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Annu. Rev. Biochem. 1992.61:721-759. Downloaded from www.annualreviews.org Access provided by University of California - Davis on 02/01/15. For personal use only.

Annu. Rev. Biochem. 1992. 6/:721-59 Copyright © 1992 by Annual Reviews Inc. All rights reserved

CONTROL OF NONMUSCLE MYOSINS BY PHOSPHORYLATION John L. Tan, Shoshana Ravid, and James A. Spudich Departments of Cell Biology and Developmental Biology, Stanford University School of Medicine, Stanford, California 94305

KEY WORDS:

cytoskeleton, cell motility, actin-based motors, protein kinases, protein phosphates

CONTENTS OVERVIEW AND PERSPECTIVES..............................................................

721

MYOSIN STRUCTURE AND FUNCTION ..................................................... The Motor Unit . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Tail Domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

723 726

MYOSIN I HEAVY CHAIN PHOSPHORYLATION .. . . .. . . .. . . . . . . . . . . .. . ... . .. . . . . . ... . .. .

728

MYOSIN II LIGHT CHAIN PHOSPHORyLATION.......................................... Phosphorylation by Myosin Light Chain Kinase. . . . .. . . . . . . . . . . . .. . . . . . ........... . . . . . . . . . Phosphorylation by Protein Kinase C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phosphorylation by Other Kinases ............................ . .......... . . . . . ... . . ...... . . . .. Dephosphorylation by Myosin Light Chain Phosphatases . .. . . . . . . . ... . . . . . .. . .. . . . . . . . . .

730 730 737 740 742

MYOSIN II HEAVY CHAIN PHOSPHORYLATION . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. .

743 746

Pho�phorylation by Myosin Heavy Chain Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phosphorylation by Other Kinases ..... . ...... . ....... . ....... . ... . . .. . . . .... . . ........ ..... .. Dephosphorylation by Myosin Heavy Chain Phosphatases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSIONS AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .

723

749 75 1 75 1

OVERVIEW AND PERSPECTIVES Myosins are enzymes capable of utilizing the chemical energy stored in ATP to support translational movement along actin filaments. These enzymes

0066-4154/92/0701-0721$02.00

721

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TAN ET AL

generally comprise two domains, a globular head domain that is common to all myosins and a tail domain that has properties unique to each type of myosin. All elemen ts necessary for force production, inc luding the sites of

ATP hydrolysis and actin binding, are contained within the relatively con­ served head domain. The structural motifs of the various tail domains presum­ ably designate the specific cellular functions of each type of myosin . Myosins have been broadly grouped into two general classes . The myosin I

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class comprises an eclectic assortment of molecules consisting of a single heavy chain with molecular weight ranging from 110,000 to 190,000 and at least one light chain with molecular weight ranging from 14,000 to 27,000. Those myosin Is that have been characterized biochemically consist of a single globular head domain and a tail domain that contains minimal con­ served regions and appears to be unique to each myosin I subclass. In contrast, members of the myosin II class have relatively homogeneous struc­ tures made up of two heavy chains, each with a molecular weight of approx­ imately 200,000, and two sets of light chains with molecular weights of approximately 16,000-20,000. These hexameric molecules consist of two identical globular head domains and a helical coiled-coil tail domain . The globular heads consist of the amino-terminal parts of the heavy chains and the two sets of light chains. The tail is formed by the intertwining of the carboxyl-terminal portions of the heavy chains. Owing to the inherent proper­ ties of the tail domains , myosin lIs are able to associate intermolecularly to form filaments . Myosins isolated from muscle tissues represent prototypical myosin II molecules . Twenty years ago, most actin-based motility in nonmuscle cells was postu­ lated to involve the two-headed filamentous myosin, now referred to as myosin II. This form of myosin is widely prevalent in eukaryotic cells and had long before been shown to drive the contraction of muscle . However, follow­ ing the discovery of myosin I molecules in Acanthamoeba 0), it has become apparent that there are potentially

multi

pl e motors

that are capable of generat­

ing actin-based movement. Studies on Dictyostel ium cells genetically en­

gineered such that they express only minor levels of myosin II (2) or a truncated form of myosin II (3) supported this notion. These cells continued to

display numerous motile activities including vesicular movement, membrane ruffling, pseudopod extension, and cell locomotion. Subsequent studies on

Dictyostelium cells in which the single-copy myosin II gene was eliminated by gene replacement, creating amoebae that do not express any myosin II molecules, confirmed that a variety of the cell's motile functions that were widely thought to be actin based do not involve myosin II (4). It is now generally speculated that these various behaviors are driven at least in part by myosin I molecules. The Dictyostel ium studies also demonstrated that myosin

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MYOSIN PHOSPHORYLATION

723

II is essential to a number of motile functions intrinsic to most nonmuscle cells. These include maintainance of cortical tension, capping of cell surface receptors, morphogenetic changes in shape associated with developmental processes, and, most dramatically, cytokinesis. Unlike skeletal muscle myosins, which are organized into relatively stable sarcomeres, nonmuscle myosin II molecules are thought to undergo a dynamic assembly/disassembly process that pennits specific spatial and temporal localization. How the various actin-based motors are regulated in vivo remains one of the fundamental questions of cell motility. This regulation must function such that the various myosins, all of which utilize tracks of actin filaments, do not interfere with each other's individual functions and may be reversibly orga­ nized in a particular cell structure at a particular time. Although the mech­ anisms that regulate these molecules are obscure, in virtually all myosins that have been biochemically characterized, phosphorylation has been shown to play a central role in regulating enzymatic activity. In addition, phosphoryla­ tion appears to control the structural and assembly properties of most myosin II molecules. Much of the work has focused on phosphorylation control of myosin II, although studies on phosphorylation of the myosin I heavy chain from Acanthamoeba have provided valuable insights into the regulation of this diverse class of myosins. This review focuses on the various kinases and phosphorylation sites thought to be involved in the regulation of myosins from nonmuscle cells, and the effects these phosphorylations have on the enzymatic and structural properties of these molecules. Several recent reviews provide excellent sum­ maries of general current knowledge of nonmuscle myosins (5-8). Although a brief summary is presented in this review on phosphorylation of smooth muscle myosin, the reader is referred to other reviews that thoroughly summarize the extensive literature on the regulation of smooth muscle (9-12), as well as recent, short reviews that concentrate on the regulation of smooth muscle and cytoplasmic myosins (13-15). MYOSIN STRUCTURE AND FUNCTION The Motor Unit Myosin from muscle tissues can be proteolytically cleaved into various fragments that distinguish the head and the tail domains. Two proteolytic products, heavy meromyosin (HMM) and subfragment I (SI), contain the globular head domain (Figure 1). HMM consists of two globular heads and approximately 113 of the a-helical coiled-coil tail domain. Sl consists of a single globular head with no tail sequences. Studies with both HMM and SI

724

TAN ET AL

HEAD

DOMAIN

TAIL DOMAIN

r---I rl------�

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MYOSIN II

HMM

LMM

Sl

MYOSIN I Figure 1

Schematic diagram of myosin domains. The globular head domain comprises th,: amino-terminal portion of the heavy chain and the light chain(s). Within the head domain, th,: heavy chain sequences are relatively conserved in both myosin Is and lIs. However, the number and composition of thc light chains of the two classes of myosin vary. Myosin II can be proteolytic ally cleaved into various fragments. Heavy meromyosin (HMM) contains the two globular heads and approximately 113 of the a-helical coiled-coil tail domain; light meromyosin (LMM) contains the terminal 2/3 of the tail domain. Subfragment I (SI), like myosin I molecules, is composed of a single head domain.

from skeletal muscle tissues have demonstrated that the globular head domain is sufficient to support movement (16--18). Similarly, fragments of the Dic­ tyostel ium myosin II that are analogous to HMM and S1 have been generated by molecular biological techniques and have also been shown to move actin filaments ( 19, 20) . Such studies have not been done with the myosin I class of molecules. However, sequence comparisons of both classes of myosins and recent map­ ping of the brush border myosin I (21, 22) suggest a similar molecular organization of the head domains of both classes of myosin; it is generally assumed that the head domain of myosin I molecules is sufficient to support movement. Numerous conserved regions of sequence in the heavy chains are present in the head domains of all myosins (for review, see 5). Indeed, with

MYOSIN PHOSPHORYLATION

725

the exception of three myosin I molecules studied in Acanthamoeba ( 1 , 23-25), the lIO-kDa brush border myosin I (26, 27), and a single member of the myosin I class in Dictyostel ium (28), which were identified by classical biochemical techniques, all other members of the myosin I class have been identified by sequence analysis of conserved regions within the head domain

(29-34). Although the myosin head is composed mainly of the heavy chain, in at

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least the myosin II class, light chains are an important component of this domain. The head domains of myosin II molecules have two light chains of approximately

16,000-20,000 daltons. One of the two light chains regulates regulat ory l ight chain. This chain is also referred to as the p hosphorylatable l ight chain, since the activity of the myosin head and is thus referred to as the

in almost all systems studied, regulation of myosin activity is mediated by phosphorylation of this chain. The notable exception is molluscan striated muscle, in which myosin activity is regulated by dircct binding of Ca2+ (35 , 36 , for review, see 37). Recent genetic studies in Drosophila have shown that cells lacking this light chain display a cytokinesis defect (38), suggesting

that the regulatory light chain is essential to the in vivo function of myo­ sin II. The other light chain, historically referred to as the

e ss enti al light chain,

does not incorporate phosphates. This light chain has been shown to constitute part of the active site of smooth muscle myosin

(39), and may be involved in (40) as well as serving as a link between the active site and the regulatory light chain (12). Binding of a monoclonal

maintaining the head structure

antibody to the essential light chain has been shown to increase the actin­ activated ATPase of smooth muscle myosin

(41).

