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Ca²⁺ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia

ABSTRACT

I. INTRODUCTION: EVIDENCE OF CALCIUM SENSITIZATION AND DESENSITIZATION

II. MYOSIN LIGHT-CHAIN KINASE ISOFORMS AND TELOKIN

III. TOOLS AND TRAPS OF THE TRADE

A. Toxins and Enzyme Inhibitors

B. ROK Inhibitors

C. Proteins Expressed, Overexpressed, Constitutively Active, and Phosphorylations: Estimating Active RhoA

IV. REGULATION UPSTREAM

A. Agonists, Receptors, Trimeric and Monomeric G Proteins, Ligands, Lipid Messengers, and Microtubules

B. GEFs, GDIs, GTPase-activating Proteins, RhoA, and ROK

1. GEFs

2. GDI and GAPs

3. ROK

C. Ephrins and Plexins: RhoA/ROK in Axonal Guidance and Angiogenesis

V. MYOSIN PHOSPHATASE

VI. CPI-17 AND PROTEIN KINASE C

VII. TYROSINE KINASES: FOCAL ADHESION KINASE

VIII. MITOGEN-ACTIVATED PROTEIN KINASE, P21-ACTIVATED KINASE, AND LIM-KINASE

A. MAPK

B. PAK

C. LIM-Kinase

IX. CALCIUM DESENSITIZATION: GUANOSINE 3',5'-CYCLIC MONOPHOSPHATE-MEDIATED PATHWAY AND ANTAGONISM TO RHOA

X. CALCIUM SENSITIZATION IN PLATELETS AND ENDOTHELIAL CELLS AND CELL MIGRATION

XI. CALCIUM SENSITIZATION BY RHOA/ROK IN HEALTH, DISEASE, AND AS A TARGET FOR THERAPY

A. Physiological Activity

B. Diseases of Smooth Muscle

C. Cancer

XII. WANTED: RHOAD MAPS THROUGH DOMAINS TORTUOUS, BUT RICH

	ABSTRACT

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	I. INTRODUCTION: EVIDENCE OF CALCIUM SENSITIZATION AND DESENSITIZATION

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Recognition that calcium is the intracellular messenger that triggers muscle contraction (144) by binding, in striated muscles, to the Ca-binding protein troponin (88) eventually led to the realization that Ca-regulated myosin II plays major roles not only in striated, but also in smooth muscle and in nonmuscle cells. Identification of other Ca-binding proteins, such as the ubiquitous calmodulin, and their effectors revealed complex, interconnected cellular signaling mechanisms, critically regulated by protein kinases (reviewed in Ref. 69) and phosphatases (reviewed in Ref. 70). In the case of smooth muscle and nonmuscle myosin II, their ATPase activity and associated motility (contraction) are activated by actin, but only when Ser-19 of the myosin regulatory light chain (RLC) is phosphorylated, usually by a calcium-calmodulin (Ca²⁺/CaM)-dependent myosin light-chain kinase (MLCK) (reviewed in Refs. 115, 357; see Fig. 1). This Ca-dependent activation of myosin II plays an essential role in a variety of processes, but regulation of cellular functions by changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) is further modulated by the Ca²⁺ sensitivity of the Ca²⁺ sensors and effectors that can also be modified by factors such as the affinity (K_CaM) of calmodulin for MLCK and the activity of the G protein-regulated myosin phosphatase.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Regulation of contraction, stress fiber formation, and cell migration through phosphorylation/dephosphorylation of the regulatory light chain (RLC20) of myosin II. Activation of myosin light-chain kinase (MLCK) by Ca2+ binding to calmodulin (CaM) leads to phosphorylation of the RLCs of myosin II to switch on cross-bridge cycling and force development by actin-activated myosin. The ratio of kinase to phosphatase activities determines the level of RLC phosphorylation and the extent of activation. [Ca2+]-independent modulation of the activities of MLCK and/or myosin light-chain phosphatase (MLCP) provides additional mechanisms for regulation of RLC20 phosphorylation.

Studies with Ca²⁺-sensitive fluorophores suggested that, as expected (363), force developed at a given global level of [Ca²⁺]_i could vary, depending on the type of excitatory stimulus: agonist-induced force is often higher than depolarization (high K)-induced force at similar, or even lower, [Ca²⁺]_i (39, 145). Studies employing cell permeabilization methods that retained G protein-coupled receptors confirmed that the underlying mechanism is agonist-induced Ca²⁺ sensitization of the contractile/regulatory apparatus, not an artifact of the Ca²⁺ reporters. When such permeabilized muscles are activated by an agonist or guanosine 5'-O-(3-thiotriphosphate) (GTPS) while [Ca²⁺]_i is clamped, they respond with increased RLC phosphorylation and force (200). The term Ca²⁺ sensitization was coined to describe this phenomenon. Interestingly, different agonists can stimulate unequal maximal Ca²⁺ sensitization (146) through yet to be identified mechanism(s), perhaps qualitatively or quantitatively variable coupling between different agonists and trimeric and monomeric (RhoA) G proteins and guanine nucleotide exchange factors (GEFs) (Fig. 4).

View larger version (37K): [in this window] [in a new window]   FIG. 4. Signaling pathways for Ca2+ sensitization in smooth muscle. Different smooth muscles respond to a large number of different agonists including catecholamines, muscarinic agonists, thromboxane, histamine, serotonin, as well as the sphingolipids: sphingosine 1-phosphate and sphingosylphosphorylcholine. Activation of their receptors initiates signaling through the illustrated cascades that inhibit myosin phosphatase (MLCP), increase RLC20 phosphorylation and contraction, stress fiber formation, and/or cell migration. The Rho/ROK pathway can also lead to activation of smooth muscle differentiation marker gene expression. GPCR, G protein-coupled receptors; GEF, guanine nucleotide exchange factor(s); RhoGDI, GDP dissociation inhibitor; RhoGAP, GTPase activating protein; ROK, Rho-kinase, ROK/ROCK II, ROK/ROCK I; C3, clostridial C3 exoenzyme ADP ribosylates and inactivates RhoA; iPLA2, Ca2+-independent phospholipase A2; CPI-17, PKC-potentiated inhibitor protein of 17 kDa; MYPT1 and PP1c, myosin phosphatase regulator and catalytic subunits; MLCK, myosin light-chain kinase; DAG, diacylglycerol; PKC, protein kinase C; AA, archidonic acid; SR, sarcoplasmic reticulum; InsP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate.

Ca²⁺ desensitization was also first recognized in similar experiments that showed a decline in force and RLC phosphorylation while [Ca²⁺]_i was unchanged during K⁺ (depolarization)-induced contractions of intact smooth muscles (147) and was confirmed by the phasic decline in RLC phosphorylation and force in permeabilized phasic smooth muscles maintained at constant [Ca²⁺]_i (202).

Ca²⁺ sensitization and desensitization are now understood to involve the major physiological mechanisms that regulate myosin II activity: phosphorylation and dephosphorylation. Phosphorylation (at Ser-19) of RLC of smooth muscle (reviewed in Ref. 181) and nonmuscle (reviewed in Ref. 342) myosin II permits their activation by actin, whereas dephosphorylation (reviewed in Refs. 141, 357) inactivates these actin-activated myosin II ATPases. Consequently, the myosin light-chain kinase (MLCK)-to-myosin phosphatase activity ratio is the major determinant of the Ca²⁺ sensitivity of myosin II.

