Identification of a new mechanism for targeting myosin II heavy chain phosphorylation by Dictyostelium myosin heavy chain kinase B
© Underwood et al; licensee BioMed Central Ltd. 2010
Received: 21 July 2009
Accepted: 3 March 2010
Published: 3 March 2010
Heavy chain phosphorylation plays a central role in regulating myosin II bipolar filament assembly in Dictyostelium, as well as in higher eukaryotic nonmuscle cells. Our previous work has demonstrated that the WD-repeat domain of Dictyostelium myosin II heavy chain kinase B (MHCK-B), unlike its counterpart in MHCK-A, is not absolutely required for targeting of the kinase to phosphorylate MHC. Thus, we tested the hypothesis that an asparagine-rich and structurally disordered region that is unique to MHCK-B can by itself function in substrate targeting.
Biochemical assays comparing the activities of full-length MHCK-B, a truncation lacking only the WD-repeat domain (B-Δ-WD), and a truncation lacking both the N-rich region and the WD-repeat domain (B-Δ-N-WD) revealed that the N-rich region targets MHCK-B to phosphorylate MHC in a manner that leads to bipolar filament disassembly. This targeting is physiologically relevant since cellular over-expression of the B-Δ-WD truncation, but not the B-Δ-N-WD truncation, leads to dramatically reduced levels of myosin II filament assembly and associated defects in cytokinesis and multicellular development.
The results presented here demonstrate that an intrinsically unstructured, and asparagine-rich, region of a MHCK-B can mediate specific targeting of the kinase to phosphorylate myosin II heavy chain. This targeting involves a direct binding interaction with myosin II filaments. In terms of regulating myosin bipolar filament assembly, our results suggest that factors affecting the activity of this unique region of MHCK-B could allow for regulation of MHCK-B in a manner that is distinct from the other MHCKs in Dictyostelium.
Background and Hypothesis
Myosin II is a molecular motor that plays a central role in facilitating a broad range of cellular activities in nonmuscle cells by driving contraction of actin filaments. In nonmuscle cells, myosin II exists in a dynamic equilibrium between bipolar filaments that can contract apposing actin filaments and monomers that are contraction incompetent. Studies in Dictyostelium discoideum , and more recently in mammalian nonmuscle cells , have demonstrated that phosphorylation of regulatory sites in the "tail" region of the myosin II heavy chain (MHC) drive bipolar filament disassembly. MHC phosphorylation in Dictyostelium is catalyzed by at least three MHC kinases (MHCK-A, -B, and -C) that share homologous α-kinase and WD-repeat domains . We have shown previously that the WD repeat domain is involved in physically targeting the catalytic domains of MHCK-A and MHCK-B to phosphorylate myosin II substrate . Even so, a truncation of MHCK-B lacking its WD-repeat domain, unlike the analogous truncation of MHCK-A, still phosphorylates myosin II up to 20% of the level observed with the full-length kinase . This suggests that there are additional mechanisms by which MHCK-B-mediated phosphorylation of MHC can be achieved.
A potentially relevant structural difference between the MHCK-A and -B proteins is that MHCK-B possesses a region of 125 amino acids between its catalytic and WD-repeat domains that is predicted to exhibit a high level of structural disorder . This region is strikingly enriched in asparagine residues (28% of the amino acids) with a stretch of 26 asparagines interrupted by a single serine residue. From this point forward we will refer to this region as the N-rich (asparagine-rich) region of MHCK-B. In the studies presented here, we extend the results from previous studies [4, 6] by exploring the hypothesis that the N-rich region of MHCK-B plays a role in facilitating WD-repeat-independent phosphorylation of MHC by the kinase. A broader goal of these studies is to examine the potential for an inherently unstructured region of a protein to play a role in substrate targeting.