In myosin I molecules, the function of the light chains is unknown. Although the light chains of myosin I molecules are commonly assumed to be an integral part of the head, this has not been established. Indeed, the light chains of the brush border myosin Is from avian and bovine intestine are thought to be carboxyl terminal of the head domain

(42). Neither the actin­

activated ATPase activity nor the phosphorylation of the heavy chain of an Acanthamoeba myosin I requires the presence of the light chain

(23).

From the few cases in which the light chains have been characterized in myosin I molecules, it is evident that the number and the composition of the light chains are very variable. The biochemically characterized Acanth a­ moeba myosin Is, myosin lA, IB, and IC, are associated with a single light chain of 15 ,000, 27,000, and 14 ,000 daItons, respectively (43). In contrast, the brush border lIO-kDa myosin I molecule binds at least three calmodulin molecules, which are the light chains of this myosin

(21) . Aside from the

calmodulin of brush border myosin 1, the biochemical properties and the primary structures of other myosin I light chains remain obscure.

726

TAN ET AL

The Tail Domain In skeletal muscle, myosin filaments are arranged in ordered arrays between actin filaments. Together with accessory proteins, these filaments constitute the sarcomeres. Myosin

II filaments similar to those present in muscle tissues Dictyos te lium, filaments composed of

are also found in nonmusc1e cells. In

II molecules have been visualized by immunocytochemical tech­ (44, 45). The importance of the assembly portion of the tail domain to

myosin

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niques

the cellular function of myosin II has been addressed by molecular genetic studies. Cells were created that express only a truncated form of myosin II instead of the intact molecule. Cells expressing this truncated form. which corresponds to the proteolytic fragment HMM and thus contains a fully functional head domain, are defective in cytokinesis and in their ability to complete development

(3). Further analysis of these mutant cells reveals that

although the cells are still motile, their general motility and chemotactic

(46). These cells also fail to cap cell sur­ (47) . Strikingly, Dictyost elium cells that do not express any myosin II exhibit the same phenotype as the HMM mutant (4). This finding indicates that the head domain, which contains all the elements necessary movement are severely impaired

face proteins

for force generation, is not sufficient for the proper cellular function of myosin II.

Analysis of the tail sequences of muscle and nonmuscle myosin

II mole­

cules reveals multiple repeating patterns throughout the entire domain that underlies the a-helical coiled-coil structure of the myosin II tail. The smallest repeating motif contains seven amino acids in which small, generally hydrophobic amino acids are usually found in the first and fourth position. The seven residues in this repeat form two turns of the a-helix with the first and fourth amino acids falling along a single aspect of the helix (48,

49). This

configuration provides the hydrophobic core essential to the formation of the a-helical coiled-coil.

A repeating pattern of four seven-residue motifs (28 are charged and

residues) is also evident along the tail. The 28-residue motifs

positioned such that they create alternating bands of positive and negative charges on the surface of the coiled-coil. These charged regions interact with those along the tails

of adjacent myosin II molecules to form the myosin

filaments. Early proteolytic studies on muscle myosin demonstrated that the carboxy}.· terminal 2/3 of the myosin II rodlike tail domain, termed light meromyosin (LMM) , contains all the properties necessary for assembly into filaments (Figure

1). More recent studies indicate that a specific region within thl�

myosin II tail domain is necessary and sufficient for filament formation (Figure

2). Cross & Vandekerckhove (50) showed that the assembly sub­

domain of smooth muscle myosin is localized to a carboxyl-terminal region

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MYOSIN PHOSPHORYLATION

727

comprising approximately 140 residues (20 nm of the tail length). In Acantha­ moeba myosin II, the carboxyl-terminal 100 amino acids of the tail contain all the elements needed for the assembly process (51) as well as the sites of heavy chain phosphorylation (52, 53). A number of studies have also mapped important regions within the tail domain of the Dictyoste lium myosin II molecule. Like the Acanthamoe ba molecule, Dictyoste lium myosin II possesses an assembly subdomain currently localized to a 34-kDa region (54, 55). This subdomain, located 34 kDa from the carboxyl terminus, is situated within the region found to be the major contact area between the two myosin II molecules in a parallel dimer (56,57). In addition,carboxyl terminal to this subdomain is a regulatory region that contains sites of heavy chain phosphorylation, which appear to play an important role in disassembling filaments. This was demonstrated by studies in which Dictyosteli um cells that

50

100

I

I

HIGHER EUKARYOTES

+

+

150

I

186nm

I I

p p

DICTYOSTELIUM

+ ACANTHAMOEBA

Figure 2

p p p

Schematic diagram of variOU$ myosin II molecules. The tail length of myosin II molecules varies depending on the source. The tail of smooth muscle myosin is 155 nm long, while those of Dictyostelium and Acanthamoeba myosin II are 1 86 and 90 nm. respectively. The positions of bends within the tail domain ate indicated by the arrows. Smooth muscle myosin is known to bend in at least two sites approximately 50 and 100 nm from the end of the tail. The Dictyoslelium myosin II bends at a site 66 nm from the end of the tail. Although the Acantha­ moeba myosin II is known to bend at a site 40 nm from the end of the tail, this bending, unlike those of higher eukatyotic and Dictyostelium myosin IIs, is not dependent on phosphorylation. The hatched boxes represent the assembly subdomains. The sites of phosphorylation are also indicated (by circled Ps). Whereas higher eukaryotic and Dictyostelium myosins ate phosphory­ lated on both the heavy and the ligh t chains, Acanthamoeba myosin is phosphorylated only on the heavy chain.

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728

TAN ET AL

express only a truncated form of myosin II containing the assembly sub­ domain but lacking sequences carboxyl terminal to this subdomain were found to exhibit severe abnormalities in their ability to disassemble myosin filaments (58). All nonmuscle myosin II molecules possess hinge regions within their tail domains. In smooth muscle and vertebrate nonmuscle myosin lIs, two hinge regions are located at approximately 1/3 and 2/3 the length of the tail from the head domain (Figure 2; 59). Bending at these sites is regulated by light chain phosphorylation. In contrast, Dictyoste li um myosin II possesses a single hinge region, located at a position similar to the second bend of the vertebrate myosin II and regulated by phosphorylation of the heavy chain (Figure 2; 57). The Acan thamoeba myosin II also contains a hinge region located at a relatively similar position as the Dictyostelium region (Figure 2). It is not known whether phosphorylation regulates bending of the Acan thamoeba myosin II. In contrast to myosin lIs, which all have very similar tail structures, myosin I tail domains are structurally very different from one another. As more myosin I molecules are characterized, this broad class is beginning to be subgrouped based on common motifs within the tail domain as well as sequence conservation within the head domain. For example, the myosin Is can be classified by the presence of recurrent motifs such as the SH3 region or a basic region (8), or by general sequence comparison as is the case with the Saccharomy ce s MY02 gene (33) and the mouse dilute gene (34). The func­ tions of the various tail domains remain unclear. However, preliminary studies indicate that the SH3 region may contribute to the ATP-independent actin-binding site within the tail domain or may anchor the myosin I molecule to a membrane-associated protein (8). Similarly, the basic region may serve to bind the myosin I molecule directly to membranes (60). MYOSIN I HEAVY CHAIN PHOSPHORYLATION Our current knowledge of phosphorylation of myosin I heavy chains is based primarily on work in Acan thamoeba. A single myosin I heavy chain kinase (MIHCK) has been purified from this organism (61) . This kinase appears to be a general protein kinase. It is capable of phosphorylating not only all biochemically characterized myosin Is from Acanthamoe ba (61) as well as a myosin I isolated from Di ctyoste li um (28), but also several other substrates such as smooth muscle myosin light chains and casein (6 1 , 62) . The sites phosphorylated by MIHCK have been identified in a number of the Acanthamoe ba myosin Is. Unlike the phosphorylation sites on the myosin

MYOSIN PHOSPHORYLATION

729

II heavy chain, the phosphorylation sites on the myosin I heavy chain are within the head domain. The sites of phosphorylation by MIHCK are serine 315 in myosin lB, serine 311 in myosin IC, and a threonine residue at a

corresponding position in myosin IA (63). These residues are located between the putative ATP- and actin-binding sites

(44, 65). In all three of these myosin

I molecules, there are two to three basic amino acids preceding the phos­ phorylated residue. Using synthetic peptides, Brzeska et al (66) further

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defined the MIHCK site and showed that a peptide composed of nine amino acids is sufficient as a substrate for MIHCK. While two basic amino acids at the amino-terminal side of the phosphorylated residue are preferred, at least one is essential. In addition, tyrosine residues situated at the carboxyl ter­ minus of the peptide appear to be essential. This phosphorylation site is conserved in a number of myosin I sequences including Dictyosteli um myosin IA (30), IB (31), IC (30), and ID (M. A. Titus, personal communication) and bovine (67) and avian (68) brush border myosin l. Phosphorylation of the heavy chain serves to increase the actin-activated

ATPase activity of myosin I molecules from Acan thamoeba (6] , 69, 70) and from Dic tyostelium (28). This phosphorylation results in a 20-fold increase in the actin-activated ATPase but has no effect on the binding of myosin I to filamentous actin (25). In addition, using the Nite lla in vitro movement assay (71, 72), Albanesi et al (73) showed that the movement of beads coated with myosin IA or lB along actin filaments depends on heavy chain phosphoryla­ tion. The

Acanthamoe ba MIHCK is regulated by autophosphorylation and by

phospholipid. Incorporation of up to approximately eight moles of phosphate per mole of kinase appears to stimulate the activity of MIHCK (74). MIHCK activity is also enhanced 20-fold by phospholipids (74). The addition of Ca2+ , however, has no effect (69, 74), suggesting that the activation of this kinase is distinct from that of PKC. Since myosin Is bind membrane lipids (75-77), the MIHCK appears to be localized such that it may regulate myosin

I molecules

that are membrane bound. Several myosin I molecules, including the

Acan thamoeba High Molecular Saccharomyc es MY02 gene product (33), the mouse di lute myosin I (34), and the Dros ophila ninaC protein (29), do not

Weight myosin I (32), the

contain the phosphorylation site for MIHCK. The mechanism of regulating these myosins is not known. The Drosoph ila n inaC protein encodes a protein kinase catalytic domain (29), which may regulate the activity of the putative motor domain. In addition, the Saccharomyces MY02 sequence contains a cdc2 phosphorylation site (33). However, further biochemical possible pp34 characterizations are necessary to elucidate the regulatory mechanisms of these myosins.

