Yosin II inside the pellet in each and every sample was quantified making use of 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 typical for purified Dictyostelium myosin, with 82 from the myosin sedimenting in the present set of assays (Figure 3B). Incubation of myosin II with MHCK-C in the presence of ATP resulted in substantial filament dis-Figure 1 Domain organization of Dictyostelium MHCKs. All 3 enzymes contain a strongly Eperisone custom synthesis conserved seven-fold WD repeat domain in the carboxyl-terminus. MHCK-A includes a one of a kind amino-terminal domain of 500 residues that forms a coiled-coil domain responsible 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 each and every MHCK for the research presented right here (at codon 2 in each case). “CAT” indicates position from 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 rich in serine, asparagine, proline, and glutamine residues.This analysis reveals striking variations in localization between these three enzymes. During cytokinesis, MHCK-A displays weak enrichment in the cell poles, although MHCKB displays a largely diffuse localization. In contrast, MHCK-C displays sturdy localization to the cleavage furrow only through the late stages of cell division. These outcomes suggest that D. discoideum cells use a family of associated 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 Hexazinone Purity established roles in the control of D. discoideum myosin filament assembly each in vitro and in vivo [16,17,24], and Egelhoff, T. T., (unpublished studies). These enzymes possess a conserved domain organization that consists of a hugely novel protein kinase catalytic domain unrelated to traditional kinases, plus a carboxyl-terminal WD repeat domain that targets these enzymes to myosin II filaments (Figure 1). Genomic sequence corresponding for the related 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 important amino-terminal domain upstream on the catalytic do-Page three of(page number not for citation purposes)BMC Cell Biology 2002,http:www.biomedcentral.com1471-21213Figure two Purification and activity of epitope-tagged MHCK-C. A. MHCK-C expression levels are indicated by western blot evaluation of total cell lysates in 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 complete length and clipped FLAG-MHCK-C (pure FLAG-MHCK-C lane). Coomassie blue stained material (Coomassie lane) indicates purity plus 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 both autophosphorylates and phosphorylates Dictyostelium myosin II around 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 inside a reaction mixtur.
