The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.     

Inhibition of zipper-interacting protein kinase function in smooth muscle by a myosin light chain kinase pseudosubstrate peptide

As a regulator of smooth muscle contractility, zipper-interacting protein kinase (ZIPK) appears to phosphorylate the regulatory myosin light chain (RLC20), directly or indirectly, at Ser19 and Thr18 in a Ca²⁺-independent manner. The calmodulin-binding and autoinhibitory domain of myosin light chain kinase (MLCK) shares similarity to a sequence found in ZIPK. This similarity in sequence prompted an investigation of the SM1 peptide, which is derived from the autoinhibitory region of MLCK, as a potential inhibitor of ZIPK. In vitro studies showed that SM1 is a competitive inhibitor of a constitutively active 32-kDa form of ZIPK with an apparent K_i value of 3.4 µM. Experiments confirmed that the SM1 peptide is also active against full-length ZIPK. In addition, ZIPK autophosphorylation was reduced by SM1. ZIPK activity is independent of calmodulin; however, calmodulin suppressed the in vitro inhibitory potential of SM1, likely as a result of nonspecific binding of the peptide to calmodulin. Treatment of ileal smooth muscle with exogenous ZIPK was accompanied by an increase in RLC20 diphosphorylation, distinguishing between ZIPK [and integrin-linked kinase (ILK)] and MLCK actions. Administration of SM1 suppressed steady-state muscle tension developed by the addition of exogenous ZIPK to Triton-skinned rat ileal muscle strips with or without calmodulin depletion by trifluoperazine. The decrease in contractile force was associated with decreases in both RLC20 mono- and diphosphorylation. In summary, we present the SM1 peptide as a novel inhibitor of ZIPK. We also conclude that the SM1 peptide, which has no effect on ILK, can be used to distinguish between ZIPK and ILK effects in smooth muscle tissues. inhibitory peptide; calcium sensitization     

Biochemistry of smooth muscle myosin light chain kinase

The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca²⁺-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.     

Limited proteolysis of smooth muscle myosin light chain kinase

Myosin light chain kinase plays a central role in the regulation of smooth muscle contraction. The activity of this enzyme is controlled by protein-protein interaction (the Ca2+-dependent binding of calmodulin) and by phosphorylation catalyzed by cAMP-dependent protein kinase. The effects of these two regulatory mechanisms on the conformation of myosin light chain kinase and the locations of the phosphorylation sites, the calmodulin-binding site, and the active site have been probed by limited proteolysis. Phosphorylated and nonphosphorylated myosin light chain kinases were subjected to limited digestion by four proteases having different peptide bond specificities (trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and thrombin), both in the presence and in the absence of bound calmodulin. The digests were compared in terms of gel electrophoretic pattern, distribution of phosphorylation sites, and Ca2+ dependence of kinase activity. A 24 500-dalton chymotryptic peptide containing both sites of phosphorylation was purified and tentatively identified as the amino-terminal peptide. The following conclusions can be drawn: neither phosphorylation nor calmodulin binding induces dramatic changes in the conformation of the kinase; the kinase contains two regions that are particularly susceptible to proteolytic cleavage, one located approximately 25 000 daltons from the amino terminus and the other near the center of the molecule; the two phosphorylation sites are located within 24 500 (probably 17 500) daltons of the amino terminus; the active site is located close to the center of the molecule; the calmodulin-binding site is located in the amino-terminal half of the molecule, between the sites of phosphorylation and the active site, and this region is very susceptible to cleavage by trypsin.     

Potent peptide inhibitors of smooth muscle myosin light chain kinase: mapping of the pseudosubstrate and calmodulin binding domains

Smooth muscle myosin light chain kinase (MLCK) is activated by calcium-calmodulin and, in turn, phosphorylates and activates the smooth muscle actomyosin ATPase, resulting in muscle contraction. The amino acid sequence of the regulatory domain of MLCK is known, and it contains a region that binds calmodulin and also bears a strong homology to the phosphorylation site in the substrate. Thus, it has been called the "pseudosubstrate". It has been proposed that calmodulin activates MLCK by binding to and reversing the autoinhibitory function of the pseudosubstrate. Synthetic peptides based on this sequence inhibit MLCK both by binding to calmodulin and by competing with the substrate at the active site. In the work reported here, we have synthesized a large number of peptides from the regulatory region of MLCK (MLCK 480-516). The region was systematically analyzed by dividing it into fragments of two to six amino acids, each containing one or more basic residues, in order to map in detail the calmodulin binding site and the autoinhibitory region. It was observed that both calmodulin binding and autoinhibition are mediated by several different fragments of the regulatory sequence. Two nonoverlapping peptides, MLCK 480-493 and MLCK 494-504, are similar in potency in inhibiting the enzyme (IC50's of 2 and 6 microM, respectively). Larger fragments, combining multiple inhibitory regions, are more potent inhibitors. For example, MLCK 480-504 is extremely potent, with an IC50 of 13 nM. The calmodulin binding site and active site directed inhibitory regions overlap, but are not identical. Residues 505-512 are important only for calmodulin binding.     