730

TAN ET AL

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MYOSIN II LIGHT CHAIN PHOSPHORYLATION Phosphorylation of the regulatory light chain was first demonstrated by Perrie et al (78) in myosin from skeletal muscle and by Adelstein et al (79) in platelet myosin. Since that time, myosin light chain phosphorylation has been shown to occur in every system in which it has been investigated with the exception of Acanthamoeba. Early work with platelet myosin demonstrated that phosphorylation of the regulatory myosin IT light chain resulted in an increase in the actin-activated ATPase of myosin (80). These works led to the characterization of specific myosin light chain kinases (MLCKs) from a variety of muscle and nonrnuscle sources (for review, see 81) including skeletal muscle, smooth muscle, brain, platelets, and more recently, fibro­ blast (82) and Dictyostelium (83, 84). Light chain phosphorylation by the Ca2+Iphospholipid-dependent protein kinase, protein kinase C (PKC) also appears to regulate myosin IT activity. In addition to MLCK and PKC, a number of other general kinases have been shown to phosphorylate the myosin IT regulatory light chains, at least in vitro. These include the Ca2+I calmodulin-dependent protein kinase type II (CaM kinase II), the cell cycle­ dependent protein kinase,pp34cdc2,the reticulocyte protease-activated protein kinase I, and the Acanthamoeba myosin I heavy chain kinase (Table I). Several other general kinases, including the cAMP-dependent protein kinase (85), epidermal growth factor receptor (86), insulin receptor (87), casein kinase I (88) and II (89,90),and phosphorylase kinase (88), also phosphory­ late isolated myosin II regulatory light chains. However, it is unlikely that these kinases regulate myosin, since they do not phosphorylate the regulatory light chain in the context of an intact myosin II molecule. Although the majority of studies focused on myosin II light chain phosphorylation have been done on smooth muscle myosin, several studies demonstrate that phosphorylation of nonmuscle myosin II molecules of higher eukaryotes results in effects essentially identical to that observed with smooth muscle myosin. In addition, molecular cloning of the cDNAs of several nonmuscle regulatory light chains from higher eukaryotic organisms (91-93) indicates that the various isoforms of the regulatory light chains are very highly homologous, conserving all characterized phosphorylation sites (Fig­ ure 3). Thus it is likely that results seen with the phosphorylation of smooth muscle myosin will hold true for the nonmuscle myosin lIs of higher eukaryo­ tic cells. However, as detailed below, this is not the case with all myosin II molecules from lower eukaryotes.

Phosphorylation by Myosin Light Chain Kinase PHOSPHORYLATION SITES There are two sites in the regulatory light chain of myosin II that are phosphorylated by MLCK, at least in vitro. These sites

MYOSIN PHOSPHORYLATION Table 1

731

Summary of myosin II regulatory light chain phosphorylation Evidence of phosphorylation

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Site of phosphorylation'

Kinase(s)b

Serl9

MLCK

Serl9

CaMK II PAPK I MIHCK

+

ThrlS

MLCK

Effects on myosin in vitro

in vivo

Increase in ATPase

Yes

Stabilization of 6S Increase in rate of movement

Further increase in ATPase

No

Further stabilization of 6S

No effect on rate of movement

Thr9

PKC

Decrease in ATPasec

Increase in Ka for actin

No

No effect on Vmax

No effect on rate of movement Decrease in rate of MLCK phosphorylation

Thr9 + Ser l or 2

PKC

Further decrease in ATPase No effect on rate of movement

Serl or 2 •

pp34Cdc2

Yes

(Ser l and 2 only)

? Decrease in ATPase

Sites listed are based on the protein sequence of chicken smooth muscle regulatory light chain.

b Myosin light chain kinase

(MLCK), Ca2+{calmodulin-dependent protein kinase type II (CaMK II), pro­ I heavy chain kinase (MllICK), protein kinase C (PKC), cell

tease-activated protein kinase I (PAPK I), myosin cycle-dependent protein kinase (pp34Cdc2).

C Effects are on myosin previously phosphorylated by myosin II.

MLCK.

Decrease in ATPase is not seen with thymus

are serine 19 (94) and threonine 18 (95-97). Although phosphorylation of each of these two sites is independent of previous phosphorylation at the other site, the rates of phosphorylation are considerably different, with phosphory­ lation at serine 19 being ap proximately 1000-fold greater than that at threonine 18 (95). Phosphorylation at threonine 18 has been de mon strated in both smooth muscle and platelet myosins and is seen on ly with high con­ centrations of MLCK (95, 97). ,

EFFECTS OF PHOSPHORYLATION ON ENZYMATIC ACTIVITY Light chain phosphorylation by MLCK has been shown to result in an increase in the a ctin activated ATPase activity of myosin lis in all systems investigated, including platelet (80), smooth muscle (98,99), and Dictyostelium (83). One notable exception is skeletal muscle myosin, which undergoes but does not require light chain phosphorylation for significant actin-activated ATPase activity (100). Light chain phosphorylation in skeletal muscle is tho ught to be -

732

TAN ET AL

MIRCK

SMOOTH MUSCLE CRICIIEN RUMAN

PJlPK I

pp34Cdc2 PRC

(M)SS It.B..AIt. A - .!. M SS !..B.. AIt. A - .K

CaM!{ II

PRC -

MLCR

T T .!..!.J!. P Q - - RATS NV F T T .K .!..!. P Q - - RAT S NV F

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NONMUSCLE CHICJJ:EN

RAT DR S O P O HLA I D C I TY O STELIUM

MSS !L�AIt. T MSS !LB..A!.. T MSS !t!.. TAG

-.!. TT .!L�J!. A Q .!. T T !...KJ!.P Q �J!. A T T.!..!..!t A Q -

-

- - RATS NV F

-

- - RATS NVF - - R·A T S NV F

MA STK.!.J!.LN RE E -SSVV L

SKELETAL MUSCLE IIAI!IlIT RAT

PKKAKRRAAAEGGSSNVF MA P KKAKRRAAAEG -SSNVF

CARDIAC MUSCLE CHICIIEN RAT

Figure 3

(MAlPKKAKKRIE - - GAN SNVF KS

P KKAKKRLE - - GGSS NVF

Phosphorylation sites of myosin II light chains.

The sequences around the

phosphorylation sites of smooth muscle and nonmuscle myosin II of higher eukaryotes are very

highly conserved. The phosphorylation s ites of MLCK, protein kinase C (PKC) , pp34cdC2, 2 Ca +lcalmodulin dependent protein kinase II (CaMK II), protease-activated protein kinase I (PAPK I), and myosin I heavy chain kinase (MIHCK) are boxed. Basic residues thought to be important in PKC phosphorylation are underlined. The Dictyostelium myosin II light chain

sequence appears more similar to those of skeletal and cardiac myosins. However, while skeletal

and cardiac myosins do not contain putative PKC sites, the Dictyostelium sequence does. The residues in parentheses are amino acids that are not contained in the mature polypeptide.

associated with increased isometric twitch tension (101). Although the phosphorylation site has not been mapped in all of these systems, it is generally assumed that the site is serine 19, or is analogous to serine 19. In Dictyostelium, the MLCK phosphorylates a serine residue (83). This site is postulated to be serine 13 or 14, sites analogous to serine 19 of higher eukaryotes (102). Studies utilizing in vitro movement assays have demonstrated that phosphorylation at serine 19 in myosins from turkey gizzard, bovine trachea, bovine aorta, and human platelet is required for force production (103-105), Similarly, light chain phosphorylation of the Dictyos te lium myosin II regula­ tory light chain is essential to movement, as measured in the Nitella in vitro movement assay (83). With this assay, beads coated with myosin II mol ecu les that incorporated approximately one mole phosphate per mole regulatory light chain moved along arrays of Nitella actin filaments at a rate of 0.2 JLmls for turkey gizzard myosin, 0.12 JLmls for bovine trachea and aorta myosin, 0.04

