BIOCHEMISTRY AND BIOLOGY OF A MYOSIN BINDING PROTEIN

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The way in which non-muscle cells regulate the important acto- myosin mediated shape changes responsible for cell division, pseudopod extension, endocytosis, and many other forms of movement are not fully understood. A great deal of knowledge exists about the biochemistry and biology of various actin binding proteins which regulate cellular actin assemblies. On the other hand, while a good deal is known about the biochemistry of myosin regulation by phosphorylation, little is known about how the cell changes the assembly state and localization of myosin during cell movement. Muscle cells have a number of myosin-associated proteins including light and heavy chain kinases, which mediate myosin assembly, and nebulin, titin, and C-protein which are part of the thick filament. However, little is known about non-muscle cell myosin-associated proteins other than the biochemistry of the kinases. Interestingly, myosin exhibits very unusual low ionic strength solubility in high speed cellular extracts, conditions where when pure it assembles into bipolar filament, which can gel with actin and its associated proteins and contract in a myosin- dependent manner. We have identified a new protein of 53 kD subunit molecular weight is sea urchin egg extracts which mediates this ususual low ionic strength solubility error directly to myosin to affect myosin low ionic strength solubility by binding directly to myosin to affect myosin low ionic strength solubility. We also have preliminary evidence that 53 kD binds to the head- rod junction of myosin and exhibits calmodulin-independent myosin light chain kinase activity. In this proposal we intend to further characterize this protein in terms of its: relatedness to other light chain kinases, functional domains, exact binding site on myosin, regulation by phosphorylation, effects on myosin ATPase and assembly, and ubiquity. A series of biological questions will be asked about 53 kD function through the use of in vitro myosin-mediated motility assays by studying the effects of myosin being bound by, or phosphorylated by, 53 kD on motility. The role of 53 kD in vivo will be studied by analyzing the effects of microinjected inhibitory monoclonal antibodies to 53 kD activity on cleavage of fertilized sea urchin eggs and by localizing 53 kD throughout the cell cycle in this and in other cells. These studies on a novel protein should help bridge the gap between our molecular knowledge of the regulation of myosin activity and how the cell controls myosin's dynamic assembly and localization for movement.