Filamentous smooth muscle myosin is regulated by phosphorylation

The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to "fix" dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody- stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1 (Cross, R. A., K. E. Cross, A. Sobieszek. 1986. EMBO [Eur. Mol. Biol. Organ.] J. 5:2637-2641) the rate of product release from dephosphorylated filaments was only 3-12-fold higher, depending on the ionic strength. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin (Sellers, J. R. 1985. J. Biol. Chem. 260:15815-15819) thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.     

Analysis of smooth muscle myosin phosphorylation with native pyrophosphate gels and its application to studies on myosin phosphorylation in contracting smooth muscle

Gizzard smooth muscle myosin, the 20,000 Mr light chain (L20) of which had been phosphorylated in vitro with a calmodulin-myosin light chain kinase system, was separated into 5 isolated bands in a pyrophosphate polyacrylamide gel. Their mobilities were in the following order: myosin with 2 unphosphorylated L20 (GM) less than myosin with 1 unphosphorylated and 1 mono-phosphorylated L20 (GMP1) less than myosin with 2 mono-phosphorylated L20 (GMP2) less than myosin with 1 mono-phosphorylated and 1 di-phosphorylated L20 (GMP3) less than myosin with 2 di-phosphorylated L20 (GMP4). We used this pyrophosphate polyacrylamide gel electrophoresis to analyze the phosphorylated state of taenia coli smooth muscle during K+-induced contraction. During the initial 2 min contraction, phosphorylated forms corresponding to GMP1 and GMP2 were detected in addition to the unphosphorylated form.     

Predominant attached state of myosin cross-bridges during contraction and relaxation at low ionic strength

The chemical states of a cross-bridge--nucleotide complex were studied using a fluorescent ATP analogue, 1-N6-etheno-2-aza-ATP(epsilon-2-aza-ATP). The fluorescence of epsilon-2-aza-ATP at specific emission wavelengths was enhanced by 12.5 times upon binding to myosin in a relaxed muscle and the fluorescence from the resultant myosin(M)-epsilon-2-aza-ADP-Pi intermediate was 2.5 times greater than that from a M-epsilon-2-aza-ADP complex. Similar enhancements of the fluorescence of epsilon-2-aza-ATP and epsilon-2-aza-ADP were observed upon binding to heavy meromyosin in solution. Binding of F-actin did not change the fluorescence of epsilon-2-aza-ATP or epsilon-2-aza-ADP bound to heavy meromyosin. When a muscle went from a relaxed state to a state of isometric contraction or contraction with shortening, the fluorescence intensity decreased only slightly or not at all, i.e. the fluorescence of nucleotides bound to most of the myosin heads during contraction is the same as that of the M-epsilon-2-aza-ADP-Pi intermediate. These results suggest that an actomyosin(AM)-epsilon-2-aza-ADP-Pi intermediate is the predominant attached state during contraction. When the ionic strength of the relaxing solution was decreased, cross-bridges formed at 6 degrees C without tension generation. At 20 degrees C, a large tension was produced although the shortening velocity was negligibly small or zero. The fluorescence intensity decreased by 15% at 20 degrees C but only a small decrease of 3% was observed at 6 degrees C, suggesting that the predominant complexes in the attached state were AM-epsilon-2-aza-ATP and/or AM-2-aza-ADP-Pi at 6 degrees C and AM-epsilon-2-aza-ADP at 20 degrees C. Thus, the identification of the actomyosin-nucleotide complexes existing before and after the force-generating step lent further support to the conclusion that the sliding force is generated by conformational changes in actomyosin when the (epsilon-2-aza-)ADP-Pi complex is bound to it.     