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MYOSIN PHOSPHORYLATION

733

/Lmis for human platelet myosin II (104), and 1 /Lmis for Di cty oste li um myosin II (83). In addition, studies of smooth muscle myosin reveal an approximately linear relationship between the amount of light chain phos­ phorylation and the speed of movement at high myosin concentration (103). These results suggest that unphosphorylated myosin produces a negative effect on the velocity of movement and may do so by an increase in the portion of the cycle time spent bound to actin, as suggested by Warshaw et al (106). This phenomenon may be analogous to that seen in smooth muscle fibers, termed the "latch state," in which tension is maintained (presumably by slowly cycling unphosphorylated myosin crossbridges) despite a return to the resting levels of light chain phosphorylation and velocity of shortening (107). Nonmuscle myosin lIs are thought to be similarly controlled. The mechanism by which light chain phosphorylation affects the enzymatic function of myosin remains controversial. Sellers & Adelstein (l08) have suggested that the kinetic step predominately affected by light chain phosphorylation involves the release of phosphate from the actin-bound myosin-ADP-Pj state, the step thought to induce the conformational changes in the myosin molecule that result in force production. This hypothesis is supported by transient kinetic experiments with smooth muscle myosin that indicate that the release of Pj from unphosphorylated HMM is slow with a rate constant of approximately 0.002 per s, and is not activated by the presence of actin (l09). However, a series of studies with smooth muscle myosin showed that filaments composed of unphosphorylated myosin exhibited a Vmax for actin-activated ATPase that was comparable to that of filaments composed of phosphorylated myosin (110, 111), suggesting that the primary effect of phosphorylation is not on the kinetic cycle of the myosin head but rather on the assembly state of the molecule. However, these results may be specific to the high Mg2+ concentration used to induce the unphosphorylated myosin into filaments (112). Using a monoclonal antibody that stabilizes filaments composed of unphosphorylated myosin at low Mg2+ concentrations, Trybus (ll2) showed that the ATPase activity of unphosphorylated myosin filaments is similar to that of unphosphorylated HMM and is not activated by actin, and that phosphorylation increases the ATPase activity 60-fold. Phosphorylation at threonine 18 has also been shown to increase the actin-activated ATPase above that attributable to phosphorylation of serine 19. In studies using smooth muscle HMM, Ikebe et al (113) determined a value of 3 .8 per s for the Vmax of the actin-activated ATPase of HMM phosphorylated at only serine 19, and 8.1 per s for the Vmax of HMM phosphorylated at both serine 19 and threonine 18, with no significant dif­ ferences in the Ka for actin. However, no effect on movement was observed by the additional phosphorylation of threonine 18 in the Nite lla assay (104).

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734

TAN ET AL

EFFECTS ON MYOSIN CONFORMATION Myosin II from smooth muscle (114) and a number of nonmuscle cells of higher eukaryotes including brush border (115), thymus (114), erythrocytes (96), and platelets (116), is known to exist in two conformations, which are determined by the phosphorylation state of the regulatory light chains. The folded state, referred to as the lOS form after its sedimentation coefficient, is favored by unphosphorylated molecules and a low-ionic-strength environment. Two bends in the myosin II tail domain, at approximately 50 and 100 nm from the head-tail junction (59), result in the lOS form. The equilibrium between this state and the extended state, the 6S form, is shifted towards the 6S form with light chain phosphorylation. Phosphorylation at serine 19 is sufficient to induce this conformational change (97, 113). However, additional phosphorylation at threonine 18 results in further stabilization of the extended 6S form, as measured by sedimentation velocity measurements and by increased sus­ ceptibility of the head-neck junction to proteolysis by papain of HMM phosphorylated at both sites (97, 113). In contrast to the extended 6S form, the IDS form is able to release the products of ATP hydrolysis at only a very slow rate, less than 0.0005 per s (117, 118), bind actin relatively weakly (119), and is unable to form filaments under physiologic conditions (Figure 4; 114). However, the release of the products of ATP hydrolysis by monomeric unphosphorylated myosin (in the lOS conformation) is approximately lO-fold slower than that of un­ phosphorylated myosin in the stabilized filaments (112). Unphosphorylated HMM, which cannot assume the folded conformation, has also been shown to release the products of ATP hydrolysis at a rate greater than that of monomer­ ic lOS myosin (118). In addition, the binding of actin to HMM is only slightly affected by phosphorylation (120). Thus, it is likely that the properties of the lOS myosin reflect the steric hindrance imposed by the folded tail onto the head domain rather than an intrinsic enzymatic property of the un­ phosphorylated molecule. Several studies suggest that the lOS conformation per se is not required for the inhibition of enzymatic activity. In one study, unphosphorylated myosin, which cannot assume the folded lOS conformation in the context of stabilized filaments, continued to exhibit minimal actin-activated ATPase activity (1 t 2). Cross et al (I 18) have also demonstrated that phosphorylated myosin in the lOS conformation releases the products of ATP hydrolysis at a greater rate: than that of unphosphorylated lOS myosin, indicating that phosphorylation can affect the enzymatic activity of myosin without any gross change in conformation. It also remains quite possible that the conformational isoforms observed for these molecules are reflections of the in vitro milieu. Despite the abundant in

MYOSIN PHOSPHORYLATION

735

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..

Figure 4



Assembly of higher eukaryotic myosin II. Molecules that are not phosphorylated by

MLCK exist mainly in a folded conformation and do not form filaments. MLCK phosphorylation

of the regulatory light chains induces a conformational change to an extended form that readily forms filaments.

vitro evidence to support the dependence of the assembly/disassembly process of myosin II on light chain phosphorylation, the presence of the folded conformation has not been observed in intact smooth muscle or in any

nonmuscle cells. Indeed, in relaxed smooth muscle, filaments composed of nonphosphorylated myosin molecules are found

(121), although experiments

with rat smooth muscle indicate that the density of myosin filaments increases with contraction

(122). In lower eukaryotic systems, phosphorylation of the

myosin II light chain does not result in the conformational changes observed with myosin

II of higher eukaryotes. Although bending within the tail domain

is evident in some of these systems, conformational changes appear to be dependent on heavy chain and not light chain phosphorylation. IN

VIVO

EVIDENCE

OF

PHOSPHORYLATION

In smooth muscle fibers,

phosphorylation of the regulatory light chain by MLCK has been shown to be

736

TAN ET AL

sufficient for contraction

(123) . Serine 19 is the primary site of phosphoryla­ (124, 125) as well as thrombin stimulation of platelets (126) . In almost all nonmuscle systems, including

tion during activation of smooth muscle fibers

lower eukaryotes, myosin II light chain phosphorylation has been shown to occur in vivo. In endothelial cells, light chain phosphorylation is involved with cell retraction

(127) . Similarly, in Dictyostelium cells, phosphorylation II light chain is stimulated by the chemoattractant cAMP (128) with a time course consistent with cell shape changes (129, 130).

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of the myosin

Diphosphorylated regulatory light chains have been observed in smooth muscle cells after neural stimulation or treatment with high concentrations of the acetylcholine agonist, carbachol. However, the maximal levels of light chains incorporating two phosphates per molecule are low;

11% for agonist 5% for neurally stimulated cells (131). Diphosphorylation, presumably at serine 19 and threonine 18, thus may not be of physiologic stimulated and

significance in smooth muscle.

With the exception of the

REGULATION OF MLCK

Dictyosteli um (83 , 84)

and possibly the Physarum MLCK (132), all characterized MLCKs are 2+ Ca Icalmodulin activated enzymes. The calmodulin-binding domain, thought to consist of a basic amphiphilic

a

helix

(133 , 134) , is situated (135, 136) . This

carboxy terminal of the protein kinase catalytic domain

domain is postulated to interact with a second regulatory domain that inhibits inherent kinase activity. Upon binding of calmodulin, MLCK is thought to undergo a conformational change in which the autoinhibitory domain is removed from the active site, thus derepressing kinase activity

(137-139).

Early peptide studies indicated that the calmodulin-binding domain overlaps the autoinhibitory domain

(137 , 139); More recent studies of smooth muscle

MLCK using site-directed mutagenesis define a minimum five-residue over­ lap of the autoinhibitory and the calmodulin-binding sites

(140) . These studies

are in contrast to deletion and mutagenesis studies done with fibroblast MLCK that suggest that the autoinhibitory and calmodulin-binding sites are distinct

(82). Whether these differences reflect the different sources of the

kinases remains to be determined. The

regulation

of

MLCK

by

calmodulin

is

itself

modulated

by

phosphorylation. Phosphorylation by cAMP-dependent protein kinase has been shown to occur at two sites (141) . One site, phosphorylated only in the absence of Ca2+ Icalmodulin, is known as the A site. The second site, the B 2+ site, is phosphorylated in either the presence or absence of Ca /calmodulin. The A site has been identified as serine

512 in smooth muscle MLCK (142).

Phosphorylation at this site results in a decreased affinity of MLCK for Ca2+/calmodulin (143) . Thus phosphorylation at this site serves to attenuate

737

MYOSIN PHOSPHORYLATION

the signal for activating MLCK. MLCK is similarly phosphorylated by the

cGMP-dependent protein kinase, resulting in decreased affinity for Ca2+ I calmodulin

(143) and PKC ( 144, 145). Recently, phosphorylation of smooth II has been observed ( 142,

muscle MLCK by the multifunctional CaM kinase

146). Phosphorylation by this kinase occurs at the same sites as those targeted by the cyclic nucleotide-dependent protein kinases, and as may be suspected,

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results in identical effects. The role of MLCK phosphorjlation in vivo, however, has not been clearly demonstrated, with data either suggesting

(147)

or disputing (148) an in vivo role for cAMP-dependent protein kinase.