Smooth muscle is particularly suitable for identifying, through measurements of force and RLC phosphorylation, mechanisms that regulate the Ca²⁺ sensitivity of myosin II, and we will use its behavior as our framework for this review. However, because nonmuscle myosin II, MLCK, and myosin light-chain phosphatase (MLCP) are ubiquitously expressed in most, if not all, nonmuscle cells, regulation of (acto)myosin II by phosphorylation/dephosphorylation is a widespread, nearly universal, cellular mechanism. In nonmuscle cells, the development of stress fibers, cell motility, migration, and RLC phosphorylation are often monitored as indices of myosin II activity (reviewed in Ref. 28).

Modulation of Ca²⁺ sensitivity by posttranslational modification of Ca²⁺ sensors is not limited to phosphorylation of myosin II but can be mediated by a variety of other mechanisms, as illustrated by the effect of cardiac troponin phosphorylation on the Ca²⁺ sensitivity of myocardial contractility (53, 247, 284), the variable Ca²⁺ sensitivity of inositol 1,4,5-trisphosphate (InsP₃) receptors (159), and the effect of phosphorylation on ryanodine receptors (171).

MLCK and the RhoA-associated Rho-kinase (RhoA/ROK) pathway are two major cellular targets for regulating Ca²⁺ sensitivity of myosin II and, as we shall suggest, they generally operate in parallel. Phenotypes attributable to RhoA/ROK and, more specifically, to ROK require basal [Ca²⁺]_i levels sufficient for "constitutive" MLCK activity. This is indicated by the inhibition of RhoA/ROK-induced effects by MLCK inhibitors and by inhibition of Ca²⁺ influx (e.g., Ref. 243) and, conversely, by the reduction of myogenic tone by ROK inhibitors in the presence of normal resting [Ca²⁺]_i (34, 394, 436). Therefore, we consider that most phenotypes of RhoA/ROK that reflect myosin II activity are manifestations of Ca²⁺ sensitization to normal, resting levels (100 nM) of [Ca²⁺]_i or to increases in [Ca²⁺]_i often stimulated by the same agonists that induce Ca²⁺ sensitization. The existence of constitutive (Ca²⁺-dependent) MLCK activity in resting cells is indicated by the decreases in resting [Ca²⁺]_i and reduced RLC phosphorylation and force induced by inhibition of Ca²⁺ influx (e.g., Ref. 148) and the inhibition of RhoA/ROK-induced stress fiber formation by MLCK inhibitors (43, 226, 281). Each of these findings suggests that phenotypes induced by ROK result from Ca²⁺-sensitizing inhibition of myosin phosphatase that also requires some activity of Ca/CaM or of a Ca²⁺-independent MLCK that phosphorylates RLC, because phosphatase inhibition will not increase RLC phosphorylation without kinase activity.

	II. MYOSIN LIGHT-CHAIN KINASE ISOFORMS AND TELOKIN

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Smooth muscle MLCK (smMLCK) is distinguished by its exquisite substrate selectivity for RLC and its ubiquitous distribution not only in smooth muscle, but also in most, if not all, nonmuscle cells in which smMLCK phosphorylates RLC Ser-19 (or its equivalent) to allow actin to activate myosin II (116, 181, 218). SmMLCK and MLCP act upon smooth muscle and nonmuscle myosin II isoforms to provide a reversible regulatory mechanism of phosphorylation/dephosphorylation that is central to physiological processes ranging from vascular, gastrointestinal, and uterine smooth muscle contraction and erectile function through platelet aggregation, cytoskeletal modeling by stress fibers, fibroblast contraction, angiogenesis, and endothelial permeability to normal and cancer cell migration, asthma, hypertension, and atherosclerosis. Each of these normal and abnormal processes involves regulation of myosin II, not only by Ca²⁺/CaM-activated smMLCK, but also by Ca²⁺-sensitizing and -desensitizing mechanisms.

The smMLCK is the product of a single gene, different from the one giving rise to skeletal muscle MLCK (skMLCK), although both are activated by Ca²⁺/CaM. Splice variant products of the smMLCK gene range from short (130-150 kDa) to long (208-214 kDa) MLCK (116, 181, 222).

A COOH-terminal Ser phosphorylation site of smMLCK provided the earliest evidence that the Ca²⁺ sensitivity of smMLCK could be regulated: phosphorylation of this site reduces the affinity of MLCK for Ca²⁺/CaM (increases K_CaM) by 10-fold. This inhibitory Ser (Ser-512 in smMLCK) can be phosphorylated by cAMP-dependent kinase (PKA; Ref. 72) and also by other kinases (3; reviewed in Refs. 116, 181), but only when Ca/CaM is not bound to MLCK. Increases in cellular cAMP are associated with MLCK phosphorylation (78), but other studies suggested that inhibitory phosphorylation of MLCK in smooth muscle in vivo (Ca²⁺ desensitization) is more likely to be an autoinhibitory mechanism mediated by Ca²⁺-dependent phosphorylation of MLCK by CaM kinase II (250, 383). In nonmuscle cells, however, inhibitory phosphorylation of MLCK may be mediated by PKA or by another kinase. Cyclic nucleotide-activated kinases can also reduce [Ca²⁺]_i (418) and inhibit the Ca²⁺-sensitizing effect of ROK (see below), complicating the identification of the specific (or major) mechanism of cyclic nucleotide-induced decrease in RLC phosphorylation in intact cells.

cGMP-dependent kinase (PKG), unlike PKA, does not phosphorylate the inhibitory site of MLCK (277), supporting the conclusion that 8-bromo-cGMP causes Ca²⁺ desensitization by, directly or indirectly, increasing MLCP activity (428, 429) rather than by inhibiting MLCK.

The range of Ca²⁺ sensitivity that can be determined by K_CaM is constrained, at least in smooth muscles, by the high cellular concentrations of CaM and MLCK, considering the very high affinity (1 nM K_CaM) for MLCK (116) and the very large bound fraction of cellular CaM (116, 444). Ca²⁺ sensitivity of MLCK may also be affected selectively by its localization relative to the relevant protein kinases, and, in nonmuscle cells, its effect on phenotype will also depend on the variable subcellular localization of myosin II heavy chain isoforms (190, 207). Binding of MLCK to native thin filaments could also affect its Ca²⁺ sensitivity and reminds one of the still unanswered question of how the low micromolar cellular MLCK, bound to thin filaments, can phosphorylate nearly 100% of the 80 µM RLC in smooth muscle (368). MLCK can also be regulated independently of K_CaM; proline-directed phosphorylation by mitogen-activated protein kinase (MAPK) increases its V_max without affecting K_CaM (205, 257). The Ca²⁺ sensitivity of MLCK may also be modulated by a G protein: GTPS increases the level of MLCK phosphorylation, possibly because a G protein-regulated phosphatase also dephosphorylates MLCK (382). However, the specific phosphatase (perhaps MLCP) that dephosphorylates MLCK has not been identified. Phosphorylation (probably at Ser-439 and Ser-991) of MLCK by p21-activated kinase (PAK) inhibits it and desensitizes nonmuscle cell myosin II to Ca²⁺ (123, 326).