Calmodulin-linked equilibria in smooth muscle myosin light chain kinase

Competition experiments using 9-anthroylcholine, a fluorescent dye that undergoes calmodulin-dependent binding by smooth muscle myosin light chain kinase [Malencik, D. A., Anderson, S. R., Bohnert, J. L., & Shalitin, Y. S. (1982) Biochemistry 21, 4031], demonstrate a strongly stabilizing interaction between the adenosine 5'-triphosphate and myosin light chain binding sites operating within the enzyme-calmodulin complex but probably not in the free enzyme. The interactions in the latter case may be even slightly destabilizing. The fluorescence enhancement in solutions containing 5.0 microM each of the enzyme and calmodulin is directly proportional to the maximum possible concentration of bound calcium on the basis of four calcium binding sites. Evidently, all four calcium binding sites of calmodulin contribute about equally to the enhanced binding of 9-anthroylcholine by the enzyme. Fluorescence titrations on solutions containing 1.0 microM enzyme plus calmodulin yield a Hill coefficient of 1.2 and K = 0.35 +/- 0.08 microM calcium. Three proteolytic fragments of smooth muscle myosin light chain kinase, apparent products of endogenous proteolysis, were isolated and characterized. All three possess calmodulin-dependent catalytic activity. Their interactions with 9-anthroylcholine, in both the presence and absence of calmodulin, are similar to those of the native enzyme. However, the stabilities of their complexes with calmodulin vary. The corresponding dissociation constants range from 2.8 nM for the native enzyme and 8.5 nM for the 96K fragment to approximately 15 nM for the 68K and 90K fragments [0.20 N KCl, 50 mM 3-(N-morpholino)propanesulfonic acid, and 1 mM CaCl2, pH 7.3, 25 degrees C]. A coupled fluorometric assay, modified from a spectrophotometric assay for adenosine cyclic 3',5'-phosphate dependent protein kinase [Cook, P. F., Neville, M. E., Vrana, K. E., Hartl, F. T., & Roskoski, R. (1982) Biochemistry 21, 5794], has provided the first continuous recordings of myosin light chain kinase phosphotransferase activity. The results show that smooth muscle myosin light chain kinase is a responsive enzyme, whose activity adjusts rapidly to changes in solution conditions.     

Stretch activates myosin light chain kinase in arterial smooth muscle

Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.     

AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase

Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser(815). Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-beta, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-beta inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which alpha1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.     

Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site.

We previously proposed a molecular mechanism for the activation of smooth muscle myosin light chain kinase (smMLCK) by calmodulin (CaM). According to this model, smMLCK is autoinhibited in the absence of Ca2+/CaM due to the interaction of a pseudosubstrate prototope, contained within the CaM binding/regulatory region, with the active site of the enzyme. Binding of Ca2+/CaM releases the autoinhibition and allows access of the protein substrate to the active site of the enzyme, resulting in phosphorylation of the myosin light chains. We now provide direct experimental evidence that the pseudosubstrate prototope can associate with the active site. We constructed a smMLCK mutant in which the five-amino acid phosphorylation site of the myosin light chain substrate was inserted into the pseudosubstrate sequence of the CaM binding domain without disrupting the ability of the enzyme to bind Ca2+/CaM. We demonstrate that this mutant undergoes intramolecular autophosphorylation at the appropriate inserted serine residue in the absence of CaM and that this autophosphorylation activates the enzyme. Binding of Ca2+/CaM to the mutant enzyme stimulated myosin light chain substrate phosphorylation but strongly inhibited autophosphorylation, presumably by removing the pseudosubstrate from the active site. These results confirm that the pseudosubstrate sequence has access to the catalytic site and that the activation of the enzyme is accompanied by its removal from this position due to Ca2+/CaM binding as predicted by the model.     

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, -actinin and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryospecific isoform with a different molecular mass was detected.     