Phosphorylation by Protein Kinase C PHOSPHORYLATION SITES

Phosphorylation by PKC has been demonstrated

in a number of systems, including smooth muscle (149), platelet

(126, 150-152), thymus (153), and brain (154). Three major sites of phosphoryla­ tion by PKC have been identified in both smooth muscle ( 155, 156) and platelet (116) myosin II light chains. These sites are threonine 9, serine 1, and serine 2 (Figure 3). Phosphorylation at these sites occurs randomly, although phosphorylation at threonine 9 occurs approximately 5 times faster than that at the other sites (156). Phosphorylation of the second site occurs without preference at either serine residue. However, phosphorylation of the third site occurs only on prolonged incubation

(156). Although these sites have not

been mapped for other systems, it is generally assumed that the sites are identical or analogous. For example, two-dimensional peptide mapping of the regulatory light chain of thymus myosin phosphorylated by PKC shows a pattern similar to that of smooth muscle myosin

(153). In addition, although

the major phosphorylation sites are identical in the smooth muscle and platelet systems, PKC phosphorylation of platelet myosin II light chains occurs 10 times faster than PKC phosphorylation of smooth muscle light chains

(116). II

No data have been collected regarding PKC phosphorylation of myosin

light chains in lower eukaryotes. However, serine 3 or threonine 4 in the Dic tyostelium regulatory myosin II light chain sequence (102) corresponds to threonine 9 in higher eukaryotic light chains. PKC phosphorylation sites are characterized by basic residues flanking, or on either side of, the phosphory­ lated residue (for review, see

157). Serine 3 and threonine 4 are situated 6 and 7), and thus represent putative PKC phosphorylation sites (Figure 3). amino terminal of three consecutive basic residues (lysine 5, arginine

EFFECTS

OF

PHOSPHORYLATION

ON

ENZYMATIC

ACTIVITY

PKC

phosphorylation of the regulatory lig!lt chains of myosin II molecules that have not been previously phosphorylated by MLCK appears to have no effects

TAN ET AL

738

on the enzymatic properties of myosin II. In smooth muscle, platelets, and thymus, PKC phosphorylation of totally dephosphorylated myosin (up to approximately

2 moles of phosphate per mole light chain) , unlike phosphory­

lation by MLCK, does not result in an increase of the actin-activated ATPase activity

( 1 43, 1 53, 1 56, 1 58). In addition, phosphorylation by PKC of a

variety of smooth muscle and platelet myosin lIs did not result in movement

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of myosin-coated beads in the

Nite lla assay (104).

However, phosphorylation by PKC of the regulatory light chain that has been previously phosphorylated by MLCK has different effects in various systems. For smooth muscle

(143 , 156, 158) and platelet (116) myosin I I

molecules prephosphorylated b y MLCK, PKC phosphorylation alters the actin-activated ATPase activity . In experiments with smooth muscle myosin, phosphorylation by PKC to one mole of phosphate per mole light chain results in a

50% decrease in the actin-activated ATPase activity of myosin II that had

incorporated two moles of phosphate per mole light chain by MLCK phosphorylation

(156). Additional phosphorylation by PKC to two moles of

phosphate per mole light chain results in a further decrease of the myosin II

25% that of myosin phosphorylated by MLCK alone (156). Determinations of the kinetic constants of myosin phosphorylated by

ATPase activity to

MLCK alone or myosin phosphorylated by both MLCK and PKC revealed that the Vmax of ATPase activity was unaffected by PKC phosphorylation , but

that the Ka for actin of myosin phosphorylated by both kinases was higher

[340 �M and 60 �M,. 63 pM and 8.8 pM respective··

than that of myosin phosphorylated by MLCK alone

respectively for smooth muscle myosin ( 1 56); ly for platelet myosin

( 1 16)] . The effect on the actin-activated ATPase

activity by PKC phosphorylation thus appears to reflect a decrease in actin affinity

(143).

In contrast, in their study of a unique embryonic smooth muscle myosin isoform , de Lanerolle

& Nishikawa (159) found that PKC phosphorylation

resulted in an increase in the actin-activated ATPase. The site of phosphoryla­ tion by PKC in this system, however, has not been identified, and it has been suggested that PKC may phosphorylate the same site as MLCK in this particular myosin

( 1 59). In addition , Carroll & Wagner ( 1 53) reported that

PKC phosphorylation of thymus myosin II resulted in no changes in the

actin-activated ATPase activity of myosin prephosphorylated by MLCK.

Despite these various observed effects on myosin ATPase activity, PKC phosphorylation of smooth muscle and platelet myosin does not seem to affect the rate of myosin-driven movement as measured in an in vitro motility assay. Using the Nite lla system , Umemoto et al

( 1 04) showed that PKC phosphOf)r­

lation of myosins from turkey gizzard , bovine trachea , bovine aorta , and human platelets that had already been phosphorylated

by MLCK

to

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MYOSIN PHOSPHORYLATION

739

1 or 2 moles phosphate per mole light chain exhibited similar rates of movement as those phosphorylated by MLCK alone. Phosphorylation by PKC may affect myosin activity indirectly by modulat­ ing MLCK phosphorylation, since in at least the smooth muscle system, PKC phosphorylation appears to affect the ability of MLCK to phosphorylate the regulatory light chain. In experiments in which smooth muscle myosin was sequentially phosphorylated by PKC and then MLCK, Nishikawa et al (143) demonstrated that the initial rate of phosphorylation by MLCK was twofold lower than with myosin that had not been prephosphorylated by PKC. This decrease in initial rate was shown to result from an approximately 10-fold decrease in affinity of MLCK for the substrate; no effect on the Vmax of MLCK phosphoryation was observed whether unphosphoryated myosin or myosin prephosphorylated by PKC was used. In addition, PKC phosphoryla­ tion may alter the site of MLCK phosphorylation ( 1 60). Prephosphorylation by MLCK has also been shown to inhibit subsequent phosphorylation by PKC. Similarly, this inhibition appears to reflect a two­ fold decrease in the affinity for myosin prephosphorylated by MLCK ( 143) , and thus at least in vitro , these two kinases appear to modulate one another. Studies of smooth muscle ( 1 6 1 ) , platelet (116), and thymus (153) myosin indicate that PKC phosphorylation does not induce the conformational changes seen with phosphorylation by MLCK . Myosin phosphorylated by PKC remains predominantly in the l OS conformation, and behaves in a similar fashion to unphosphorylated myosin under various ionic strength conditions. However, smooth muscle myosin that had been phosphorylated by both MLCK and PKC exhibits a sedimentation coefficient of 7 . 3 S , intermediate to that of unphosphorylated myosin or myosin phosphorylated by either PKC or MLCK ( 1 61). Analysis of electron micrographS of myosin phosphorylated by both MLCK and PKC revealed that the percent of molecules in the folded conformation (59%) in 0. 1 5-0.08 M ammonium acetate was intermediate to that observed with myosin phosphory­ lated by MLCK alone (44%) and by PKC alone (93%) (161). Whether this change in the equilibrium between the l OS and the 65 forms is significant remains unclear. Using viscosity measurements to assess the conformation state of platelet myosin, Ikebe & Reardon (116) observed no differences in viscosity of myosin phosphorylated by MLCK alone from that phosphorylated by both MLCK and PKC . However, no direct visualization of myosin con­ formation was done in this study. The discrepancy between the results seen with smooth muscle and platelet myosins may reflect differences in these systems, but more likely reflects differences in ionic strength conditions and in the assays themselves . EFFECTS ON MYOSIN CONFORMATION

740 IN

TAN ET AL

VIVO EVIDENCE OF PHOSPHORYLATION

The results of studies of PKC

phosphorylation of the myosin II regulatory light chain in intact cells differ in many respects from those obtained from in vitro experiments. Initial studies with smooth muscle tissue from porcine carotid artery suggested that phorbol ester-induced contraction was mediated by low levels, approximately 0 .05 mole of phosphate per mole light chain, of PKC phosphorylation of the myosin light chain ( 1 62) . However, more recent studies with intact and

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glycerinated smooth muscle tissue from the same source indicate that PKC phosphorylation of myosin light chains, even to relatively high levels (greater than one mole phosphate per mole light chain) is not sufficient to induce contraction or to modulate the isotonic shortening or the isometric force produced by muscle fibers consisting of myosin phosphorylated at the MLCK sites ( 1 63). In addition, muscarinic stimulation of intact bovine tracheal smooth muscle failed to reveal any evidence of PKC phosphorylation of the myosin light chains (9) . Thus in the smooth muscle system , no compelling evidence exists for the role of PKC phosphorylation in intact cells . Phosphorylation of the myosin II light chains at the PKC sites in human platelets in response to phorbol ester stimulation ( 1 52 , 1 64) and thrombin ( 1 26), and in rat basophilic leukemia cells in response to antigen stimulation

(165), have also been demonstrated. However, the possible effects of phosphorylation at the PKC sites in intact nonmuscle cells have yet to be elucidated . In addition, mapping of the PKC sites indicates that only the serine residues are phosphorylated in vivo ( 1 26, 1 5 2 , 1 65 , 1 66) . No evidence exists that the

threonine residue that is preferentially phosphorylated by PKC in vitro is

targeted by PKC in vivo. The effects of PKC phosphorylation in vivo remain obscure , since in vitro experiments that imply that phosphorylation by PKC inhibits the actin-activated ATPase may reflect the effects of phosphorylation at the threonine residue .