Long smMLCK isoforms (208-214 kDa; Ref. 181) are alternatively spliced products of the same gene as the short smMLCK and telokin. The NH₂-terminal extension of long MLCKs contributes to its strong actin binding with higher affinity than that of short MLCK (353). Several smooth muscles contain both short and long MLCK (29). Long MLCK is prominently expressed in embryonic smooth and nonmuscle cells (181), particularly in endothelium (402), where both forms are present in mature cells, although the long MLCK tends to predominate in cultured endothelial or smooth muscle cells (29), and its expression increases with passage in culture. NH₂-terminal Tyr phosphorylation of endothelial long smMLCK by p60_src has been suggested to be regulatory, possibly by affecting its subcellular localization (26, 27), but it remains to be determined whether it occurs in vivo and affects Ca²⁺ sensitivity. Localization of enzymes relative to their substrates, such as different localization of, respectively, long and smMLCK (29) and different subcellular Ca²⁺ domains are likely to result in spatially selective modulation of Ca²⁺ signaling.

Downregulation of short smMLCK in cultured smooth muscle cells does not reduce basal or stimulated RLC phosphorylation (19), suggesting that either long smMLCK can substitute for the small isoform in these cells or that the <10% remaining short MLCK is sufficient to carry out normal RLC phosphorylation. This possibility cannot be excluded, considering that <1% of total CaM in smooth muscle is required to support maximal force (444), and the two isoforms have comparable kinase functions (29). The potential of other kinase to compensate, at least partially, for Ca²⁺/CaM-MLCK is indicated by contraction of smooth muscles in which the gene has been deleted and each isoform is absent (H. Wang, A. P. Somlyo, and A. V. Somlyo, unpublished data).

Telokin, a 17-kDa acidic peptide whose sequence is identical to the COOH terminus of MLCK, is expressed independently through a promoter located in an intron of the MLCK gene (74, 167). It is a PKG substrate in vivo implicated in Ca²⁺ desensitization (see below).

skMLCK, a somewhat smaller (77-90 kDa) product of a different gene than smMLCK (181), is also regulated by Ca²⁺/CaM and can be Ser-phosphorylated by PKA, but this phosphorylation does not affect its Ca²⁺/CaM sensitivity (27, 89).

	III. TOOLS AND TRAPS OF THE TRADE

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Much has been learned about the RhoA/ROK pathway by relatively new research tools and methods, only some of which can be discussed here.

A. Toxins and Enzyme Inhibitors

Bacterial toxins and exoenzymes that affect Rho subfamily small GTPases (reviewed in Refs. 36, 37, 44) have been valuable not only to the pathogens utilizing them, but to investigators of the small GTPases targeted. The C3-exoenzyme (reviewed in Ref. 36) and the Escherichia coli protein EDIN (130, 369) are ADP-ribose transferases that ADP-ribosylate the Asn-41 residue of Rho, but not Rac or Cdc42. ADP-ribosylation inactivates RhoA and inhibits Ca²⁺ sensitization (108, 130). The high specificity of C3 led to its extensive use for inhibiting the RhoA/ROK pathway upstream without directly affecting Rac or Cdc42. Experimental use of the relatively cell impermeant C3 is facilitated by the chimeric protein DC3B that utilizes the B fragment of diphtheria toxin to allow intracellular penetration of C3 in whole tissues (15, 108). Clostridium difficile toxin B, a major cause of severe diarrhea, also inactivates RhoA (233), but is less specific, as it glycosylates not only Thr-37 of RhoA, but also the equivalent (Thr-35) residues of Rac and Cdc42. Toxin B inactivates these proteins by "immobilizing" the glycosylated residues that contribute to Mg²⁺ coordination required for nucleotide binding (417) and so prevents the GTP/GDP exchange necessary for activity. The Yersinia enterocolytica toxin (YopT) uncouples RhoA from its effectors, detaches it from membranes, and prevents its interaction with the Rho-binding domain of rhotekin (364, 446). YopT is a Cys proteinase that cleaves RhoA by removing the prenylated COOH-terminal methyl between Gly-189 and Cys-190 of RhoA (344). This finding, combined with the inability of cleaved RhoA to interact with the Rho-binding domain of its effector, also suggests that the interaction between RhoA and its effectors normally occurs at a lipid membrane. The observation that Rho-guanine nucleotide dissociation inhibitor (GDI) interacts with RhoA even after treatment with YopT (364) is surprising, because insertion of the prenylated RhoA COOH terminus into the hydrophobic cavity of GDI provides the strong binding for the complex. It may be reconciled by the possibility that weak binding of the NH₂ terminus of GDI to RhoA (231) is sufficient for coimmunoprecipitation, because YopT can extract endogenous RhoA from its complex with GDI (4), and GDI can interact with nonprenylated RhoA in a yeast two-hybrid screen (99).

In contrast to inhibitory toxins, the cytotoxic necrotizing factor 1 (CNF-1) activates Rho, Rac, and Cdc42 by deamidating Gln-63 (of Rho) or Gln-61 (Rac and CDC42), converting a Gln to a glutamate. This "mutation" blocks the GTPase activity of the Rho proteins, rendering them constitutively active, causing not only major changes in cytoskeletal architecture, but also disturbance of cell to cell interactions and epithelial barrier function; E. coli producing this toxin are implicated in urinary tract infections (37, 154).

B. ROK Inhibitors

Highly selective inhibitors against ROK, in particular, Y-27632, discovered by Narumiya and co-workers (107, 393), have been extremely useful in assessing specific phenotypes and consequences of ROK inhibition. In addition to Y-27632 and another pyridine derivative, Wf-536, two other chemically unrelated compounds, HA-1077 and H-1152P (328), show high selectivity against ROK. The inhibitory constant (K_i) of Y-27632 is 0.2-0.3 µM for ROK and 10 µM for protein kinase C (PKC)-; an important property of Y-27632 is that, although it competes with ATP for binding, it is highly effective in cells (containing high ATP levels), with the same IC₅₀ as its K_i (165). The most recent compound, H-1152P, has a K_i of 0.0016 µM for ROK and 9.27 µM for PKC (328). Although ROK inhibitors are not perfectly selective, their significantly greater (100-fold) activity against ROK than against conventional PKCs is sufficient, in most cases, for evaluating specific ROK functions. Minor caveats are that against the novel PKC, PKC-, the IC₅₀ of Y-27632 is 14 µM in the presence of 2 mM ATP (94), whereas 10 µM Y-27632 inhibits very significantly ROK-mediated Ca²⁺ sensitization in the presence of 4.5 mM MgATP. The extent to which inhibition of novel PKCs by Y-27632 could interfere with the interpretation of its effects may also depend on the expression levels, in different smooth muscles, of novel PKCs and CPI-17, activated by PKCs (see below). The second caveat is that, at concentrations (e.g., 100 µM) usually higher than required to inhibit Ca²⁺ sensitization of force, Y-27632 reduces the fura 2 signal used to measure agonist-induced increases in the cytoplasmic Ca²⁺ signal (168). In most cases Y-27632 at 10 µM concentrations or less is a sufficiently specific inhibitor of ROK, and the few uncertainties about its specificity can be resolved by upstream inhibition of RhoA itself with C3 or EDIN.

Inhibition of geranyl-geranyl transferase is another in vivo, albeit nonspecific (51), method for inhibiting RhoA upstream (224), because COOH-terminal geranyl-geranylation is required for membrane localization involved in Ca²⁺-sensitizing Rho activity (108, 127, 130).