Location of the inhibitory region of smooth muscle myosin light chain kinase

Proteolysis by trypsin of gizzard myosin light chain kinase (MLC kinase) in the absence of Ca2+-calmodulin produced a 64,000-dalton inactive fragment which was converted to a 61,000-dalton Ca2+-calmodulin-independent active fragment. This confirmed previous results (Ikebe, M., Stepinska, M., Kemp, B. E., Means, A. R., and Hartshorne, D. J. (1987) J. Biol. Chem. 262, 13828-13834). On the other hand, proteolysis of MLC kinase in the presence of Ca2+-calmodulin initially produced a 66,000-dalton Ca2+-calmodulin-dependent active fragment which was converted to a 61,000-dalton Ca2+-calmodulin-independent active fragment with further proteolysis. The amino acid sequences from the N terminus of the 66,000-dalton, 64,000-dalton, and 61,000-dalton fragments were determined. The sequence was not found in the reported partial amino acid sequence of MLC kinase (C-terminal 60% of whole sequence) (Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381), and, therefore, the cleavage sites are in the remaining 40% N-terminal portion of the sequence of MLC kinase. The C terminus of these MLC kinase fragments was determined by employing the carboxypeptidases A, B, and Y digestion followed by the amino acid analysis of the released amino acids. As a result, it was concluded that the C terminus of the 66,000-dalton, 64,000-dalton, and 61,000-dalton MLC kinase fragments are arginine 522, lysine 490 and arginine 494, and lysine 473, respectively. These results show that the inhibitory domain is in the amino acid sequence of 474-490, and that the amino acid sequence 494-522 confers the calmodulin-dependent kinase activity.     

Substrate based inhibitors of smooth muscle myosin light chain kinase.

Activation of myosin light chain kinase is a prerequisite for smooth muscle activation. In this study, short peptide analogs of the phosphorylation site of the myosin light chain were studied for their effects on several contractile protein systems. The peptides inhibited phosphorylation of isolated ventricular and smooth muscle myosin light chains by smooth muscle myosin light chain kinase, but they were only weak inhibitors of phosphorylation of intact myosin and actomyosin. The peptides were also unable to block force development or myosin light chain phosphorylation in glycerol permeabilized fibers of swine carotid media. Apparently, the association of the myosin light chain with myosin changes its conformation such that substrate analogs which are potent inhibitors of the phosphorylation of isolated myosin light chains by myosin light chain kinase are ineffective at blocking phosphorylation of the intact molecule.     

Molecular cloning and sequencing of the chicken smooth muscle myosin regulatory light chain

A cDNA probe was constructed from a chicken skeletal muscle regulatory light chain cDNA and was used to screen a chicken gizzard cDNA library. A clone containing the entire coding region of the chicken gizzard regulatory light chain was isolated and sequenced. The deduced protein sequence is identical to the most recently reported chemical sequence of the chicken smooth muscle regulatory light chain, and has homologies with other troponin C-like calcium-binding proteins.     

Functional role of the C-terminal domain of smooth muscle myosin light chain kinase on the phosphorylation of smooth muscle myosin

Smooth muscle myosin light chain kinase (MLCK) is known to bind to thin filaments and myosin filaments. Telokin, an independently expressed protein with an identical amino acid sequence to that of the C-terminal domain of MLCK, has been shown to bind to unphosphorylated smooth muscle myosin. Thus, the functional significance of the C-terminal domain and the molecular morphology of MLCK were examined in detail. The C-terminal domain was removed from MLCK by alpha-chymotryptic digestion, and the activity of the digested MLCK was measured using myosin or the isolated 20-kDa light chain (LC20) as a substrate. The results showed that the digestion increased K(m) for myosin 3-fold whereas it did not change the value for LC20. In addition, telokin inhibited the phosphorylation of myosin by MLCK by increasing K(m) but only slightly increased K(m) for LC20. Electron microscopy indicated that MLCK was an elongated molecule but was flexible so as to form folded conformations. MLCK was crosslinked to unphosphorylated heavy meromyosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the absence of Ca(2+)/calmodulin (CaM), and electron microscopic observation of the products revealed that the MLCK molecule bound to the head-tail junction of heavy meromyosin. These results suggest that MLCK binds to the head-tail junction of unphosphorylated myosin through its C-terminal domain, where LC20 can be promptly phosphorylated through its catalytic domain following the Ca(2+)/CaM-dependent activation.     

Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin

At relatively high concentrations of myosin light chain kinase, a second site on the 20,000-dalton light chain of smooth muscle myosin is phosphorylated (Ikebe, M., and Hartshorne, D. J. (1985) J. Biol. Chem. 260, 10027-10031). In this communication the site is identified and kinetics associated with its phosphorylation and dephosphorylation are described. The doubly phosphorylated 20,000-dalton light chain from turkey gizzard myosin was hydrolyzed with alpha-chymotrypsin and the phosphorylated peptide was isolated by reverse phase chromatography. Following amino acid analyses and partial sequence determinations the second site of phosphorylation is shown to be threonine 18. This site is distinct from the threonine residue phosphorylated by protein kinase C. The time courses of phosphorylation of serine 19 and threonine 18 in isolated light chains follow a single exponential indicating a random process, although the phosphorylation rates differ considerably. The values of kcat/Km for serine 19 and threonine 18 for isolated light chains are 550 and 0.2 min-1 microM-1, respectively. With intact myosin, phosphorylation of serine 19 is biphasic; kcat/Km values are 22.5 and 7.5 min-1 microM-1 for the fast and slow phases, respectively. In contrast, phosphorylation of threonine 18 in intact myosin is a random, but markedly slower process, kcat/Km = 0.44 min-1 microM-1. Dephosphorylation of doubly phosphorylated myosin (approximately 4 mol of phosphate/mol of myosin) and isolated light chains (approximately 2 mol of phosphate/mol of light chain) follows a random process and dephosphorylation of the serine 19 and threonine 18 sites occurs at similar rates.     

Functional interactions between smooth muscle myosin light chain kinase and calmodulin

Calmodulin (CaM) binding by turkey gizzard myosin light chain kinase (MLCK) causes subtle changes in the fluorescence emission and polarization excitation spectra of the enzyme. Fluorescence experiments using 9-anthroyl-choline (9AC), which competes with ATP in binding, demonstrate mutually stabilizing interactions between the CaM and ATP binding sites corresponding to delta G = -0.6 to -0.7 kcal/mol. Fluorescence titrations in the presence of 9AC or 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] confirm the stoichiometry of 1 mol of CaM/MLCK. Phosphorylation of MLCK has no effect on either the protein fluorescence or the binding of ATP and 9AC. The dissociation constant for the MLCL-CaM complex is increased approximately 500-fold on phosphorylation. Values of Kd for the phosphorylated enzyme range from 0.5 to 1.1 microM in 0.2 N KCl, pH 7.3, 25 degrees C. We showed competition between MLCK and other CaM binding proteins and peptides by using both fluorescence and catalytic activity measurements. Competition for CaM occurs with ACTH, beta-endorphin, substance P, glucagon, poly(L-arginine), myelin basic protein, troponin I, and histone H2A. Phosphorylation of the last three proteins by the adenosine cyclic 3',5'-phosphate dependent protein kinase diminishes their ability to compete. Phosphorylation of MLCK by the protein kinase gives 0.95 +/- 0.04 and 2.2 +/- 0.4 mol of incorporated 32P in the presence and absence of CaM, respectively. These stoichiometries agree with those recently reported [Conti, M. A. & Adelstein, R. S. (1981) J. Biol. Chem. 256, 3178].     

Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase

The 20,000-dalton light chain of turkey gizzard myosin is phosphorylated at two sites. Dual phosphorylation is observed when both intact myosin and isolated light chains are used as substrates. Phosphorylation of the second site is not observed at higher ionic strength (e.g. 0.35 M KCl). The first phosphorylation site (serine 19) is phosphorylated preferentially to the second site. The latter is phosphorylated more slowly than the first site, and its phosphorylation requires relatively high concentrations of myosin light chain kinase. It is suggested that myosin light chain kinase catalyzes the phosphorylation of both sites on the light chain, and several reasons are cited that make it unlikely that a contaminant kinase is involved. The second phosphorylation site is a threonine residue. Based on the results of limited proteolysis of the light chain, it is concluded that the threonine residue is close to serine 19, and possible locations are threonines 9, 10, and 18. At all concentrations of MgCl2, phosphorylation of the second site markedly increases the actin-activated ATPase activity of myosin and accelerates the superprecipitation response of myosin plus actin.     

Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain

Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction [IUBMB Life 51 (2001) 337, for review]. The well-established mode for its regulation is to phosphorylate the 20 kDa myosin light chain (MLC 20) to activate myosin ATPase activity. MLCK exhibits myosin-binding activity in addition to this kinase activity. The myosin-binding activity also stimulates myosin ATPase activity without phosphorylating MLC 20 [Proc. Natl. Acad. Sci. USA 96 (1999) 6666]. We engineered an MLCK fragment containing the myosin-binding domain but devoid of a catalytic domain to explore how myosin is stimulated by this non-kinase pathway. The recombinant fragment thus obtained stimulated myosin ATPase activity by V(max)=5.53+/-0.63-fold with K(m)=4.22+/-0.58 microM (n=4). Similar stimulation figures were obtained by measuring the ATPase activity of HMM and S1. Binding of the fragment to both HMM and S1 was also verified, indicating that the fragment exerts stimulation through the myosin heads. Since S1 is in an active form regardless of the phosphorylated state of MLC 20, we conclude that the non-kinase stimulation is independent of the phosphorylating mode for activation of myosin.