Phosphorylation by Other Kinases PHOSPHORYLATION BY THE CaM KINASE II

Early studies on the substrates

of CaM kinase II indicated that this general protein kinase is able to phos­

phorylate isolated myosin 11 regulatory light chain ( 167, 1 68) but not intact

myosin ( 1 69 , 170). However, more recent studies have demonstrated that myosin from brain (1 7 1 ) , sea urchin egg (1 72 ), and smooth muscle (173 ) are

phosphorylated by CaM kinase II. The discrepancy between these results and previous results may reflect the different tissue sources of the kinase and, for smooth muscle myosin, the calmodulin concentrations used to assay kinase activity . In the early studies using smooth muscle myosin, activation of CaM kinase II was achieved by the addition of approximately stoichiometric

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MYOSIN PHOSPHORYLATION

741

amounts of calmodulin with respect to kinase. However. since then smooth muscle myosin has been reported to bind Ca2-t-tcalmodulin with high affinity ( 174) . This affinity is apparently higher than that of CaM Kinase II for Ca2+Icalmodulin, since phosphorylation of myosin by this kinase requires a calmodulin concentration greater than that of myosin ( 173). This phenomenon is not observed with the Ca2-t-Icalmodulin-dependent MLCK, since the affin­ ity of MLCK for Ca2+ Icalmodulin is approximately lOa-fold greater than that of CaM kinase II ( 173). The site of phosphorylation by CaM kinase II has been determined for smooth muscle myosin and appears to be serine 1 9 . This was inferred from experiments that showed that the phosphorylation site is a serine residue, that two-dimensional mapping of tryptic digests of myosin phosphorylated by CaM kinase II and MLCK are identical, and that phosphorylation by CaM kinase II is not seen after phosphorylation by MLCK ( 1 73). As would be expected, phosphorylation by CaM kinase II results in an increase in the actin-activated myosin ATPase activity ( 17 1 , 173). What role the CaM kinase II may play in vivo remains to b e determined . The greater rate of phosphorylation of the myosin II regulatory light chain by MLCK and the greater affinity for Ca2-t-/calmodulin of MLCK imply that phosphorylation by CaM kinase II may not be significant in vivo. HowevE;r, Edelman et al ( 1 73) suggest that light chain phosphorylation by CaM kinase II may be important during the phase following the intracellular Ca2-t- wave since, unlike MLCK , CaM kinase II remains active as a result of an initial Ca2+/calmodulin-dependent autophosphorylation. In addition, in certain nonmuscle tissues such as brain. the concentration of CaM kinase II has been estimated to be at least IOOO-fold greater than that of MLCK ( 1 73). Thus, the higher concentration of CaM kinase II may offset the lower rate of light chain phosphorylation. Preliminary studies using smooth muscle pp 34cdc2 myosin indicate that myosin II molecules may be a substrate for the cell cycle-dependent protein kinase pp34cdc2 ( 175). The phosphorylation site is believed to be serine 1 or serine 2, sites that are also phosphorylated by PKC ( 1 76). The effects of this phosphorylation, however, have not been con­ clusively determined. Although the sites are also those targeted by PKC, at least in vitro, PKC phosphorylates threonine 9 with faster kinetics than it phosphorylates either serine residue ( 1 56) . It remains unclear whether the decrease in the actin-activated ATPase activity seen with PKC phosphoryla­ tion of myosin previously phosphorylated by MLCK results from phosphorylation of threonine 9 or whether phosphorylation of serine 1 or serine 2 alone is sufficient to affect the actin-activated ATPase. Thus no PHOSPHORYLATION BY

742

TAN ET AL

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evidence exists to indicate that phosphorylation by the pp34cdc2 results in the same in vitro effects on myosin activity observed with PKC phosphorylation. It would be of great interest to see what effects phosphorylation of the regulatory light chain by pp34cdC2 has on myosin II function in vitro. This should provide some insights into the in vivo effects of phosphorylation not only by this kinase but also by PKC , since the in vivo sites of PKC phosphorylation appear to be only at serine 1 and 2 ( 1 26, 152, 1 66, 1 67) . PHOSPHORYLATION BY PROTEASE-ACTIVATED PROTEIN KINASE I The phosphorylation of the regulatory light chain from skeletal muscle ( 1 77, 1 78) and smooth muscle (90) by the protease-activated protein kinase J from skeletal muscle , reticulocytes, and chicken gizzard has been reported. This kinase , which phosphorylates myosin light chain in a Ca2+ -independent manner and is activated by limited proteolysis by trypsin, appears to phos­ phorylate the regulatory light chain at serine 1 9 , as assessed by two­ dimensional tryptic and chymotryptic peptide mapping of light chain phos­ phorylated by this kinase and MLCK, and by phosphoamino acid determina­ tion (90 ) . Activation of the myosin II actin-activated ATPase activity has been demonstrated with phosphorylation of the regulatory light chain by the pro­ tease-activated protein kinase (90) . However, no physiological significance of light chain phosphorylation by this kinase has been established.

The myosin I heavy chain kinase isolated from Acanthamoeba has been shown to phos ­ phorylate smooth muscle myosin regulatory light chains (61) at the same residues phosphorylated by MLCK and with comparable rates, resulting in an increase in the actin-activated ATPase activity (62) . However, this phosphorylation is most likely fortuitous since the Acanthamoeba myosin I heavy chain kinase does not phosphorylate the Acanthamoeba myosin II regulatory light chain. Thus although it is intriguing to consider the role of a single protein kinase able to activate both the myosin I and the myosin II systems, no evidence exists that such a kinase exists , although several general protein kinases, in particular the membrane-associated PKC, may be candi­ dates. PHOSPHORYLATION BY MYOSIN I HEAVY CHAIN KINASE

Dephosphorylation by Myosin Light Chain Phosphatases

A number of general protein phosphatases isolated from skeletal and smooth muscle tissues are known to remove the phosphate from the regulatory myosin light chain (for review , see 179) . Protein phosphatase type 1M (PP- I M) appears to be the major enzyme responsible for dephosphorylating myosin

743

MYOSIN PHOSPHORYLATION

60% 90% of the total myosin light chain phosphatase activity, respectively ( 179-1 8 1 ) . Two protein phosphatases with high myosin light chain de­

light chain in skeletal and cardiac muscle, accounting for approximately and

phosphorylating activity have also been identified from turkey gizzard. These

(1 82). ( 1 8 3 , 1 84) , and is thought to be

enzymes have been termed smooth muscle phosphatases III and IV Smooth muscle phosphatase IV binds myosin analogous to PP- I M

( 179) . In addition, a phosphatase associated with smooth

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muscle myosin that appears distinct from the smooth muscle phosphatase IV has been recently identified

( 1 85).

Since the general protein phosphatases appear t o b e ubiquitous , i t is presumed that an enzyme equivalent to PP- I M also functions to dephos­ phorylate the myosin II regulatory light chain in nonmuscIe cells. A myo­ sin II light chain phosphatase activity has been partially purified from

Die­ tyoste li um cells. This phosphatase dephosphorylates the light chain but not the heavy chain of Dictyostelium myosin II (83). It would be interesting to

determine whether it is a nonmuscIe isoform of PP- l M .

MYOSIN II HEAVY CHAIN PHOSPHORYLATION Phosphorylation of myosin II heavy chain has been found to occur in a variety of nonmusc1e cells as well as in the catch muscle of mollusks (Table

2), and

appears to be a general mechanism of regulating myosin II function. In most nonmuscle cells, heavy chain phosphorylation occurs in addition to light chain phosphoryl ation . Much of the work on the molecular details of myosin

II heavy chain phosphorylation, including the purification and characteriza­ tion of specific myosin II heavy chain kinases (MHCKs) , has focused on lower eukaryotic systems . The myosin II heavy chain has been shown to be a substrate for several protein kinases. In addition to substrate-specific MHCKs, the heavy chain can be phosphorylated by a number of general kinases , including PKC as well as casein kinase II (CK II) and a Ca2+ Icalmodulin dependent protein kinase (CaM kinase) . With the exception of myosin II

from Physarum, phosphoryla­

tion of the heavy chain by endogenous kinases results in a decrease of the actin-activated ATPase (Table

2) . In all myosin lIs studied, the phosphoryla­

tion sites on the heavy chain are located within the tail domain , and in most cases, are in close proximity to the carboxyl terminus (Figure

2). The

mechanism by which heavy chain phosphorylation regulates the activity of the head domain is not known . Heavy chain phosphorylation regulates the assem­ bly state of the

Dietyoste lium myosin II molecule, which may in tum affect ( 1 86, 1 87) . Heavy chain phosphoryla-

the measured actin-activated ATPase

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-...I

t

Table 2

Summary of myosin II heavy chain phosphorylation P/heavy chain

Phosphory-

Phospho-

Myosin source

Kinase'

lation

amino acid

Fibroblast

endogenous

in vitro

0. 3 - 0.5

Leukemic myo-

endogenous

in vivo

1 .4

endogenous

in vivo

Phosphorylation site

(mole/mole)

Effect on myosin

References

Decrease in ATPase

244

Decrease in ATPase

211

2 [()

blast Lymphocyte

& in vitro

Physarum

endogenous

in vitro

Thr

Pancreatic acinar

endogenous

in vivo

Ser & Thr

MHCK

in vivo

Ser

Increase in ATPase

212, 245 246

0.05

cell Acanthamoeba

1489, 1 494, 1 499

3

& in vitro

Decrease in ATPase Decrease in filament

5 2 , 53, 199, 201

formation

Dictyostelium

MHCK

in vivo & in vitro

Ser & Thr

1 823, 1 833 , 2029

2-4

Decrease in ATPase Decrease in filament formation

1 28, 1 86 , 1 92 , 197, 1 98 , 207, 226, 247

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Brush border

CaMK

in vitro

Ehrlich ascites

CK II

in vivo & in vitro

tumor cell Macrophage

CK II

Thr COOH-terminal 10 kDa

in vivo

Ser

COOH-terminal 10 kDa

& in vitro

Brain

CK II

in vitro

Ser

COOH-terminal 5 kDa

Human platelet

PKC

in vivo

Ser

COOH-terminal 50 ami-

& in vitro

2

No effectb

22 1-224

No effect

219

No effect

2 1 8 , 248 , 249

No effect

220, 250-252 1 65 , 2 14 ,

no acids

Chicken epithe-

PKC

in vitro

Ser

1 91 5

250

Rat basophilic

PKC

in vivo

Ser

last 50 amino acids

165 , 2 1 7

lium

215

leukemia cells Bovine platelet

PKC

in vitro

Thymus

PKC

in vitro

Molluscan catch

cAMPK

in vivo

muscle

& in vitro

1 16 153

rod portion

Endogenous kinases refer to identified activities that have not been characterized. Myosin heavy kinase II (CK II), protein kinase C (PKC), cAMP-dependent protein kinase (cAMPK). b No effect on ATPase activity or filament formation was detected. •