C. Proteins Expressed, Overexpressed, Constitutively Active, and Phosphorylations: Estimating Active RhoA

The valuable tools of molecular biology can also lay traps for the unwary. The activity and target specificity of constitutively active kinases may differ from those of the endogenous forms, and expression of transfected proteins and, even more so, overexpression may result in subcellular distributions and activities that are not representative of physiological pathways. Unexpected cross-reactivity of antibodies (e.g., Ref. 132), unless validated by protein sequencing when first used, could also mislead. We suggest that criteria, like those developed by Krebs and Beavo (216), should be extended to include stringent controls for these powerful, newer methods. Their criteria (216) for verifying that an enzyme undergoes physiologically significant phosphorylation/dephosphorylation are still valid, and it is particularly important in the case of signaling mechanisms to demonstrate that a kinase substrate is phosphorylated in an intact cell system with a time course consistent with the anticipated functional change. We also suggest that localization of an enzyme relative to both its substrate and its activating mechanism should be consistent with its effect on integrated cell function. If the function of a given G protein, kinase, or phosphatase requires translocation to its substrate (or vice versa), such as the case of RhoA and ROK translocation to the plasma membrane (108, 127, 254, 375) and the cellular traffic of MLCP (34, 349) or CPI-17 between ROK and myosin filaments, the time course of this relocalization should precede and parallel the change in Ca²⁺ sensitivity.

Failure to identify the specific phosphorylation site(s) in a kinase or phosphatase can complicate interpretation of findings. For example, results obtained with, respectively, ³²P incorporation and site-specific antibodies to phosphopeptides may lead to differing conclusions. Radioisotopic assay showing increased radioactivity in a protein may reflect turnover or incorporation into a nonfunctional site rather than stoichiometric change in a functional residue, whereas a site-specific antiphosphopeptide antibody will not detect potentially relevant phosphorylation of a residue other than the one against which the antibody was generated. This problem can arise when evaluating Thr-696 phosphorylation of myosin phosphatase inhibitor (MYPT1) that contains more than one ROK phosphorylation site (10). In case of negative results, such as contraction without detectable RLC phosphorylation (reviewed in Ref. 337), the time course of phosphorylation should be determined using rapid kinetic methods: RLC phosphorylation in smooth muscle can peak within 3 s of stimulation and return close to resting levels within 30 s while significant force is maintained (147).

Active GTP · RhoA is the major determinant of Ca²⁺-sensitizing ROK activity, rather than the total cellular RhoA that in nonstimulated cells exists largely as the inactive GDP · RhoA · GDI complex (Fig. 2). Methods of estimating the cellular concentration of GTP · RhoA are based on the binding of GTP · RhoA, but not GDP · RhoA, to specific regions of Rho effectors: Rho binding domains (RBDs) that can precipitate GTP · RhoA from cell homogenates while leaving GDP · RhoA (complexed with GDI) in solution. The RBDs of rhotekin (308) and of p140 mDia (194) have been valuable in detecting changes in RhoA activity induced by Ca²⁺-sensitizing agonists and inhibitors.

View larger version (76K): [in this window] [in a new window]   FIG. 2. Scheme of regulation of the small GTPase RhoA. In the resting state, RhoA · GDP exists in the cytosol with its prenylated, hydrophobic tail buried within its partner, GDP dissociation inhibitor (RhoGDI). Activation of receptors coupled to certain trimeric G proteins (Gq, G12,13) and receptor Tyr kinases leads, through activity of guanine nucleotide exchange factors (GEFs), to the exchange of GTP for GDP on RhoA. RhoGDI dissociates and RhoA · GTP associates with the membrane where it interacts with Rho-kinase (ROK) to initiate signaling cascades. Rho GTPase activating proteins (RhoGAPs) catalyze hydrolysis of GTP bound to RhoA and RhoA · GDP reassociates with RhoAGDI.

Ideally, the time course of RhoA activation and of the downstream effect of phosphorylation by activated ROK should be determined. This would not only validate the role of RhoA/ROK in a given process, but would also provide information about intervening steps. For example, the kinetics that include a long lag phase (6 s) between photolysis of caged GTP in the nucleotide binding pocket of the RhoA/GDI complex and the onset of contraction suggests the intervention of a multistep, slow process preceding RLC phosphorylation (Fig. 3).

View larger version (27K): [in this window] [in a new window]   FIG. 3. Kinetics of activation of Ca2+-sensitized force upon photolysis of caged GTP-G14VRhoA/GDI complex in a -escin permeabilized strip of rabbit portal vein smooth muscle at pCa 6.5. Addition of the caged GTP complex had no effect before photolysis. Upon photolysis (arrow), 6 µM, a saturating concentration of free GTPG14VRhoA/GDI, was released at a rate of 60 s-1. Back extrapolation of the fast, rising phase to the prephotolysis baseline shows a lag phase of 6 s (inset). The lag between phosphorylation of RLC to the onset of force (444) accounts for only <0.5 s. Thus the 5.5-s lag phase triggered with this saturating concentration of the complex reflects the time course of dissociation of RhoA · GTP from GDI, translocation of GTP · RhoA to the membrane, activation of ROK, and inhibition of MLCP.

	IV. REGULATION UPSTREAM

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A. Agonists, Receptors, Trimeric and Monomeric G Proteins, Ligands, Lipid Messengers, and Microtubules

A variety of agonists can induce Ca²⁺ sensitization through RhoA and its downstream effectors (Fig. 4). RhoA, a small (20 kDa) monomeric GTPase (reviewed in Ref. 28), was identified as a major messenger of Ca²⁺ sensitization in two studies initiated by us that showed that activated RhoA (GTP · RhoA) Ca²⁺-sensitized force (130, 149) and RLC phosphorylation (130) in permeabilized smooth muscle. Agonists can activate RhoA through numerous G protein-coupled receptors: -adrenergic, muscarinic, prostanoid (75, 146, 166, 325), purinergic (332), endothelin (412), thrombin (90, 241, 398), vasopressin (220, 376), oxytocin, epidermal growth factor, purinergic, ephrin, semaphorin; angiotensin and EDG lysophospholipid (365, 386) receptors (e.g., Refs. 9, 16, 68, 75, 87, 90, 114, 130, 146, 150, 153, 155, 166, 241, 263, 293, 332, 379, 406, 411, 437 and reviewed in Refs. 321, 338, 357, 358). Most G_q-coupled receptors activate both RhoA and phospholipase C (PLC) that induces InsP₃ production and Ca²⁺ release from the sarcoplasmic/endoplasmic reticulum, increase Ca²⁺ influx through receptor-operated or voltage-gated channels (reviewed in Refs. 184, 359) and inhibit maxiK channels resulting in depolarization (8). Therefore, under physiological conditions, the three mechanisms, Ca²⁺ release, influx, and sensitization, often act in concert (1, 357, 358). However, InsP₃ does not affect Ca²⁺ sensitivity and to what extent diacylglycerol, the other product of PLC, has a significant physiological role in Ca²⁺ sensitization is still under examination (see sect. VI). Furthermore, agonists acting on thromboxane A₂ receptors and linked through G_12,13 can induce high force through Ca²⁺ sensitization without (or with only minimal) activation of PLC, at least as indicated by their limited ability to release Ca²⁺ (39, 146, 280). Receptors coupled to G_q and G_12,13 family trimeric G proteins are upstream initiators of RhoA/ROK-mediated Ca²⁺ sensitization through complex downstream mechanisms that are only now being unraveled (see below and Refs. 124, 204).

Trimeric G protein(s) have been implicated in RhoA/ROK-mediated Ca²⁺ sensitization by the demonstration that transfected -subunits of G_12,13 activate RhoA (140, 186, 240) and by the Ca²⁺-sensitizing effect of aluminum fluoride (AlF₄^-) (130, 189, 438). Because early work (177) did not show direct interaction of AlF₄^- with monomeric GTPases, whereas a GGDP · AlF₄^- adduct interacts with GEFs that activate RhoA (e.g., Refs. 35, 45, 60, 111, 140), it had been thought that AlF₄^- cannot directly activate RhoA. However, recently observed structural interactions between AlF₄^- and small G proteins (56, 152, 313) revive the possibility, entertained earlier (130), of a direct functional interaction (activation) between AlF₄^- and RhoA.

