Novel Myosin Heavy Chain Kinase Involved in Disassembly of Myosin II Filaments and Efficient Cleavage in MitoticDictyosteliumCells

Abstract

We have cloned a full-length cDNA encoding a novel myosin II heavy chain kinase (mhckC) from Dictyostelium. Like other members of the myosin heavy chain kinase family, the mhckC gene product, MHCK C, has a kinase domain in its N-terminal half and six WD repeats in the C-terminal half. GFP-MHCK C fusion protein localized to the cortex of interphase cells, to the cleavage furrow of mitotic cells, and to the posterior of migrating cells. These distributions of GFP-MHCK C always corresponded with that of myosin II filaments and were not observed in myosin II-null cells, where GFP-MHCK C was diffusely distributed in the cytoplasm. Thus, localization of MHCK C seems to be myosin II-dependent. Cells lacking the mhckC gene exhibited excessive aggregation of myosin II filaments in the cleavage furrows and in the posteriors of the daughter cells once cleavage was complete. The cleavage process of these cells took longer than that of wild-type cells. Taken together, these findings suggest MHCK C drives the disassembly of myosin II filaments for efficient cytokinesis and recycling of myosin II that occurs during cytokinesis. INTRODUCTIONDuring cytokinesis, cells are pinched into two parts by constriction of contractile rings. The contractile rings contain parallel filaments of actin and myosin II, a configuration suitable for constriction of the ring, in animal cells (Mabuchi and Okuno, 1977;Mabuchi, 1986;Glotzer, 1997;Robinson and Spudich, 2000) and in the amoeba cells of the cellular slime mold Dictyostelium discoideum (Yumura and Fukui, 1985;Fukui and Inoue, 1991). It is known that disassembly of the contractile ring components, including the myosin II filaments, accompanies the progression of cytokinesis, ultimately leading to fusion of the opposing plasma membranes and separation of the two daughter cells (Yumura et al., 1984). Little is known, however, about how disassembly of myosin II filaments is regulated during this process.D. discoideum is a powerful experimental system that enables functional analysis of various myosin II mutants in the absence of the wild-type form (De Lozanne and Spudich, 1987;Manstein et al., 1989;Uyeda and Yumura, 2000). Through the use of such myosin II mutants, for instance, the functional significance of the phosphorylation of the three threonine residues at positions 1823, 1833, and 2029 in the tail region of Dictyostelium myosin II was demonstrated: their phosphorylation state regulates the assembly and disassembly of myosin II filaments (Kuczmarski and Spudich, 1980;Egelhoff et al., 1991Egelhoff et al., , 1993. One mutant in which alanine residues were substituted for the three threonines (3XALA myosin) mimics the dephosphorylated state and accumulates excessively in the equatorial region of mitotic cells (Egelhoff et al., 1993). In contrast, 3XASP myosin, in which the threonine residues are substituted with aspartate residues, mimics the phosphorylated state, cannot form bipolar filaments, and shows markedly reduced accumulation in the cleavage furrows . More recently, systematic mut...

Identification and Characterization of a Novel α-Kinase with a von Willebrand Factor A-like Motif Localized to the Contractile Vacuole and Golgi Complex inDictyostelium discoideum

“…The thoroughly analyzed members to date include MHCK A, MHCK B, and MHCK C, and at both the biochemical level and in vivo all three seem to be involved in the control of myosin II filament assembly and localization within the cell (Steimle et al, 2001b;Liang et al, 2002;Nagasaki et al, 2002;Rico and Egelhoff, 2003). We have identified a fourth member of this family in Dictyostelium with similar domain organization that we refer to as MHCK D (our unpublished data).…”

“…Using synthesized cDNA as template, PCR amplifications were performed with VwkA-specific primers [5Ј-CAACAGGTA-AACAACGTAGTGAACGTG-3Ј] and vWFA-AS(4928-03) [5Ј-CCAGGTTTAC-CAAGGTTACAACTTCC-3Ј]. As controls, PCR was performed on the same template samples with primers specific to the developmentally induced gene Car2 (Saxe et al, 1993) and the constitutively expressed gene IG7 (Nagasaki et al, 2002), by using primers as follows: …”

“…Earlier studies showed that GFP-tagged MHCK A and MHCK C are enriched in the cell cortex, whereas MHCK B is mostly distributed throughout the cytoplasm of nonmigrating cells (Liang et al, 2002;Nagasaki et al, 2002). The low endogenous expression levels of VwkA precluded immunocytochemical localization analysis, so we developed stable cell lines expressing N-terminal GFP-tagged VwkA (GFPVwkA) and C-terminal GFP-tagged VwkA (VwkA-GFP) to allow dynamic localization studies to be performed on live cells ( Figure 10A).…”

Myosin heavy chain kinases play essential roles in Ca2+, but not cAMP, chemotaxis and the natural aggregation of Dictyostelium discoideum

“…To investigate the role of the myosin heavy chain kinases, the behavior of the individual Mhc null mutants mhckA 2 (Côte and Bukiejko, 1987), mhckB 2 (Clancy et al, 1997;Rico and Egelhoff, 2003), mhckC 2 (Nagasaki et al, 2002) and mhckD 2 (Yumura et al, 2005) ), has been analyzed by computer-assisted live-cell reconstruction and motion analysis systems. We fully expected to find that one or more of the mutants would exhibit behavioral defects similar to those of the mutant 3XALA (Heid et al, 2004).…”

Autophosphorylation Activates Dictyostelium Myosin II Heavy Chain Kinase A by Providing a Ligand for an Allosteric Binding Site in the α-Kinase Domain

“…Three of the proteins, termed MHCK B, C, and D, are closely related to MHCK A and at least two to them, MHCK B and C, function cooperatively to regulate myosin II filament assembly in vivo (13)(14)(15). The other two ␣-kinases, AK1 and VwkA, have domain structures unrelated to MHCK A. AK1 contains an Arf GTPase-activating protein domain and VwkA contains an N-terminal von Willebrand factor A-like domain.…”