1 .3 - 1 . 5

Decrease i n filament

253 , 254

formation chain kinase (MHCK), Ca2+lcalmodulin-dependent kinase (CaMK), casein

-..l � Ul

746

TAN ET AL

tion of the Acanthamoe ba myosin II, however, does not appear to affect the assembly state of the molecule ( 188) but inhibits the actin-activated ATPase (52). Phosphorylation by Myosin Heavy Chain Kinases SITES In the lower eukaryotic systems in which MHCKs have been characterized, there are multiple sites of phosphorylation by MHCK. In Acanthamoe ba, three sites within the tail domain have been

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PHOSPHORYLATION

identified (53). These sites lie within the nonhelical carboxyl-terminal region of the tail and have been mapped to serine residues 1489, 1494, and 1499 ( 1 89). Dictyost elium myosin II also contains multiple sites of phosphorylation near the carboxyl terminus of the tail domain (54, 55, 190-194). Three sites of phosphorylation have been mapped to threonine residues 1823, 1833, and 2029 (54, 195, 196). Serine phosphorylation of the Dictyoste lium myosin II heavy chain has also been shown (128, 197, 198), but the site(s) of serine phosphorylation remain to be determined. In Acanthamoe ba , myosin II heavy chain phosphorylation results in a decrease of the actin-activated ATPase (199). Phosphorylation of the three serines near the carboxyl terminus by a partially purified MHCK completely inhibits the myosin ATPase activity (52). Phosphorylation of only serine 1489 appears to be sufficient for this inhibition (5 1 , 200). Early studies suggested that heavy chain phosphorylation of the Acantha­ moe ba myosin II resulted in destabilization of myosin filaments (20 1 , 202). However, more recent studies indicate that heavy chain phosphorylation has no effect on the assembly properties of Acan thamoeba myosin II (188). In addition, studies using a monoclonal antibody that binds near the tip of the Acanthamoeba myosin II tail and depolymerizes filaments indicate that dis­ assembly of filaments is associated with an inhibition of the actin-activated ATPase activity (203-205). Thus, although phosphorylation and assembly do not appear to be directly related, both dephosphorylation of the heavy chain and filament formation appear to be necessary for maximal actin-activated ATPase activity. Other studies suggest that the overall phosphorylation level of the filament rather than an intramolecular change induced by phosphorylation at the tip of the tail is what affects the ATPase activity. These studies showed that heteropolymers of dephosphorylated and phosphOlylated myosin (202) and of dephosphorylated myosin and phosphorylated 28-kDa tryptic fragment de­ rived from the carboxyl terminus of the tail (200) exhibited decreased ATPasl� EFFECTS OF PHOSPHORYLATION

MYOSIN PHOSPHORYLATION

747

activity as compared to filaments exclusively composed of dephosphorylated myosin. Atkinson et al (2 06) have proposed that the state of phosphorylation

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of the carboxyl-terminal nonhelical region regulates the actin-activated ATPase activity by altering the conformation of the filament in such a way that the myosin heads, although still able to bind filamentous actin, cannot proc eed through a normal catalytic c ycle . In Dictyoste lium, phosphorylation of the myosin II heavy chain also results in a decrease in the actin-activated ATPase of the molecule. In initial studies, phosphorylation by a partially purified MHCK to one mole of phosphate incorporated per mole of heavy chain resulted in an 80% inhibition of the A TPase activity, which is reversible with dephosphorylation (191, 197). In contrast to phosphorylation of the Acanthamoeba myosin II, however, phosphorylation of the Dictyoste lium myosin II heavy chain has a dramatic effect on the ability of the molecule to assemble into filaments, which may be the cause of the apparent increase in the actin-activated ATPase

( 1 86, 1 87 ,

192, 207). Although unphosphorylated Dictyostelium myosin II will form filaments at salt concentrations less than 200--250 mM, phosphorylated molecules will not assemble at any salt concentration (187, 192). Studies utilizing rotary shadowing techniques to visualize myosin II molecules reveal that phosphory­ lation of the heavy chain promotes a folding of myosin II at approximately 2/3 the length of the tail from the head domain (57), within the 34-kDa assembly subdomain defined by Q'Halloran et al (55) . Under conditions that promote filament assembly , molecules in this bent conformation are excluded from the myosin filament. In the bent conformation, the assembly subdomain is pre­ sumably sequestered by steric hindrance or by association with an adjacent

region as a result of local conformational changes induced by the introduction of negative charges at the nearby phosphorylation sites. Thus, unlike assem­

bly of higher eukaryoti c m yosin lIs, which is predominately dependent on

light chain ph osphorylation , assembly of the Dictyos telium myosin II appears

to be regulated by heavy ch ain phosph orylation , which favors a folded

monomeric conformation that is unable to incorporate into dimers or filaments (Figure 5). IN

VIVO EVIDENCE O F PHOSPHORYLATION

Phosphorylation of the myosin

II heavy chain has been demonstrated in a number of cell systems (Table 2). In Dicty ostelium cells, both serine and threonine phosphorylation of the myosin II heavy chain occurs in vivo (128, 197). Stimulation of chemoattrac­ tant-competent cells by cAMP results in an increase in the phosphorylation levels of the myosin II regulatory light chain and heavy chain (128, 208 , 209). This increase appears to be predominantly due to an increase in threonine

TAN ET AL

748

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/ Figure 5

Assembly of Dictyostelium myosin

II. Such assembly is depcndcnt on heavy chain

dephosphorylation in the tail domain. Phosphorylation of the tail induces a bend within the tail.

These bent molecules cannot assemble into filaments. Dephosphorylated molecules exist mainly in an extended form and can form filaments.

phosphorylation and parallels myosin translocation to the cytoskeleton and cell sh ape ch anges ( 1 30) . However, little is known about the precise in vivo phosphorylation sites. Thus whether the observed in vivo phosphorylation

reflects known MHCK activity or other kinase activity is still unclear . In Acanthamoeba , at least two of the three serine sites identified by in vitro phosphorylation by MHCK are known to occur in vivo (52) . REGULATION OF MHCK and

MHCK from fibroblasts (2 1 0) , l ymphocy tes (2 1 1) , Physarum (21 2) w as initially described as a protein kinase activity that

copurifies with actomyosin. These kinases phosphorylate only the myosin II heavy chain and require only Mg2+ and ATP as cofactors. Similarly , a partially purified MHCK from Acanthamoeba (52) appears to be specific for the Acanthamoeba myosin II heavy chain and also requires only Mg2+ and ATP for activity The Acanthamoeba MHCK is not regulated by cAMP or .

cGMP and is only slightly inhibited by Ca2+ Icalmodulin.

At least four distinct MHCKs have been identified in Dictyostel ium (187,

MYOSIN PHOSPHORYLATION

7 49

19 1 , 198, 207). At least two have been studied from growing cells . Maruta et al (191) reported the partial purification of a MHCK activity from growing

Dic tyos teli um cells that phosphorylates only threonine residues and is not regulated by known kinase effectors such as Ca2+ Icalmodulin, cAMP, and cGMP. Kuczmarski (198) identified at least two MHCK activities from growing cells. Both are specific for the Dictyoste lium myosin II heavy chain

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and require only Mg2+ and ATP as cofactors . One activity phosphorylates

both serine and threonine residues while the other phosphorylates only threonine residues. In addition, Cote & B ukiejko (207) have purified to near homogeneity a MHCK from the soluble fraction of growing cells. This kinase, which is specific for Dictyosteli um myosin II heavy chain, phosphory­ lates only threonine residues . Its activity is unaffected by the presence Ca2+ I calmodulin, cAMP, or cGMP. However, this kinase appears to be activated by autophosphorylation (21 3 ) . It is unclear whether the threonine-specific activity partially purified by Maruta et al ( 1 9 1 ) and Kuczmarski (198)

represents the MHCK characterized by Cote & Bukiejko (207).

Two additional MHCKs have been purified from aggregation-competent

Dictyostelium cells. One has been shown to phosphorylate Dictyoste li um as well as Physarum myosin II heavy chain on threonine residues and is inhibited by the presence of Ca2+Icalmodulin ( 1 9 1 ) . A second MHCK, purified from the membrane-associated fraction, phosphorylates only threonine residues in

the Di ctyosteli um myosin II heavy chain and undergoes autophosphorylation

(187). What effect, if any, autophosphorylation has on the activity of this

kinase is unknown. As with most of the various MHCKs studied in Di c­

tyoste lium, this MHCK is not affected by Ca2+ Icalmodulin, cAMP, or cGMP. Recently, a cDNA clone corresponding to this MHCK has been isolated and characterized (S . Ravid and J. A. Spudich, personal communica­

tion) . The primary structure of this MHCK is very reminiscent of that of PKC.

In addition to the highly conserved protein kinase catalytic domain , this ' MHCK contains the cysteine repeat motif characteristic of the PKC familY . It is intriguing to speculate that this MHCK may be a substrate-specific member of the PKC family.

Phosphorylation by Other Kinases PHOSPHORYLATION BY PKC

The initial observation that PKC phosphory­

lates myosin II heavy chain came from experiments with rat basophilic leukemia (RBL) cells and human platelets . In RBL cells, aggregation of IgE receptors present on the plasma membrane results in hydrolysis of inositol phospholipids and activation of PKC . This activation correlates with an

increase in the level of myosin II heavy chain phosphorylation ( 1 66) . Similar

results were observed in human platelets treated with phorhol esters (2 1 4) .

