HL23615 to DJH). A part of this review was presented at the 2nd Annual Meeting of IACS Japan Section.

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Myosin phosphatase: Structure, regulation andfunction