Sphingosine 1-phosphate (S1P), sphingosylphosphorylcholine (SPC), and lysophosphatidic acid (LPA) are lipid Ca²⁺-sensitizing agonists that activate the RhoA/ROK pathway (9, 270, 386; reviewed in Refs. 234, 320, 360, 364). SPC increases the force-to-[Ca²⁺]_i ratio in intact and force at constant [Ca²⁺]_i in permeabilized coronary artery smooth muscle (386). In rat microvessels the effect of SPC is partially inhibited by PLC inhibitors (9). However, it is difficult to identify the mechanisms through which the PLC inhibitor acts without evaluating changes in Ca²⁺ in nonpermeabilized preparations. It is likely that Ca²⁺ sensitization by SPC involves Tyr phosphorylation, most likely of a RhoGEF. This is suggested by the ability of SPC to induce translocation of the Tyr kinase Fyn from the cytosol to the cell membrane and the inhibition of the SPC-induced phosphorylation of MLCP (in coronary artery smooth muscle) by the Tyr kinase inhibitor PP1 (270). The Tyr kinase inhibitor also inhibits translocation of ROK to the membrane and relaxes the Ca²⁺-sensitized contraction (270). SPC-induced localization of ROK and possibly RhoGEF may be related to its ability to induce Tyr phosphorylation of focal adhesion proteins: p125^FAK, p^130CAS, and paxillin (reviewed in Ref. 318). S1P-induced Tyr phosphorylation of FAK may also be a downstream effect of RhoA/ROK: it is inhibited by C3 in 3T3 fibroblasts (411). C3 does not inhibit Tyr phosphorylation of another focal adhesion protein, paxillin in permeabilized smooth muscle (249), or in neuroblastoma cells in which C3 inhibits phosphorylation of FAK (260).

Phosphatidylinositol 4,5-bisphosphate (PIP₂) and products of phosphatidylinositol 3-kinase (PI3K) have also been implicated in interactions with RhoA/ROK (282, 442), but a clear picture has yet to emerge to show whether PIP₂ and/or PI3K products contribute to physiological regulation of RhoA/ROK. In Swiss 3T3 cells, the PI3K inhibitor wortmannin inhibits activation of Rac by platelet-derived growth factor (PDGF) and insulin receptors, but does not affect the induction of stress fibers by RhoA/ROK (282), whereas the latter effect is inhibited by the Tyr kinase inhibitor tyrphostin. Similarly, inhibition of PI3K does not affect cytoskeletal changes induced by the GEF Vav3, whereas PI3K-dependent stimulation of cell motility is mediated by Rac1 and CDC42, not by Rho (320, 380). Although PIP₂ can stimulate GDP release from both CDC42 and Rho in vitro (442), it is uncertain whether this effect of PIP₂ is physiological. Integrin-mediated activation of nuclear factor B (NF-B) is inhibited by wortmannin, but this mechanism is also mediated by Rac, not RhoA (310). The available evidence suggests that PI3K products are more likely to be involved in interactions with Rac and CDC42 than with the RhoA/ROK pathway. Given the hydrophobicity of phosphoinositides, the suggestion that their stimulation of GEFs, at least for Rac, occurs when both are localized to the lipid membrane (354) is consistent with the converging evidence that RhoA, RhoGEFs, and ROK are recruited to the plasma membrane during activation of the RhoA/ROK pathway. RhoA, Rac, and CDC42 bind to the same GDI isoform, and it is yet to be determined how phosphoinositides can act selectively on Rac · GDI, presumably sensing subtle differences between the GDP · Rac · GDI and the GDP · RhoA · GDI interface. It may be the result of utilization of different GEFs activated by, respectively, phosphoinositides or Tyr phosphorylation. Alternatively, different Tyr kinases (or in different cellular localizations) may be employed.

Microtubule depolymerizing agents, nocodazole or colchicine, induce contractions of smooth muscle that are relaxed by Y-27632 (64, 441). Disassembly of microtubules in neutrophil granulocytes also increases ROK activity and RLC phosphorylation inhibited by Y-27632 (274). The release of tubulin or of a microtubule-associated protein (possibly a microtubule-bound GEF; Refs. 122, 217, 309, 396) during disassembly may activate RhoA, consistent with the interpretation that microtubular depolymerization induces Ca²⁺ sensitization by activating RhoA/ROK. These findings provide a mechanistic explanation of why nocodazole and colchicines induce an, albeit, small (4-6% of maximal contractile responses) contraction of mature smooth muscles (441) that contain few microtubules. If this mechanism has a physiological role it is more likely to be in dividing and proliferating cells.

B. GEFs, GDIs, GTPase-activating Proteins, RhoA, and ROK

1. GEFs

Activation of RhoA, the Ca²⁺-sensitizing step that follows engagement of the trimeric G protein with its coupled receptor, requires active GEFs (reviewed in Ref. 336) that catalyze the exchange of cytoplasmic GDP · RhoA complexed with GDI (reviewed in Ref. 289) into the active GTP · RhoA that associates with the plasma membrane and activates ROK (108, 127) (Fig. 2). Numerous GEFs have been identified in the human genome (reviewed in Ref. 178), with the most information available about PDZ-RhoGEF, LARG, and p115 RhoGEF (35, 60, 62, 140, 212, 215, 219, 411, 420). A common feature of these GEFs is the presence of a DBL homology (DH) domain responsible for GEF activity, followed in tandem by a pleckstrin homology (PH) domain involved in protein-phosphoinositide lipid, and protein-protein interactions (317; reviewed in Ref. 151). Thus the PH domain also directs subcellular localization to regions other than the lipid membrane: it is required for localizing the RhoGEF, Lbc to actin stress fibers as well as for transformation of NIH 3T3 cells, although the DH domain of Lbc is sufficient for stimulating DNA synthesis (290). Trimeric G proteins, G_12,13 (60, 110) and G_q (35), are coupled to GEF(s), LARG, PDZ-RhoGEF and p115 RhoGEF, through an RGS (regulators of G protein signaling) like domain (RGSL) of the GEF; this domain has GTPase-activating protein (GAP) activity toward the associated G (214, 215) and strong binding of p115 RhoGEF to G₁₃ requires both the RGSL and the DH and PH domains, although the RGSL domain is sufficient for GAP activity (420). Association of PDZ-GEF with G₁₂ family proteins occurs also through an Lsc homology (LH; the murine homolog of the RGSL; Refs. 56, 230) domain (111).

A Tyr kinase inhibitor, tyrphostin, can inhibit activation of RhoA by G₁₃ (186), but not the effect of G₁₂ on neuronal morphology (372; reviewed in Ref. 110; see below). In addition to p115 RhoGEF, three other RhoGEFs, Vav, LARG, and PDZ-RhoGEF, may be activated by Tyr phosphorylation that appears to be a common, although probably not the only, mechanism activating of PDZ-Rho-GEF, LARG (372, reviewed in Ref. 110), Vav (reviewed in Refs. 45, 336), and vascular smooth muscle RhoGEF (285). The Tyr kinase(s) that Tyr phosphorylates PDZRhoGEF and LARG in response to stimulation with thrombin (60) has been identified in some instances as c-Src (45, 336, 405) and in others FAK. The variable inhibition of RhoA activation by different Tyr kinase inhibitors suggests that more than one Tyr kinase (and phosphatase) is involved in RhoGEF regulation, depending on cell type and Ca²⁺-sensitizing agonist.

