ИСТИНА

High and low molecular weight myosin light chain kinases (long MLCK and short MLCK, respectively) are highly specialized enzymes to phosphorylate Ser19 / Ser19, Thr18 of myosin II regulatory light chain (RLC). MLCK isoforms are involved in a large variety of cellular motile events and show different developmental and tissue distribution as well as localization in cells. Both MLCKs are believed to reside on actin-containing filaments, however localization in different cell types may be dependent on the relative abundance of actin-containing stress fibers. In short MLCK, three DFRxxL motifs at the very N-terminus of the enzyme are responsible for its binding to actin. Long MLCK shows higher affinity to actin-containing filaments both in vitro and in cells due to two additional DF/VRxxL motifs and additional amino acid sequence(s) present in the unique N-terminal extension of long MLCK. Recent studies have confined the N-terminal actin-binding site to two IgG-like domains while the first IgG-like domain retained a reduced ability to bind actin. Moreover, for long MLCK, there is a growing list of protein partners, which may serve to target long MLCK to specific areas in non-muscle cells. Still, there are large gaps in our understanding of the long MLCK domain structure and molecular mechanisms that are involved in regulation of MLCK-partner interactions in cells and direct MLCK to subcellular compartments where MLCK enzymatic and/or scaffolding activity is needed. In this study, we attempted to gain further insights into molecular anatomy of long MLCK by using long MLCK truncation mutants to investigate their contribution in MLCK subcellular localization. In CV-1 cells, full-length EGFP-tagged long MLCK localized to stress fibers. However, truncated EGFP-tagged ΔN245-long MLCK, which lacks two N-terminal IgG-domains involved in actin binding, showed a significant reduction in its actin-binding properties. Instead, ΔN245-long MLCK readily redistributed to a submembrane compartment and accumulated in focal adhesions. Moreover, as revealed by imaging of living transfected CV-1 cells, ΔN245-long MLCK induced multiple podosomes. Interestingly, a FLAG-tagged MLCK fragment consisting of two IgG-like domains also demonstrated a reduced binding to stress fibers and redistributed to the submembrane compartment. However, in this case, no active podosome formation was evident. Collectively, our findings demonstrate that several distinct MLCK actin-binding domains should play in concert to provide for a stress-fiber residence of long MLCK and override the effects of distinct active sequences that target MLCK to other partners / localization. These findings are inconsistent with the widely accepted model, which assumes that long MLCK is tightly bound to actin through its DFRxxL-domains, while its N-terminal domain may search for transient interactions with other actin filaments or microtubules. The relative affinities of actin-binding regions of long MLCK to microfilaments may vary within the context of full-length MLCK and, perhaps, depend on type of cells, in which long MLCK is expressed. Our results provide a rationale for further elucidation of mechanisms of fine-tuning MLCK transient localization (and activity) in cells through regulation of MLCK affinity to actin and/or other protein partners. For a cell, one possible way of such regulation is by means of posttranslational modifications. Indeed, recently we demonstrated that long MLCK N-terminal actin-binding site might be a subject for Ser / Thr phosphorylation-dependent regulation. Additionally, in this study, we found that upon in-vitro phosphorylation at Ser25 and Thr56, recombinant human short MLCK DFRxxL-domain demonstrated reduced ability to bind and bundle filamentous actin in vitro. Thus, it is tempting to hypothesize that phosphorylation might represent a “switching mechanism” to regulate long MLCK partnership in cells. We suggest that in attempts to identify regulatory sites, it is reasonable to rely on MLCK phosphorylation discovered in vivo by mass-spectrometry techniques.