Mechanism of smooth muscle myosin phosphorylation

In vertebrate smooth muscles, phosphorylation of the regulatory light chain appears to be necessary for actin activation of the Mg-ATPase activity and for the in vitro assembly of myosin into filaments. From a correlation between the degree of phosphorylation and enzymatic activity, it was suggested that both myosin heads must be phosphorylated before either head could be activated by actin, and that phosphorylation of filamentous myosin occurred in a negatively cooperative manner (Persechini, A., and Hartshorne, D. J. (1981) Science 213, 1383-1385; Ikebe, M., Ogihara, S., and Tonomura, Y. (1982) J. Biochem. (Tokyo) 91, 1809-1812; Sellers, J. R., Chock, P. B., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14181-14188). Here we have determined the mechanism of phosphorylation by separating dephosphorylated and phosphorylated myosin species based on their different structural properties in the minifilament buffer system (5 mM citrate, 22 mM Tris). Fully phosphorylated myosin remained assembled as minifilaments in 1 mM Mg-ATP, but dephosphorylated myosin dissociated to a mixture of folded monomers and dimers. Gel filtration was used to separate these two structures. At intermediate levels of phosphorylation, the relative amount of myosin that formed minifilament and dimer and the degree of phosphorylation of the separated species relative to the initial level of phosphorylation was measured. From these data, it was possible to deduce that singly and doubly phosphorylated myosin remained assembled in the presence of nucleotide. Myosin molecules with 0, 1, or 2 heads phosphorylated could also be separated by nondenaturing gel electrophoresis. The amount of myosin which formed each species was quantitated as a function of phosphorylation. Results from the combined approaches are consistent with a model in which light chain kinase randomly phosphorylates myosin, independent of the state of aggregation of the myosin.     

The action of PKA on smooth muscle myosin phosphorylation

The aim of the study is to reveal the characterization of PKA acting on myosin. We found: (a) in the absence of Ca(2+)/CaM, PKA slightly phosphorylated MLC(20) and stimulated the Mg(2+)-ATPase activity of myosin, which was strengthened significantly by arachidonic acid (ACAD); (b) Ca(2+)-independent phosphorylation of myosin by PKA was obviously less efficient than both Ca(2+)-dependent and independent phosphorylation of myosin by MLCK; (c) micro-amount of calponin could not increase the precipitation of myosin phosphorylated by PKA, but it increased the precipitation of myosin phosphorylated by MLCK, suggesting the presence of conformational differences between the myosins phosphorylated by PKA and by MLCK.     

The heavy-chain stoichiometry of smooth muscle myosin is a characteristic of smooth muscle tissues

The stoichiometry of the two heavy chains of myosin in smooth muscle was determined by electrophoresing extracts of native myosin and of dissociated myosin on sodium dodecyl sulfate (SDS) 4%-polyacrylamide gels. The slower migrating heavy chain was 3.6 times more abundant in toad stomach, 2.3 in rabbit myometrium, 2.0 in rat femoral artery, 1.3 in guinea pig ileum, 0.93 in pig trachea and 0.69 in human bronchus, than the more rapidly migrating chain. Both heavy chains were identified as smooth muscle myosin by immunoblotting using antibodies to smooth muscle and non-muscle myosin. The unequal proportion of heavy chains suggested the possibility of native isoforms of myosin comprised of heavy-chain homodimers. To test this, native myosin extracts wer electrophoresed on non-dissociating (pyrophosphate) gels. When each band was individually analysed on SDS-polyacrylamide gel the slowest was found to be filamin and the other bands were myosin in which the relative proportion of the heavy chains was unchanged from that found in the original tissue extracts. Since this is incompatible with either a heterodimeric or a homodimeric arrangement it suggests that pyrophosphate gel electrophoresis is incapable of separating putative isoforms of native myosin.     

Physical integrity of smooth muscle myosin filaments is enhanced by phosphorylation of the regulatory myosin light chain.

BACKGROUND AND AIMS: Smooth muscle myosin monomers self-assemble in solution to form filaments. Phosphorylation of the 20-kD regulatory myosin light chain (MLC20) enhances filament formation. It is not known whether the phosphorylated and non-phosphorylated filaments possess the same structural integrity. METHODS: We purified myosin from bovine trachealis to form filaments, in ATP-containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of MLC20 within 20-40s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. RESULTS: MLC20 phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. CONCLUSION: Modification of the structural and physical properties of myosin filaments by MLC20 phosphorylation may be a key regulation step in smooth muscle where formation and dissolution of the filaments are required in the cells' adaptation to different cell length.     

Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin

Smooth muscle myosin was purified from turkey gizzards with the 20,000-dalton light chains in the unphosphorylated state. The actin-activated MgATPase activity was 4 nmol/min/mg at 25 degrees C. When the myosin was phosphorylated to 2 mol of Pi/mol of myosin using purified myosin light chain kinase, calmodulin, and ATP, the actin-activated MgATPase activity rose to 51 nmol/min/mg. Complete dephosphorylation of the same myosin by a purified phosphatase lowered the activity to 5 nmol/min/mg, and complete rephosphorylation of the myosin following inhibition of the phosphatase raised it again to 46 nmol/min/mg. Human platelet myosin could be substituted for turkey gizzard myosin, with similar results. A chymotryptic fragment of smooth muscle myosin which retains the phosphorylated site on the 20,000-dalton light chain of myosin was prepared. Using the same scheme for reversible phosphorylation, this smooth muscle heavy meromyosin was found to show the same positive correlation between phosphorylation of the myosin light chain and the actin-activated MgATPase activity. The results with smooth muscle heavy meromyosin show that the effect of phosphorylation on the actin-activated MgATPase activity can be separated from the effects of phosphorylation on myosin filament assembly.     

Ca2+-phospholipid dependent phosphorylation of smooth muscle myosin

Isolated myosin light chain from chicken gizzard has been shown to serve as a substrate for Ca²⁺-activated phospholipid-dependent protein kinase. Autoradiography showed that Ca²⁺-activated phospholipid-dependent protein kinase phosphorylated mainly the 20,000-dalton light chain of chicken gizzard myosin. Exogenously added calmodulin had no effect on myosin light chain phosphorylation catalyzed by the enzyme. The 20,000-dalton myosin light chain, both in the isolated form and in the whole myosin form, served as the substrate for this enzyme. In contrast to the isolated myosin light chain, the light chain of whole myosin was phosphorylated to a lesser extent by the Ca²⁺-activated phospholipid dependent kinase. Our results suggest the involvement of phospholipid in regulating Ca²⁺-dependent phosphorylation of the 20,000-dalton light chain of smooth muscle myosin.     

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.     

Cytoplasmic Ca2+ is a primary determinant for myosin phosphorylation in smooth muscle cells

Initiation of smooth muscle contraction is associated with Ca2+/calmodulin activation of myosin light chain kinase which catalyzes the phosphorylation of the 20-kDa light chain of myosin. In tracheal smooth muscle cells in culture, the extent of myosin light chain phosphorylation is less than 10% at basal cytosolic free Ca2+ concentrations of 150 nM. Stimulation of these cells with serotonin, histamine, carbachol, or the Ca2+ ionophore, ionomycin, increases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation. Light chain phosphorylation reaches a maximal value of 67% at Ca2+ concentrations below 1 microM. The relationship between the extent of light chain phosphorylation and cytosolic free Ca2+ concentration is apparently independent of the source of free intracellular Ca2+ or the agent used to stimulate the cells and is not altered by pre-exposure of the contractile apparatus to high concentrations of free Ca2+. Pretreatment of cells with 8-bromo-cyclic GMP or forskolin decreases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation in response to histamine or ionomycin. Pretreatment with 8-bromo-cyclic GMP also decreases the maximal extent of light chain phosphorylation. These results indicate that cytosolic free Ca2+ concentration, per se, is a primary determinant for myosin light chain phosphorylation in tracheal smooth muscle cells.     

Correlation of conformation and phosphorylation and dephosphorylation of smooth muscle myosin

The rate of phosphorylation and dephosphorylation of smooth muscle myosin by myosin light chain kinase and by two myosin light chain phosphatases (gizzard phosphatase IV and aorta phosphatase) are measured in various conditions; the relationship between the rate of phosphorylation and dephosphorylation of myosin and the myosin conformation is also studied. The rate of dephosphorylation of myosin was completely inhibited in the presence of 1 mM MgCl2 and ATP at low ionic strength where phosphorylated myosin forms a folded conformation. The inhibition was released when myosin formed either an extended monomer or filaments. The rate of phosphorylation of myosin was also affected by the conformation of myosin. The rate for a folded myosin was slower than those for an extended monomer and filamentous myosin. The phosphorylation and dephosphorylation of heavy meromyosin, subfragment-1, and the isolated 20,000-dalton light chain are not inhibited at low ionic strength, and the rate of phosphorylation and dephosphorylation was decreased with increasing ionic strength. KCl dependence of the rate of phosphorylation and dephosphorylation of myosin was normalized by using KCl dependence of subfragment-1, and it was found that the marked inhibition of the rate of phosphorylation and dephosphorylation of myosin is closely related to the change from an extended to a folded conformation of myosin.