Light Chain Kinase Inhibitors

The identification and development of specific inhibitors of MLCK could provide valuable experimental tools for exploring physiological functions of MLCK and myosin phosphorylation in smooth muscle and nonmuscle cells. Although inhibitors that act by binding calmodulin have been used experimentally, their usefulness is limited because they inhibit other calmodulin-dependent enzymes in tissues and cells (Asano and Stull, 1985 Nakanishi et al., 1992). Therefore, attempts have been made to develop novel reagents that inhibit MLCK activity directly. ML-9 [l-(5-chloronaphthalenesulfonyl)-lH-hexahydro-l, 4-diazepine] inhibits purified smooth muscle MLCK competitively with respect to ATP (Saitoh et al., 1987). Predictably, it inhibits RLC phosphorylation in ac-tomyosin, skinned fibers, and smooth muscle strips (Ishikawa et al., 1988). However, ML-9 also inhibits other protein kinases, so its specificity under different experimental conditions needs to be established. 

New experimental strategies for inhibiting kinases have included the use of autoinhibitory domains from specific protein kinases (Kemp et al., 1991). The autoinhibitory sequences are similar in amino acid composition to the substrates of the kinase and block catalytic activity in the presence of Ca +Zcalmodulin. Such pep- 

Wortmannin is produced by the fungal strain Tal-aromyces wortmannin KY12420, which inhibits smooth muscle MLCK competitively and irreversibly with respect to ATP (Nakanishi etal, 1992). At concentrations less than 1 xM, MLCK activity was inhibited, whereas at a 10-fold higher concentration there was no effect on the activities of other protein kinases. Wortmannin inhibited myosin RLC phosphorylation and contraction in rat aorta. These initial experiments demonstrated the potential usefulness of wortmannin as an experimental reagent that selectively inhibits MLCK and not other protein kinases. Unfortunately, it also inhibits other enzymes, including, in particular, phospha-tidylinositol3-kinase (Nakanishietal., 1994 Kimura et al, 1994). 

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Nakanishi, S. Kakita, S. Takahashi, I. Kawahara, K. Tsukada, E. Sano, T. Yamada, K. Yoshida, M. Kase, H. Matsuda, Y. Hashimoto, Y. Nonomura, Y. Wortmannin, a microbial product inhibitor of myosin light chain kinase. J. Biol. Chem., 267, 2157-2163 (1992)... 

Nakanishi, S. Ando, K. Kawamoto, I. Matsuda, Y. MS-347a, a new inhibitor of myosin light chain kinase from Aspergillus sp. KY52178. J. Antibiot., 46, 1775-1781 (1989)... 

Nakanishi S, Kakita S, Takahashi I, Kawahara K, T sukuda E, Sano T, Y amada K, Y oshida M, Kase H, Matsuda Y, et al (1992) Wortmaimin, a microbial product inhibitor of myosin light chain kinase. J Biol Chem 267 2157-2163... 

Tokui T, Brozovich, F, Ando S, Ikebe M (1996) Enhancement of smooth muscle contraction with protein phosphatase inhibitor 1 Activation of inhibitor 1 by cGMP dependent protein kinase. Biochem Biophys Res Commun 220 777-783 Tdrbk K, Trentham DR (1994) Mechanism of 2-chloro-(epsilon-amino-Lys75)-[6-[4-(Nd -diethylamino)phenyl]-l,3,5-triazin-4-yl]calmodulin interactions with smooth muscle myosin light chain kinase and derived peptides. Biochemistry 33 12807-12820... 

If MLCK activates contraction by increasing myosin phosphorylation, then an increase in the activity of myosin light chain phosphatase, MLCP, by decreasing the fraction of myosin which is phosphorylated, should lead to relaxation from the active (contractile) state. Cyclic adenosine monophosphate (AMP) is a strong inhibitor of smooth muscle contraction and it has been suggested that activation of MLCP could result from its phosphorylation via cAMP activated protein kinase (see Figure 5). 

In the Chinese hamster ovary cell line ts20, containing a thermosensitive ubiquitin-activating enzyme, El, Sachse et al. (2002) showed that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. The coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes API, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. 

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