Cytoplasmic dynein functions at several sites during mitosis; however, the basis of targeting to each site remains unclear. Tandem mass spectrometry analysis of mitotic dynein revealed a phosphorylation site in the dynein intermediate chains (ICs) that mediates binding to kinetochores. IC phosphorylation directs binding to zw10 rather than dynactin, and this interaction is needed for kinetochore dynein localization. Phosphodynein associates with kinetochores from nuclear envelope breakdown to metaphase, but bioriented microtubule (MT) attachment and chromosome alignment induce IC dephosphorylation. IC dephosphorylation stimulates binding to dynactin and poleward streaming. MT depolymerization, release of kinetochore tension, and a PP1-γ mutant each inhibited IC dephosphorylation, leading to the retention of phosphodynein at kinetochores and reduced poleward streaming. The depletion of kinetochore dynactin by moderate levels of p50(dynamitin) expression disrupted the ability of dynein to remove checkpoint proteins by streaming at metaphase but not other aspects of kinetochore dynein activity. Together, these results suggest a new model for localization of kinetochore dynein and the contribution of kinetochore dynactin.

Although the conventional myosins (myosins-II) responsible for processes such as muscle contraction and cytokinesis have been intensively studied, relatively little is known about the properties of the unconventional myosins. One widely distributed group of unconventional myosins, the class V myosins, have been suggested to play a role in organelle transport or membrane targeting (reviewed in Refs. 1 and 2). Brain myosin-V (BM-V 1 ), which was originally identified as a calmodulin (CaM)-binding protein in vertebrate brain, is the only member of this class of unconventional myosins to be purified and characterized biochemically (3-7). The class V myosins share a common domain structure consisting of an N-terminal head domain containing the actin-binding and ATP hydrolysis sites, an extended neck domain containing six IQ motifs (which form binding sites for CaM and/or related light chains), and a tail domain consisting of a region predicted to form coiled coils attached to a globular region of unknown function (7,8). The hypothetical functions of the class V myosins and their novel domain structure, particularly the extended neck domain, raise the obvious question of how the basic biochemical properties of the class V myosins compare with those of other types of myosins.Among the critically important properties of a molecular motor are its steady-state ATPase activity and the factors which regulate this activity. Known myosins are regulated either indirectly via actin-associated proteins such as troponin/ tropomyosin (as in vertebrate skeletal muscle myosin) or directly via the subunits of the myosin molecule itself. In the latter type of direct "myosin-linked" regulation, the light chains associated with the neck domain often function as the regulating subunits. The myosin light chains are all members of the CaM superfamily of proteins, although not all of them have retained the ability to bind to Ca 2ϩ . Vertebrate non-muscle and smooth muscle myosins-II are "turned on" by phosphorylation of their regulatory light chains by Ca 2ϩ /CaM-dependent myosin light chain kinase (9), whereas molluscan myosin-II and vertebrate brush border (BB) myosin-I are regulated by the direct binding of Ca 2ϩ to their light chains (10, 11). Like BB myosin-I, BM-V has multiple CaM light chains that remain bound in the absence of Ca 2ϩ (3,5,7). The neck domain of BM-V has been shown to be the precise site of CaM binding (8), suggesting that this myosin might also be directly regulated by Ca 2ϩ binding. The initial reports of BM-V ATPase activity also indicated that this protein is activated by Ca 2ϩ (5, 7), although the K ATPase for Ca 2ϩ and F-actin were not determined. The tail domains of myosins are unique to each class in the myosin superfamily and probably reflect the specific functions

This study identifies zwint-1 as a novel substrate for AurB during mitosis. Phosphorylation is required for outer kinetochore assembly during prometaphase. However, zwint-1 dephosphorylation is required at metaphase for checkpoint silencing.

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