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TAN ET AL

Phosphopeptide mapping of myosin II from activated RBL cells and human platelets revealed that the phosphorylated peptides are similar, suggesting that sites of phosphorylation in these systems are analogous if not identical. Phosphorylation by PKC has also been demonstrated in vitro with myosin II heavy chain from human platelet (2 14) , chicken intestinal epithelial cells (2 1 5 ) , bovine platelets ( 1 1 6) , and thymus ( 153) . The phosphorylated amino acid is a serine residue, corresponding to serine 1 9 1 5 of the myosin heavy chain sequence of chicken intestinal epithial cell (2 1 6) , which is located within the a-helical region about 50 amino acids from the carboxyl terminus (2 1 5 , 2 17). Although the role of PKC phosphorylation of myosin II heavy chain is unknown, Adelstein et al (2 1 7) have suggested that this phosphoryla­ tion destabilizes myosin filaments in RBL cells , a role similar to MHCK phosphorylation of Dictyostelium myosin II heavy chain. Changes in myosin localization occur after stimulation of RBL cells with kinetics that are con­ sistent with the myosin phosphorylation (A. Spudich, personal communica­ tion) . In RBL cells, destabilization of the myosin filaments may also promote disruption of actin filaments and allow histamine granules to approach the cell membrane where their contents can be released (166, 2 17). Myosin II heavy chains from macrophages (2 1 8) , Ehrlich ascites tumor cells (21 9) , and bovine brain (220) have been shown to be phosphorylated by CK II. This phosphorylation is known to occur in vivo in at least the macrophage and the Ehrlich ascites tumor cell system. The site of phosphorylation by CK II is located near the tip of the tail but is distinct from the site phosphorylated by PKC (2 1 5 ) . In brain myosin II, the site of CK II phosphorylation is a serine residue found in the nonhelical region at the carboxyl terminus of the tail (220). Interestingly, smooth muscle myosins from chicken gizzard and rat aorta contain neither the PKC nor the CK II phosphorylation sites (2 15). However, phosphorylation by CK II does not appear to have any effect on the actin-activated ATPase activity or the assembly properties of myosin II in vitro. Whether CK I I phos­ phorylation affects some aspect of myosin 11 function in vivo remains to be determined. PHOSPHORYLATION BY CK II

PHOSPHORYLATION BY CaM KINASE Ca2+/calmodulin-dependent phos­ phorylation of the myosin II hcavy chain from brush border by an endogenous kinase (22 1 , 222) has been demonstrated in vitro (22 3 , 224) . This phosphorylation occurs on a threonine residue. However, like CK 11 phosphorylation, no effects on the properties of myosin II have been detected as a consequence of phosphorylation by the CaM kinase .

MYOSIN PHOSPHORYLATION

751

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Dephosphorylation by Myosin Heavy Chain Phosphatases Phosphatases capable of dephosphorylating myosin II heavy chains have been identified from Acanthamoeba and Dictyostelium . The phosphatase from Acanthamoeba has an apparent molecular weight of 39 ,000 and has broad substrate specificity (225). This enzyme appears to be a type 2A phosphatase ( 179) in that it is inhibited by ATP, pyrophosphate , and NaP but is stabilized by Mn2+ (225). Dephosphorylation of the Acanthamoeba myosin II heavy chain using this phosphatase results in an increase of the actin-activated myosin ATPase . Two phosphatases have also been partially purified from Dictyostelium (226). One phosphatase dephosphorylates the myosin II heavy chain as well as the regulatory light chain. Both also dephosphorylate histone and casein but at a much slower rates. These phosphatases require Mg2+ for their activity but are unaffected by Ca2+ . Dephosphorylation of Dictyostelium myosin II by these phosphatases increases the actin-activated myosin ATPase and induces filament formation.

CONCLUSIONS AND FUTURE DIRECTIONS Virtually all nonmuscle myosins , with the possible exception of brush border myosin I , are controlled by phosphorylation. The specific mechanisms by which phosphorylation controls the enzymatic and structural properties of these motor proteins vary . In higher eukaryotes, phosphorylation of myosin II molecules on the regulatory light chain appears to be necessary for both the actin-activated myosin ATPase as well as the ability to form filaments. Although phosphorylation of the heavy chain is also known to occur, the specific effects of this phosphorylation on the properties of myosin II are unclear. In contrast, the properties of Dictyostelium myosin II are clearly modulated by regulatory pathways involving phosphorylation of the regulatory light chain and the heavy chain. Distinct kinases phosphorylate these two subunits. In this system, phosphorylation of the regulatory light chain enhances motor activity while that of the heavy chain inhibits filament formation. These phosphorylations thus must be spatially and temporally coordinated in vivo to organize the apparatus essential to the cellular function of myosin II in this cell. In Physarum, phosphorylation also appears to increase myosin activity . Although phosphorylation of both polypeptide chains occurs , the specific effects of the individual phosphorylations remain to be determined. In addi­ tion , binding of Caz+ to the essential light chain regulates the Physarum myosin II by negating the activating influence of phosphorylation (2 10, 227).

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TAN ET AL

Unlike these myosin lIs, Acanthamoeba myosin II is predominantly con­ trolled by phosphorylation of the heavy chain. However, in contrast to the effects of this phosphorylation in Dictyostelium, it appears that heavy chain phosphorylation at the tip of the tail domain does not affect the assembly properties of the Acanthamoeba myosin II but rather may affect the quater­ nary structure of the filament to modulate ATPase activity . Limited data is available on the regulation of the very diverse class of myosin I molecules. Phosphorylation of the heavy chain within the head domain activates amoeboid myosin Is . However, whether this mechanism underlies the regulation of other types of myosin I is unknown . The brush border myosin I binds Ca2+ Icalmodulin and thus seems to be a Ca2+_ regulated enzyme. However, studies on the Ca2+ dependence of brush border myosin I-based motility have arrived at inconsistent results . Whereas one study (228) demonstrated that the brush border myosin I is active at low Ca2+ concentrations, another study suggested that the enzyme is active at high Ca2+ concentrations (229). Although this discrepancy may be explained by a number of possibilities, it is intriguing to speculate that the difference in the two preparations of brush border myosin I may reflect the phosphorylation state of the molecule and that the brush border myosin I , like Physarum myosin II, is dually regulated by a direct effect of Ca2+ and by phosphoryla­ tion. The sequences of brush border myosin I from both bovine and avian sources contain the phosphorylation site recognized by the Acanthamoeba MIHCK . It would thus be interesting to determine whether the Acanthamoeba MIHCK will phosphorylate brush border myosin I or if an analogous kinase: exists in brush border. Over the past several years , it has become apparent that the molecular basis of cell motility involves a multitude of motor molecules . Novel myosins are being identified not only in animal cells, but also in a number of plant systems including Nitella (230) , Egeria (23 1 ) , Lycopersicon (232) , Heracleum (233), Chara (234, 235), Pisum (236), and Ernodermis (237). In addition, several reports have suggested the presence of novel myosin molecules in bacterial cells (238-240) . However, a number of these newly identified myosins , especially those from eukaryotic sources, have only been characterized at a molecular genetic level. Little is known regarding their cellular functions , their structure, their enzymatic properties , or the regulatory mechanisms of these molecules . In addition to the myriad of myosins that are now seemingly present in all nonmuscle (and possibly muscle) cells, a number of signalling pathways may control the activity of each motor molecule. In higher eukaryotes and in Dictyostelium and Physarum, several kinases may regulate at least myosin 11 activity in vivo by multiple phosphorylations on both the regulatory light

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753

chain and the heavy chain. These phosphorylations are presumably coordin­ ated such that the myosin II molecule is appropriately localized and activated for a particular cellular function. For example, phosphorylation of the light chain may regulate the role of myosin II in cell division and cell shape changes. Early studies involving microinjection of antibodies specific to smooth muscle MLCK into sea urchin eggs suggest that MLCK activity and presumably light chain phosphorylation are necessary for cell division (24 1). More recent studies using antisense oligonucleotides indicate that a decrease in MLCK results in morphological cell shape changes and a possibly slowed cell proliferation (82). In contrast, PKC phosphorylation may modulate myosin II in such a way that vesicular fusion to the plasma membrane and thus secretion may occur. However, the precise relationships between particu­ lar phosphorylation events and specific cellular functions of myosin II remain obscure. The determination of such a relationship can only come about from map­ ping of in vivo phosphorylation sites coupled with the study of cells that have been specifically mutated such that they no longer express the specific kinases , or that contain myosin that does not possess the phosphorylation sites. One system amenable to such studies is Dic tyosteli um. Both the myosin heavy chain (242) and the regulatory light chain ( 1 02) as well as the com­ plementary DNA of the MLCK (243) and the MHCK ( S . Ravid and J. A . Spudich, personal communication) have been characterized. The disruption of the Dictyoste li um myosin heavy chain has already led to new insights into myosin II function. It is hoped that new insights will also be gained from disruption of the myosin kinase genes. Specific mutations that eliminate the phosphorylation site in both the heavy and the light chain can also be made. Such mutated molecules can subsequently be introduced into cells genetically engineered such that they do not express the particular myosin polypeptide chain. This approach would be better suited in determining the role of myosin II phosphorylation by general protein kinases such as PKC or pp34cdc2 • The availability of gene targeting techniques and the feasibility of biochemical characterizations of myosins in organisms such as Dictyoste li um as well as in mammalian cells should allow for a better understanding of the role of myosin phosphorylations in controlling these motor proteins. ACKNOWLEDGMENTS

We are indebted to Kathy Ruppel for critical review of the manuscript and for extensive assistance in its preparation. We would also like to thank Tom Egelhoff, Hans Warrick, Meg Titus, Mitsuo Ikebe, and Janet Smith for many helpful discussions .

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Control of nonmuscle myosins by phosphorylation.

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