The ability of G_q to activate the RhoA/ROK pathway was considered likely, because agonists activating receptors coupled to G_q usually also cause Ca²⁺ sensitization (358) that can be dissociated from the Ca²⁺-releasing activity of InsP₃ (206), and the upstream role of G_q was directly established in cells lacking G_12,13 (62, 403). The G_q-coupled mechanism is inhibited by a dominant-negative mutant of LARG, further indicating that this GEF can be coupled to G_q, although in these cells (HEK 293T) G_12,13 activates RhoA still more effectively than G_q does. The mechanism and/or protein domains involved in activation of RhoGEFs by, respectively, G₁₃ and G_q, however, are different. Activated G_q and stimulation of G_q-coupled receptors can strongly activate endogenous RhoA in HEK 293T cells, but whereas PDZ-RhoGEF, LARG, and p115 RhoGEF coimmunoprecipitate with G_12,13, they do not coimmunoprecipitate with G_q (62). Furthermore, in the same type of cell, G₁₃, but not G_q, recruits p115 RhoGEF to the plasma membrane and can activate it both directly and as the result of upstream activation of a thromboxane A₂ receptor (25). Strong plasma membrane-recruiting activity requires both the RGSL and PH domains (25). GEF activity of LARG in NIH 3T3 cells is stimulated by active G_q as well as by G_12,13 (35). Although it may be early to conclude, but these studies, performed on transfected cells, suggest that whereas LARG and PDZ-RhoGEF can be activated by both G_q,11 and by G_12,13, activation of p115 RhoGEF is more limited to the G_12,13 family. It remains to be seen whether these implications will be found valid about the functions of the endogenous proteins in nontransfected and noncultured cells and tissues. The Ca²⁺-sensitizing effectiveness of a given agonist probably depends on the cellular location of different receptors, trimeric G proteins, and RhoGEFs. The very high Ca²⁺-sensitizing activity of thromboxane A₂ receptors (146) that are coupled to G_12,13 does suggest that in other systems, as in the transfected cell (403), this family of trimeric G proteins mediates Ca²⁺ sensitization most potently.

Stimulation of thromboxane A₂ receptors linked to G₁₃ and stimulation of lysophosphatidic acid (LPA) receptors recruit cytoplasmic p115-RhoGEF to the plasma membrane of, respectively, HEK 293T and NIH 3T3 and COS cells (25, 419). Such observations and the translocation of both RhoA and ROK to the cell membrane during Ca²⁺ sensitization (108, 127) suggest that Ca²⁺ sensitization-signaling complexes are assembled at (and/or form) docking sites on the plasma membrane where RhoA, ROK, and GEFs are translocated (25, 108, 127, 419) and, probably, activated.

The multiplicity of possible permutations between the many agonists, receptors, trimeric G proteins, GEFs and, not to mention, cell types, complicates and, so far, eludes identification of the agonist- and cell-specific GEFs mediating Ca²⁺ sensitization of a given cell type in vivo. GEF activity in solution is usually assessed by measuring GEF-catalyzed acceleration of nucleotide exchange on nonprenylated Rho-family G proteins, rather than on the endogenous form: the prenylated GDP · RhoA · GDI complex. Expression (often overexpression) of these GEFs in vivo is often assayed by determining their ability to induce malignant transformation of cells. The pathway mediating this response is not necessarily identical, or even utilizes the same domains as induction of stress fibers (336), and does not identify (with the possible exception of an integrin-related process with Rac GDI, see Ref. 79) the mechanism that dissociates, at least temporarily, the tightly bound GDP · RhoA · GDI complex to release GDP (231, 304; reviewed in Ref. 289) and allow the exchange of GTP for GDP.

It is likely that nucleotide exchange on RhoA, and probably also Rac (314) as well as ROK activation are facilitated by lipid-protein (or protein-protein) interactions at the plasma membrane, because a preformed GTP · RhoA in a GTP · RhoA · GDI complex can Ca²⁺-sensitize smooth muscle and "spontaneously" translocates in vitro to liposomes (129, 304). Furthermore, prenylated RhoA is a much better substrate for p115 RhoGEF than nonprenylated RhoA (420).

The importance of cellular specificity and localization of RhoGEFs is indicated by recent reports showing expression of an ephrin-receptor coupled GEF that is selectively expressed in vascular smooth muscle, Vsm-RhoGEF (285), another GEF specifically at epithelial tight junctions (23) and the human ECT2 GEF localized to nuclei (384). It will be interesting to find out if Vsm-RhoGEF docks preferentially on the smooth muscle selective protein LPP (132).

Rnd1 is a recently discovered small GTP binding protein, related to RhoA, that inhibits RhoA-mediated stress fiber formation (283) and Ca²⁺ sensitization of smooth muscle (229). Rnd1 shares 45-49% identity with Rho but has no significant GTPase activity and appears to be in a permanently GTP-bound form (283). It is farnesylated and associated with the membrane, hence its ability to inhibit RhoA also supports the notion that some step(s) of RhoA/ROK activation occurs at the cell membrane. The absence of RhoA/ROK-mediated Ca²⁺ sensitization in midterm myometrium correlates well with the concurrent increase in Rnd3 expression. Ca²⁺ sensitization can be restored by farnesyl transferase inhibitors that prevent membrane localization of Rnd3 (47). These results are consistent with a physiological role of Rnd proteins in regulating the RhoA/ROK pathway either negatively, as in this instance, or positively, when Rnd1 associates with plexin (288).

Prenylated GTP · RhoA_vall4 Ca²⁺ sensitizes smooth muscle permeabilized with -escin-, but not with Triton (130), whereas nonprenylated GTP · RhoA_vall4 fails to Ca²⁺-sensitize the more mildly (-escin)-permeabilized preparations. The unexpected observation that when it associates with the cytoplasmic tail of plexin, an intramembrane protein, Rnd1 activates RhoA/ROK, instead of inhibiting it (288) suggests catalytic activity to occur at membranes. These findings are consistent with the conclusion that association with a relatively intact (not Triton-treated) membrane is required for Ca²⁺ sensitization by RhoA.

2. GDI and GAPs

GDI has the important role of maintaining the otherwise hydrophobic RhoA in an inactive cytosolic, ternary GDP · RhoA · GDI complex by capturing the prenylated COOH terminus of RhoA in its hydrophobic pocket while its NH₂-terminal region interacts with the switch-1, switch-2 regions of RhoA and inhibits nucleotide exchange (231; reviewed in Ref. 289) until activated by GEFs. High concentrations of GDI relax force Ca²⁺-sensitized by GTPS, recombinant GTP · G14V RhoA or by -adrenergic and muscarinic agonists and can extract GTP · RhoA translocated to membranes (129). The very high concentration of GDI required for these effects suggests that the likely in vivo function of GDI is not to interact directly with active GTP · RhoA, but to prevent perpetual recycling of GDP · RhoA, following hydrolysis of GTP · RhoA, to GDP · RhoA, in the presence of significantly higher cytosolic GTP than GDP. ADP-ribosylation of cytosolic RhoA occurs in vivo (108, 118) and increases its binding to GDI (118), contributing to the inhibition of RhoA activity by C3. However, the lack of activity of GTP · RhoA that was ADP-ribosylated in vitro (130) suggests that ADP-ribosylation inhibits RhoA through more than one mechanism.

