Abstract
Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction. A soybean isoform of CaM, SCaM-4 (77% identical to human CaM) fails to activate MLCK, whereas SCaM-1 (90.5% identical to human CaM) is as effective as CaM. We exploited this difference to gain insights into the structural requirements in CaM for activation of MLCK. A chimera (domain I of SCaM-4 and domains II-IV of SCaM-1) behaved like SCaM4, and analysis of site-specific mutants of SCaM-1 indicated that K30E and G40D mutations were responsible for the reduction in activation of MLCK. Competition experiments showed that SCaM-4 binds to the CaM-binding site of MLCK with high affinity. Replacement of CaM in skinned smooth muscle by exogenous CaM or SCaM-1, but not SCaM-4, restored Ca(2+)-dependent contraction. K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca(2+)-induced contraction to CaM-depleted skinned smooth muscle, consistent with their capacity to activate MLCK. Interpretation of these results in light of the high-resolution structures of (Ca(2+))(4)-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys(30) and Gly(40) and residues from the C-terminal domain is created upon binding to MLCK, formation of which is required for activation of MLCK. Interactions between this activation domain and a region of MLCK distinct from the known CaM-binding domain are required for removal of the autoinhibitory domain from the active site, i.e., activation of MLCK, or this domain may be required to stabilize the conformation of (Ca(2+))(4)-CaM necessary for MLCK activation.
Highlights
Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction
We demonstrated previously that SCaM-1 is as potent as brain CaM (bCaM) in activation of smooth muscle MLCK and activates MLCK to the same extent as bCaM: half-maximal activation of MLCK (KCaM) occurred at 1.7 nM bCaM with a Hill coefficient of 1.1 and at 1.8 nM SCaM-1, with a Hill coefficient of 1.1, whereas SCaM-4 caused very little activation of MLCK even at 1 M (Fig. 2A) [9]
Similar results were obtained in assays using MLC-(11–23) as substrate; in this case, half-maximal activation of MLCK occurred at 8 nM bCaM and 5 nM SCaM-1, and no activation was observed with SCaM-4
Summary
CaM, calmodulin; SCaM, soybean calmodulin; hCaM, human calmodulin; bCaM, bovine brain calmodulin; MLCK, myosin light chain kinase; LC20, 20-kDa light chain of myosin; MLC-(11–23), synthetic peptide corresponding to residues 11–23 of LC20; TFP, trifluoperazine; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; RTA, rat tail artery. The availability of the different plant CaM isoforms provides very useful tools for studying the mechanism of activation of MLCK and other CaM target enzymes. We show that SCaM-4 behaves as a CaM antagonist, confirming earlier findings that binding of CaM to MLCK and activation of the enzyme are distinct events [18]. Two mechanisms could explain these results: (i) saturation of the Ca2ϩbinding sites of CaM induces a conformational change in MLCK that permits interactions between this newly created surface on CaM and an unspecified region of the kinase distinct from the well known CaM-binding site, and these interactions are required for removal of the autoinhibitory domain from the active site; or (ii) the newly formed surface is required to stabilize the conformation of (Ca2ϩ)41⁄7CaM necessary for MLCK activation
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