Cell Culture and Cell-Based Assays
Dictyostelium cells were cultured as described previously  in HL5 supplemented with penicillin and streptomycin. Cell lines harbouring recombinant expression plasmid were selected at 50 μg/ml Geneticin (G418) for fusion protein over-expression. Cell lines over-expressing full-length or truncated versions of MHCK-B were analyzed for defects in cytokinesis and multicellular development as described previously . Cell lines were analyzed for their levels of myosin II filament assembly as described by Rico and Egelhoff , with the exception that the MHCK-B proteins were over-expressed in a mhkA/B/C-null background . AX2, mhkB-null , and mhkA/B/C-null cell lines were obtained from the Dicty Stock Centre .
Fusion Protein Construction, Expression, and Purification
Kinase Phosphorylation Assays, Myosin II Assembly Assays, and Myosin II Co-Sedimentation Assays
Phosphorylation assays for MHC, MH-1 peptide, and myelin basic protein (MBP) were performed as described previously, except that kinase was added at 50 nM  and the concentration of myosin II included in the assay was 1.0 μM instead of 0.42 μM as in previous studies. Myosin II assembly assays were performed exactly as detailed by Rico and Egelhoff , except that kinase was added at 80 nM. Experiments analyzing MHCK-B fusion proteins for co-sedimentation with myosin II filaments were carried out as described previously .
The N-Rich Region Facilitates MHCK-B Phosphorylation of MHC In Vitro
In the current studies, we tested the hypothesis that the N-rich region of MHCK-B is able to function in substrate targeting. If this is the case, then the ability of an MHCK-B truncation lacking both the N-rich and WD-repeat domains (B-Δ-N-WD) to phosphorylate MHC will be greatly reduced or absent. To explore this possibility, purified GST-tagged MHCK-B (full-length), B-Δ-WD, and B-Δ-N-WD proteins (Figure 1A) were assayed for kinase activity toward Dictyostelium MHC, as well as toward a peptide substrate (MH-1). MH-1 has been shown previously to be phosphorylated by alpha kinase catalytic domains in a WD-repeat-independent manner , and thus its phosphorylation is a useful measure of the basal kinase activity of the catalytic domain.
We found that removal of both the N-rich and WD-repeat domains (GST-B-Δ-N-WD) renders the catalytic domain barely able to phosphorylate MHC above detectable levels, whereas the truncation containing the N-rich region (GST-B-Δ-WD) can still use MHC as a substrate, albeit at about 30% of that displayed by the full-length kinase (Figure 1B). Taken together, these results suggest that removal of the N-rich region severely compromises MHC phosphorylation by the catalytic domain. By contrast, the innate kinase activity of the catalytic domain is not lost upon removal of the N-rich region and/or the WD-repeat domain of MHCK-B since all three versions of MHCK-B phosphorylated MH-1 peptide to the same level (Figure 1C). Moreover, we found that the presence of the N-rich region has no effect on the phosphorylation of another protein substrate, MBP (Additional file 1), suggesting that the targeting activity of the N-rich region is specific for MHC.
Targeting by the N-Rich Region Leads to Myosin II Filament Disassembly In Vitro
Cells Over-Expressing MHCK-B-Δ-WD Exhibit Cytokinesis Defects and Decreased Myosin II Assembly
The results presented here extend our previous studies of WD-repeat domain mediated targeting of enzyme activity  and have revealed that a highly disordered  and asparagine-rich region of MHCK-B can guide the catalytic domain to phosphorylate MHC and drive myosin II filament disassembly The demonstrated importance of the N-region in defining the catalytic activity of MHCK-B suggests that factors targeting this unique region could provide a means of regulating the kinase in a manner that is distinct from the other MHCKs in Dictyostelium. In a broader context, our findings support the idea that highly specific substrate targeting can be mediated by a region of an enzyme that lacks a recognizable motif or predicted fold. These findings may be of particular significance to studies of other Dictyostelium proteins in which asparagine-rich regions are fairly common, but their functions are largely unknown .
This work was supported by an NIH grant to P.A.S. (2R15GM066789-02).
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