Hydrolysis of RhoA-bound GTP (and other Rho family proteins) is catalyzed by GAPs (reviewed in Refs. 255, 297, 338; see Fig. 2). GAPs are required to accelerate the otherwise slow rate of hydrolysis by Rho family proteins, but information about their mechanism of activation or constitutive activity is limited. Interestingly, Tyr phosphorylation of p190 GAP increases its GAP activity that decreases the active GTP · RhoA (142), whereas Src-mediated Tyr phosphorylation also activates GEFs to increase RhoA/ROK activity. Given that there are 53 Rho · GAP domain-containing proteins encoded in the human genome and GAP specificity for their Rho family proteins is different in vitro and in vivo (297), identification of which GAP functions under specific physiological conditions is going to be challenging.

3. ROK

Activation of the Ser/Thr kinase ROK by GTP · RhoA is the next step of Ca²⁺ sensitization. The two major isoforms of ROK are Rho-kinase /ROK II and Rho-kinase /ROK 1 (10, 163, 225, 244). Binding of GTP · RhoA to the RBD of ROK leads, through a conformational change, to autophosphorylation and activation of the kinase (55; reviewed in Ref. 178) that is the major effector of Ca²⁺ sensitization of myosin II (reviewed in Ref. 358). The physiological importance of regulation of myosin II by RhoA/ROK is shown by numerous studies indicating that Ca²⁺ sensitization can be inhibited both upstream, through ADP ribosylation by the bacterial exoenzymes C3 and EDIN (108, 130) or glycosylation with C. difficile toxin B (233) to inhibit RhoA, as well as downstream with the selective ROK inhibitors Y-27632 (107, 165, 393) or HA-1077 (265, 328). Point mutations of RhoA that cause loss of its GTPase activity (e.g., G_14V, Q_63L) yield constitutively active proteins, albeit with variable GDI binding properties (reviewed in Ref. 232). We note that ROK mediates Ca²⁺ sensitization (MLCP inhibition) and induces focal adhesions, but other RhoA effectors mediate actin polymerization (164; but see sect. VIIIC).

Not only RhoA, but arachidonic acid can also activate ROK in solution (101) and possibly also in vivo, as indicated by the reversal of arachidonic acid-induced Ca²⁺ sensitization by Y-27632 (14, 107). Agonists can increase cellular arachidonic acid (131, 137) that may contribute to Ca²⁺ sensitization by activating ROK (14, 107) and/or by dissociating the regulatory from the catalytic subunit of myosin phosphatase (14, 126, 401) and/or by activating an atypical PKC (112).

Direct phosphorylation of the myosin RLC by ROK is not a physiologically significant mechanism, at least in smooth muscle, although ROK can phosphorylate both smooth muscle and nonmuscle myosin RLC in solution, and exogenous ROK can contract isolated stress fibers (187). However, activation of RhoA/ROK by GTPS in the absence of Ca²⁺ and MLCK activity fails to cause RLC phosphorylation or significant force development by smooth muscle (161, 356, 373) and the specificity constant k_cat/K_m of MLCK for RLC is much higher than that of ROK (40). It is possible that ROK that is concentrated to specific sites such as the cleavage furrow, may, if activated by RhoA or by some yet unrecognized mechanism, directly phosphorylate RLC. Cytokinesis is accompanied by RLC phosphorylation and accumulation of ROK and citron kinase at the cleavage furrow (165, 210, 245). However, inhibition of ROK with Y-27632 does not block cytokinesis, implicating another kinase (165, 210, 245), such as another Rho effector, citron kinase (433).

Spatial differences in RLC phosphorylation between, respectively, cortical and central regions of a cell (254), may reflect different levels of myosin phosphatase inhibition influenced by localization of the RhoA/ROK system, including MYPT1 (34, 389) or recruitment of MLCK (58), rather than direct phosphorylation of RLC by ROK. Differences in spatial control by MLCK, ROK, and other kinases and myosin phosphatase may also be influenced by localized sources of Ca²⁺, inhomogeneous influx through Ca²⁺ channels, and release from the sarcoplasmic reticulum (32, 171, 211, 359, 400).

Spatial control of function in cultured nonmuscle cells may also be affected by the different subcellular distributions, activities, and turnover of nonmuscle myosin II isoforms (207). Related to whether ROK directly phosphorylates RLC is the issue of diphosphorylation of RLC. In mature, contracting smooth muscle, RLC diphosphorylation (Thr-18 in addition to Ser-19 of RLC) by MLCK is observed only when phosphorylation levels exceed 40% (199). In vitro, ROK is more effective than MLCK in diphosphorylating RLC and may be responsible for localized accumulations of diphosphorylated RLC (245) that is also increased in cells transfected with ROK and colocalizes with the latter (392). The extent and in vivo functional consequences of RLC diphosphorylation (41) by ROK, by citron kinase (433), or by other kinases (261, 275) remain to be further explored.

C. Ephrins and Plexins: RhoA/ROK in Axonal Guidance and Angiogenesis

Ephrins are a special class of receptor Tyr kinases whose ligands are ephrins bound to cell membranes by GPI glycosylphosphatidylinositol (GPI) anchoring (A-ephrins) or having a membrane-spanning region (ephrin B); they are important upstream regulators of RhoA/ROK and play major roles in morphogenesis (reviewed in Ref. 153). The ligands, ephrins, are obligatory membrane dwellers and, therefore, act on their receptors, ephrins, on adjacent contacting cells. The cytoplasmic domains of ephrins allow for bidirectional signaling.

Ephrin signaling plays important roles in both axon guidance and morphogenesis. In neuronal cells activation of RhoA/ROK causes repulsion and growth cone collapse, whereas in endothelial cells ephrins induce endothelial de-adhesion and migration. Activation of RhoA/ROK provides a repulsive signal that results in collapse of growth cones seeking the appropriate dendrite, whereas activation of Rac provides attractive cues by activating PAK and inhibiting MLCK (295). Ephrin signaling through Rho-GTPases can be mediated by a GEF; a specific GEF, ephexin, directly interacts with the cytoplasmic tail of the Eph-A4 receptor (reviewed in Ref. 295). Given the opposing roles of, respectively, Rac and RhoA on neuronal growth cones, it is interesting that activated ephexin mediates converging signals; it reduces the activation of Rac while it activates RhoA. Activation of the ephrin-A5 pathway in retinal ganglion cells results in increased concentration of active GTP · RhoA, active ROK, and increased RLC phosphorylation, each inhibited by C3 or Y-27632 (406), clearly revealing cytoplasmic myosin II as the target effector of RhoA/ROK.

During early angiogenesis, ephrin-B2 ligand is a marker of the arterial endothelium (114, 348), whereas Eph-B4 is expressed in venous endothelium. The adult ephrin-B2, in addition to continuing to be a marker of arterial endothelium, also becomes expressed in the smooth muscles cells and pericytes investing the endothelium (114, 348). The persistence of arterial expression of ephrin-B2 in adult blood vessels and during tumor angiogenesis suggests the continued importance of ephrin signaling in the adult (114, 348) and, given expression of an appropriate receptor, ephrin-B2, may even mediate signaling between endothelium and smooth muscle.

The importance of the persistence of ephrin signaling into adulthood is indicated by the ephrin-B2 expression during neovascularization and tumor angiogenesis (114, 348). Signaling to the RhoA/ROK pathway is indicated not only by the ephrin-induced activation of RhoA/ROK in neuronal cells (406), but also by the inhibition of the earliest stage of angiogenesis, endothelial vacuole formations, as well as in vivo tumor angiogenesis by ROK inhibitors (361, 362). Because ephrin-B2 deficiency of null (^-/^-) mutants (413, reviewed in Ref. 435) is lethal (348), it would be interesting to determine the effect of targeting this gene product to inhibit tumor angiogenesis.

