Effects of phosphorylation by myosin light chain kinase on the structure of Limulus thick filaments.

Abstract

Abstract. The results discussed in the preceding paper (Levine, R. J. C., J. L. Woodhead, and H. A. King. 1991. J. Cell Biol. 113:563-572.)indicate that A-band shortening in Limulus muscle is a thick filament response to activation that occurs largely by fragmentation of filament ends. To assess the effect of biochemical changes directly associated with activation on the length and structure of thick filaments from Limulus telson muscle, a dually regulated tissue (Lehman, W., J. Kendrick-Jones, and A. G. Szent Gyorgyi. 1973. Cold Spring Harbor Symp. Quant. Biol. 37: 319-330.) we have examined the thick filament response to phosphorylation of myosin regulatory light chains. In agreement with the previous work of J. Sellers (1981. J. Biol. Chem. 256:9274-9278), Limulus myosin, incubated with partially purified chicken gizzard myosin light chain kinase (MLCK) and [y32p]_ ATP, binds 2 mol phosphate/mole protein. On autoradiographs of SDS-PAGE, the label is restricted to the two regulatory light chains, LC~ and LC2. Incubation of long (>/4.0/~m) thick filaments, separated from Limulus telson muscle under relaxing conditions, with either intact MLCK in the presence of Ca ~÷ and calmodulin, or Ca2÷-independent MLCK obtained by brief chymotryptic digestion (Walsh, M. P., R. Dabrowska, S. Hinkins, and D. J. Hartshorne. 1982. Biochemistry. 21:1919-1925, causes significant changes in their structure. These include: disordering of the helical surface arrangement of myosin heads as they move away from the filament backbone; the presence of distal bends and breaks, with loss of some surface myosin molecules, in each polar filament half; and the production of shorter filaments and end-fragments. The latter structures are similar to those produced by Ca2÷-activation of skinned fibers (Levine, R. J. C., J. L. Woodhead, and H. A. King. J. Cell Biol. 113:563-572). Rinsing experimental filament preparations with relaxing solution before staining restores some degree of order of the helical surface array, but not filament length. We propose that outward movement of myosin heads and thick filament shortening in Limulus muscle are responses to activation that are dependent on phosphorylation of regulatory myosin light chains. Filament shortening may be due, in large part, to breakage at the filament ends. W ~. have previously reported that thick filaments separated from intact Limulus telson muscle, maintained in a relaxed state, have an average length >4/zm, whereas those obtained from similarly intact muscle, stimulated to contract isotonically either by exposure to high K ÷ or electrically, have a mean length of 3/~m. This decrease in thick filament length is not the result of a rearrangement of component myosin and paramyosin molecules, since both long and shortened filaments have (a) identical helical surface arrays of myosin heads: threefold screw symmetry and fourfold rotational symmetry, and (b) identical diameters: the centers of mass of the myosin heads in both cases lie at a radius of 15 nm from the center of the filamen...

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

“…The best supported hypothesis stems from observations that thick filament shortening 1) is not associated with changes in thick filament helical structure but 2) is associated with changes in thick filament charge , phosphorylation state , and the appearance of unattached thick filament end fragments (Levine et al, 1991b) and 3) regulatory light chain phosphorylation causes the release of similar end fragments in vitro (Levine et al, 1991a). These observations are consistent with thick filament shortening occurring via a reversible, phosphorylation dependent, disaggregation of thick filament ends.…”

The chicken muscle thick filament: temperature and the relaxed cross-bridge arrangement

“…Many of these questions, such as the step size per ATP hydrolysis (Yanagida et al, 1985; *To whom correspondence should be addressed. Ishijima et al, 1991;Uyeda et al, 1991), the effects of phosphorylation of the thick filaments (Sweeney & Stull, 1986;Craig et al, 1987;Levine et al, 1991Levine et al, , 1992 and the role of the various accessory proteins in the thick filament (Craig & Offer, 1976;Starr et al, 1985;Bennett et al, 1986), relate to the structure of the thick filament or the constraints that its structure may place on the interaction of the myosin head with actin.…”

Biochemical and Physiological Regulation of Cardiac Myocyte Contraction by Cardiac-Specific Myosin Light Chain Kinase

“…The phosphorylation of MLC2 at Ser-15 results in the addition of a negative charge to the N-terminal region of MLC2, which induces the myosin head to swing out from a position close to the thick filament's backbone toward the actin filament, and this structural change increases the rate through which the myosin-actin interaction occurs and promotes force generation at a given level of Ca 2+ (Figures 1,3). 30 …”