Yosin II within the pellet in each and every sample was quantified applying SDS-PAGE and Coomassie blue staining as a measure of filament assembly (Figure 3A). Incubation of myosin II with MHCK-C in the absence of ATP resulted in assembly levels standard for PS10 custom synthesis purified Dictyostelium myosin, with 82 in the myosin sedimenting within the present set of assays (Figure 3B). Incubation of myosin II with MHCK-C within the presence of ATP resulted in substantial filament dis-Figure 1 Domain organization of Dictyostelium MHCKs. All three enzymes contain a strongly conserved seven-fold WD repeat domain at the carboxyl-terminus. MHCK-A has a exceptional amino-terminal domain of 500 residues that forms a coiled-coil domain accountable for oligomerization and for localization to anterior actin-rich cell extensions. MHCK-B has an amino-terminal segment of 115 residues of presently unknown function. GFP was fused at the amino-terminus of every MHCK for the studies presented right here (at codon 2 in every single case). “CAT” indicates position on the conserved protein kinase catalytic domain in each and every enzyme. “SNPQ” (black boxes) indicates position of segments of MHCK-B and MHCK-C that display low amino acid complexity and are wealthy in serine, asparagine, proline, and glutamine residues.This analysis reveals striking differences in localization among these 3 enzymes. In the course of cytokinesis, MHCK-A displays weak enrichment at the cell poles, whilst MHCKB displays a largely diffuse localization. In contrast, MHCK-C displays powerful localization for the cleavage furrow only during the late stages of cell division. These outcomes suggest that D. discoideum cells use a family members of related MHCKs to modulate myosin II filament assembly, each with distinct roles.ResultsMHCK domain organization and MHCK C biochemical activity The enzymes MHCK-A and MHCK-B have established roles inside the manage of D. discoideum myosin filament assembly both in vitro and in vivo [16,17,24], and Bromopropylate Inhibitor Egelhoff, T. T., (unpublished studies). These enzymes possess a conserved domain organization that contains a highly novel protein kinase catalytic domain unrelated to standard kinases, in addition to a carboxyl-terminal WD repeat domain that targets these enzymes to myosin II filaments (Figure 1). Genomic sequence corresponding to the connected enzyme MHCK-C was deposited in GenBank by Loomis and colleagues (accession number AAC31918). MHCK-C differs from MHCK-A and MHCK-B in that it lacks any significant amino-terminal domain upstream on the catalytic do-Page three of(web page number not for citation purposes)BMC Cell Biology 2002,http:www.biomedcentral.com1471-21213Figure 2 Purification and activity of epitope-tagged MHCK-C. A. MHCK-C expression levels are indicated by western blot analysis of total cell lysates with the 3xALA parental cell line (3XALA lane) and lysates of 3xALA cell overexpressing FLAG-MHCK-C (3xFLAG-MHCK-C lane). Immunoreactivity of purified FLAG-MHCK-C indicates presence of full length and clipped FLAG-MHCK-C (pure FLAG-MHCK-C lane). Coomassie blue stained material (Coomassie lane) indicates purity as well as the presence of a clipped breakdown catalytic domain fragment migrating at 35 kDa. Western blot performed with polyclonal antisera generated against the catalytic domain of MHCK-C. B. FLAG-MHCK-C each autophosphorylates and phosphorylates Dictyostelium myosin II on the heavy chain. C. Kinetics and stoichiometry of myosin heavy chain (MHC) phosphorylation by FLAG-MHCK-C. For panels B and C phosphorylation was performed in a reaction mixtur.