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 [4], 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.
Further analyses revealed that the B-Δ-N-WD truncation exhibited reduced levels of autophosphorylation compared to the full-length kinase and the B-Δ-WD truncation, (Figure 2A). This suggests that a portion of the 15 to 20 autophosphorylation sites in MHCK-B [4, 6] reside in the N-rich region of the kinase. A recent study of the mammalian alpha-kinases TRPM6/TRPM7 revealed that the ability of these kinases to phosphorylate myosin II heavy chain is dependent on the autophosphorylation of unstructured regions of these kinases [11]. We examined the B-Δ-WD truncation for a similar mode of regulation and found that pre-autophosphorylation had no apparent effect on the ability of the truncation to phosphorylate MHC substrate (Figure 2B). This result is consistent with previous studies demonstrating that autophosphorylation of full-length MHCK-B has no effect on the kinase activity of the enzyme [6].
Targeting by the N-Rich Region Leads to Myosin II Filament Disassembly In Vitro
Our results thus far demonstrate that the N-rich region alone can serve as a MHC targeting domain; however, it is not clear if MHC phosphorylation via this mechanism indeed drives myosin II filament disassembly [12]. To explore this possibility, we examined the ability of purified myosin II to assemble into sedimentable filaments after phosphorylation by GST-tagged MHCK-B or its truncations. We found MHC phosphorylation by both MHCK-B (full-length) and MHCK-B-Δ-WD promotes myosin II filament disassembly, whereas incubation with MHCK-B-Δ-N-WD has no effect on the ability of myosin II to form filaments (Figure 1D). We have shown previously that removal of the WD-repeat domain of MHCK-B reduces the ability of the kinase to interact directly with myosin II filaments to about 30% of that exhibited by the full-length kinase [4]. In the current study, we found that the B-Δ-N-WD truncation did not co-sediment with myosin II filaments (Figure 3A and 3B), suggesting that the N-rich region facilitates phosphorylation of MHC by binding directly to myosin II filaments.
Cells Over-Expressing MHCK-B-Δ-WD Exhibit Cytokinesis Defects and Decreased Myosin II Assembly
We next tested the hypothesis that if substrate targeting by the N-rich region is physiologically significant, then over-expression of the MHCK-B-Δ-WD truncation in Dictyostelium cells should lead to an increase in the amount of cellular myosin II in the disassembled state. In turn, myosin II dependent processes, such as cytokinesis in suspension culture and multicellular development, should be compromised [3]. To this end, we compared the suspension culture growth rates of Dictyostelium cells over-expressing full-length MHCK-B, MHCK-B-Δ-WD or MHCK-B-Δ-N-WD (Additional file 2) with that of wildtype AX2 cells. Indeed we found that cells over-expressing MHCK-B-Δ-WD proliferate in suspension culture at a much slower rate than AX2 cells and become increasingly large and multinucleated over time (Figure 4A, B, and 4C). By contrast, cells over-expressing the MHCK-B-Δ-N-WD truncation grow normally in suspension culture. Likewise, we found that cells over-expressing either full-length MHCK-B or the B-Δ-WD truncation stalled at the mound stage of multicellular development, whereas those with elevated levels of the B-Δ-N-WD truncation completed the developmental cycle normally (Additional file 3).
The cellular defects observed with B-Δ-WD over-expression are consistent with a decreased ability of the cell to form myosin II bipolar filaments. To explore this possibility we examined the assembly state of cellular myosin II by analyzing the levels of myosin II associated with the detergent-insoluble fraction of cells over-expressing full-length MHCK-B or its truncations. These experiments were performed as described previously [6, 8, 13] except that the MHCK-B proteins were over-expressed in the mhck A/B/C-null background [8], as a means of increasing the sensitivity of this assay. In these cells, myosin II is constitutively over-assembled due to the absence of MHCK-A, -B, and -C activities [8] (Figure 5A). As a result, decreased assembly of myosin II filaments is more evident than in AX2 cells where the level of cytoskeleton associated myosin II is relatively low in vegetative cells. We found that over-expression of the full-length or B-Δ-WD versions of MHCK-B in the mhck A/B/C-null background resulted in an approximately 85% and 46% reduction in the amount of assembled myosin, respectively (Figure 5A and 5B). By contrast, over-expression of the B-Δ-N-WD truncation did not lead to a decrease in the level of myosin II associated with the cytoskeleton-enriched pellet of the cell (Figure 5A and 5B); thus indicating that the N-rich region can target the MHCK-B catalytic domain to phosphorylate MHC and drive filament disassembly in the cell.