DIRECT BINDING OF MYOSIN-II TO PHOSPHOLIPID-VESICLES VIA TAIL REGIONSAND PHOSPHORYLATION OF THE HEAVY-CHAINS BY PROTEIN-KINASE-C

Recent cloning and sequencing studies suggest that heavy chains of all non-muscle myosins II have a protein kinase C (PKC) phosphorylation s ite within their tail regions. A fragment of human macrophage myosin h eavy chain, encompassing its COOH-terminal 396 amino acids (MIIA(F46)) , was expressed in Escherichia coli to provide a model system for stud y of PKC-mediated phosphorylation. PKC phosphorylated this fragment wh en phosphatidylserine (PS) liposomes were present, but not when liposo mes made from PS/phosphatidylcholine (PC) were used. The reaction requ ired Ca2+, but not other activators such as diacylglycerol (DG) or pho sphatidylinositol 4,5-bisphosphate. Phosphorylation of MIIA(F46),as no t observed in the presence of micelles of PS or PS/DG. Similar results were obtained using native myosin II purified from bovine brain and c hicken intestine brush border. Phosphorylation of light chains, in con trast, occurred even with PS/PC liposomes if DG was present. Addition of the PS and PS/DG liposomes significantly increased the turbidities at 340 nm of MIIA(F46) and native myosin II, and the extent of increas e depended upon the type of myosin used. Also, PS and PS/DG liposomes shifted the gel filtration elution positions of MIIA(F48) and myosin I I. In contrast, liposomes of PS/PC and PS/PC/DG gave only a slight inc rease in turbidity with all myosins and fragments and did not noticeab ly shift their gel filtration elution positions. These results suggest that myosins II bind to PS liposomes via the COOH-terminal regions of their heavy chains with affinities specific to each myosin isoform, t hat the binding is dependent upon the PS composition, and that PKC pho sphorylates the PS-bound heavy chains.