Dynamic MLCK activation

Myosin II regulatory light chain (RLC) is the convergent point of multiple signaling cascades as shown on the left. It is unclear why non-muscle cells devise so many seemingly overlapped pathways to phosphorylate a handful of RLC residues. These signals, however, pose great challenge in our effort to understand the molecular events leading to myosin regulation in vivo.

Since RLC is targeted by a multitude of kinase, we propose the hypothesis that the compartmentalization of cellular signals lead to regional myosin function and organization. This hypothesis argues that signal leading to myosin activation (or deactivation) cannot be understood as simple, linear pathways. Instead, they are molecular events that occur at specific spatial and temporal dimension.

The difficulty in addressing this problem is compounded by the fact that numerous signaling cascades that converge on RLC display significant crosstalk. For example, p21-activated kinase (PAK) mono-phosphorylates RLC at Ser-19 yet prevents MLCK from di-phosphorylating RLC at Ser-19/Thr-18. PAK does so by directly inhibiting MLCK through phosphorylation. Rho kinase, on the other hand, not only phosphorylate RLC at Ser-19, but also inactivates myosin phosphatase, thus further augmenting its myosin-activating capacity. The wealth of biochemical information obtained on these pathways, however, has not fully elucidated how they affect myosin organization and function in vivo. This hurdle is due in large part to our inability to characterize signal transduction with the proper spatial-temporal regulation. It is therefore conceivable that one preliminary approach would be to correlate myosin function and organization with the activities of these kinases. In this study, we focus our attention on the best characterized RLC-targeting kinase - myosin light chain kinase (MLCK), a [Ca²⁺]₄/calmodulin-dependent enzyme. We have devised a novel biosensor, MLCK-FIP, that allows us to simultaneously monitor the localization of MLCK as well as its state of activation in live cells.

MLCK-FIP is a chimeric protein generated by fusing the fluorescent indicator protein (FIP) (J. Biol. Chem. 272: 13270-4) to the C-terminus of myosin light chain kinase (MLCK). The FIP comprises of a donor fluorophore (blue fluorescent protein, BFP), linked by an MLCK-derived calmodulin-binding domain, to an acceptor fluorophore (red-shifted green fluorescent protein, GFP). Fluorescent resonant energy transfer (FRET) occurs when the two fluorophores are brought to close proximity by the coiled calmodulin binding domain in the absence of Ca²⁺/calmodulin. In the presence of Ca²⁺, Ca²⁺/calmodulin binds to both MLCK as well as the FIP. The calmodulin binding domain uncoils, and increases the distance between the two fluorophores, thus disrupting the FRET. This molecule therefore serves as biosensor to simultaneously detect the localization as well as the activation state of MLCK in live cells. The biochemical characterization of this biosensor and the detailed analysis of MLCK activity using this sensor has been published (J. Cell Biol. 156: 543-553, 2002). This web page therefore serves to further supplement the published data, and keeps an update of our latest, unpublished results. Please click on thumbnails below to view larger version of the image or the videoclips.

	MLCK-FIP detects ionomycin-induced [Ca²⁺]₄/calmodulin changes in vivo

	MLCK-FIP demonstrates MLCK activation and localization dynamics during cell contraction. First part of the videoclip show the two-dimensional ratio image, while the second half shows the three-dimensional ratio image in which the intensity of MLCK (hence the 'amount' of kinase) is displayed as peak heights

	Another example of MLCK activation along stress fibers during contraction

	The MLCK activity profile correlates with RLC phosphorylation pattern in vivo, and the 4-color fluorescent imaging delineates myosin activation mediated by MLCK or potentially by some other kinases

	Myosin organization into clusters of bipolar filaments within the lamella, corresponds to the high MLCK activity in this region. Taken together, these results suggest that the MLCK-mediated regulation may have taken place before the clustering of myosin reaches the level detectable by the resolution of fluorescent microscopy.

	Active MLCK display association with actin meshwork within the protruding lamella.

	The biosensor displays differential distribution of MLCK activity in migrating cell - MLCK is significantly more active in the leading lamella than the retracting tail.

	MLCK shows bimodal distribution during cell division. Note the enrichment of MLCK in the cleavage furrow prior to the maximal activation leading to the contraction of the furrow.