Plexins are large transmembrane protein receptors whose ligands are transmembrane or secreted semaphorins (reviewed in Ref. 379); like ephrins, they participate in axon guidance and angiogenesis. Plexins associate with neuropilins, another class of semaphorin receptors (reviewed in Ref. 271). Plexins, like ephrins, play a repulsive role in axon guidance. The COOH terminus of activated Plexin-B1 associates with PDZ-RhoGEF in 3T3 cells (87) and with LARG and PDZ-RhoGEF in HEK293 cells and activates RhoA (16). The interaction with the cytoplasmic tail of plexin-B1 is thought to contribute to recruitment of LARG and PDZ-RhoGEF to the plasma membrane during semaphorin 4D-plexin-B1-mediated activation of RhoA (150), similar to the interactions of the COOH terminus of the insulin-like growth factor I with PDZ domains (385). These membrane-associated interactions involve both the RGSL and PDZ domains of the GEFs (374). Mutation of the COOH-terminal amino acids of plexin-B1 that are required for interaction with the PDZ domain or expression of a dominant negative PDZ-RhoGEF blocks activation of RhoA by plexin-B1 and inhibit the RhoA-mediated neurite contraction and retraction. Activation of RhoA by endogenous plexin-B1 activated by semaphorin 4D is also inhibited by overexpression of the PDZ domain of Rho-GEF, although not by expression of the RGSL domain (298).

A surprising, recently reported aspect of the activation of RhoA by plexin-B1 has all the earmarks of RhoA/

ROK activation, including contraction of COS-7 cells that is inhibited by Y-27632. However, robust activation of RhoA/ROK occurs not by solely activating plexin with the semaphorin ligand, but also requires the association of plexin-B1 with Rnd1 (288), the Rho family small GTPases that hitherto has been only known as an inhibitor or RhoA/ROK (see above). The question of whether plexin-B1 deinhibits constitutive negative regulation of RhoA by Rnd1 is yet to be answered.

	V. MYOSIN PHOSPHATASE

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The physiologically relevant myosin phosphatase (MLCP) responsible for inactivating smooth muscle and nonmuscle II by dephosphorylating its highly specific substrate, RLC bound to myosin heavy chain, is a trimeric enzyme in both avian (6, 345) and mammalian (351) smooth muscle. It consists of a catalytic 37- to 38-kDa PP1c, an associated 110- to 130-kDa regulatory targeting subunit (MYPT1), and a tightly bound 20-kDa subunit of unknown function (reviewed in Refs. 42, 141, 358; Fig. 5). Several other protein phosphatases can dephosphorylate isolated, but not the heavy chain-bound, RLC.

View larger version (57K): [in this window] [in a new window]   FIG. 5. Myosin light-chain phosphatase (MLCP) is associated with myosin II and consists of three subunits: the catalytic subunit PP1c, the regulatory subunit MYPT1 that targets the catalytic subunit PP1c phosphatase to myosin to confer substrate specificity, and a 20-kDa subunit of unknown function. The MYPT1 shown is the human M133 isoform. The binding of PP1c to MYPT1 increases the enzymatic activity of PP1c toward RLC. Exon 13 of MYPT1 is deleted in the human and exon 12 in the chicken, leading to isoforms with or without central inserts, which gives rise to different numbering of the phosphorylated amino acids shown below. Some isoforms have a COOH-terminal leucine zipper (LZ), a site for protein kinase G (PKG) binding. Four amino acids, KVKF, at the NH2 terminus confer strong binding to PP1c. The six ankyrin repeats may serve as a docking site for protein-protein interactions (141). The following kinases phosphorylate sites on MYPT1 and/or CPI-17, a phosphorylation-potentiated 17-kDa phosphatase inhibitor resulting in MLCP inhibition: protein kinase C (PKC); MYPT kinase, also known as Zip-like kinase; integrin-linked kinase (ILK); Rho-kinase (ROK); and myotonic dystrophy kinase (DMPK). The dotted lines reflect phosphorylation that has been detected in vitro, and the solid lines indicate phosphorylation documented in vitro and in vivo.

Long thought to be an unregulated "housekeeping enzyme," we suggested, based on the Ca²⁺-independent contractile effect of GTPS on permeabilized smooth muscle, that smooth muscle MLCP is a G protein-regulated enzyme (356) and verified it by demonstrating that GTPS and agonists increase RLC phosphorylation independently of changes in [Ca²⁺]_i, by inhibiting dephosphorylation of RLC (199, 201). Recognition of MLCP inhibition as the major mechanism of Ca²⁺ sensitization (199, 201, 356), combined with the demonstration that pretreatment of smooth muscle with adenosine 5'-O-(3-thiotriphosphate) (ATPS) under Ca²⁺-free conditions caused thiophosphorylation of MYPT1 and increased the Ca²⁺ sensitivity of force (391) focused attention on MYPT1 as a ROK substrate (193). MYPT1, through its NH₂ terminus, binds the MLCP complex to and enhances its catalytic activity for myosin II (113, 143). The inhibitory site phosphorylated by ROK was identified as Thr-696 (133-kDa isoform; Thr-654 of the 130-kDa isoform) in the human sequence (102, 158), although ROK can also phosphorylate other NH₂-terminal threonines (102; reviewed in Ref. 10).

Phosphorylation (or thiophosphorylation) of Thr-696 MYPT1 through the RhoA/ROK pathway has been detected in a variety of cells, including platelets (271), smooth muscle (166, 301, 373), and human endothelial and prostate cancer cells (362) but is not invariably detectable in intact Ca²⁺-sensitized tissues and may be developmentally regulated (reviewed in Ref. 42). MYPT1 undergoes a developmental isoform switch in chicken gizzard smooth muscle (83). The adult gizzard contains an isoform (M130) from which a central region (exon 13) that is present in the embryonic form (M133) is spliced out. The mature aortic smooth muscle retains this sequence (286) that is near to, but does not include, the regulatory phosphorylation site. GTPS does not Ca²⁺-sensitize adult gizzard (311, but cf. Ref. 12), in spite of detectable MYPT1 thiophosphorylation (although its site has not been determined). In contrast, the tonic chicken aortic smooth muscle that contains the spliced in (M133) MYPT1 isoform is Ca²⁺-sensitized by GTPS without a detectable increase in phosphorylation (311). We have also been unable to detect changes in MYPT1/Thr-696 phosphorylation in chicken amnion smooth muscle Ca²⁺-sensitized with GTPS, although the same site-specific antibody against phospho-Thr-696 readily detected increased MYPT1 phosphorylation in cultured amnion smooth muscle (A. Stevenson, M. Eto, J. D. Matthew, A. P. Somlyo, and, A. V. Somlyo, unpublished observation) and other cells (362). More recently agonist-induced increases in phosphorylation of Thr-853, but not the regulatory Thr-696 site, as well as Thr-38 of CPI-17 was detected in several smooth muscles (198). Thr-853 is not an inhibitory site in vitro (102), but its phosphorylation state could affect localization of MLCP and its catalytic subunit on myosin (401), and thereby, in conjunction with Thr-696 phosphorylation, may be responsible for Ca²⁺-sensitizing inhibition of MLCP when Thr-696 is constitutively phosphorylated. It remains to be determined whether the difficulty