Figure 3

Phosphorylation of myosin II by FLAG-MHCK-C drives filament disassembly. Myosin II was subjected to phosphorylation by FLAG-MHCK-C as for experiments in figure 2. A. Samples containing myosin II (500 nM MHC concentration), FLAG-MHCK-C (100 nM), and BSA (1 μg/μl) were incubated either without ATP (-) or with ATP (+) for 30 minutes, adjusted to 50 mM NaCl for optimal myosin II filament assembly, then subjected to sedimentation at 90,000 ×g for 10 min to pellet assembled filaments. Equal fractions of pellets (P) and supernatants (S) were subjected to SDS-PAGE and Coomassie blue stain. Disassembly is reflected as a loss of MHC in the pellet fractions. No disassembly of myosin occurs if ATP is added in the absence of FLAG-MHCK-C (not shown). B. Densitometric quantification of the percent myosin II in the pellet fractions. Error bars represent S.E.M., n = 5.