Inhibition of Calmodulin-Activated Smooth-Muscle Myosin Light-Chain Kinase by Calmodulin-Binding Peptides and Fluorescent (Phosphodiesterase-Activating) Calmodulin Derivatives

Abstract

Aspects of the biochemistry of calmodulin have been addressed that bear on its cell biological role as a mediator of Ca2+ regulation. Calmodulin-binding peptides derived from the amino acid sequence of smooth-muscle myosin light-chain kinase (MLCK) were characterized as inhibitors of calmodulin activation of MLCK-catalyzed phosphorylation of the smooth-muscle regulatory light chain (MLC). MLCK activity was determined by measuring the rate of formation of one of the reaction products, ADP, in a coupled enzymatic assay by continuous fluorimetric monitoring of NADH removal in 100 microM CaCl2 at ionic strength 0.15 M, pH 7.0 and 21 degreesC. The Km value of calmodulin was 3.5 nM, a value 16-35-fold greater than the Kd value of calmodulin for MLCK [Török, K., and Trentham D. R. (1994) Biochemistry 33, 12807-12820]. The different Km and Kd values are most likely associated with the rate-limiting step in MLC phosphorylation being associated with product release from MLCK. The values of the inhibition constants, Ki, were the following: Ac-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-CONH2 (Trp peptide), 8.6 (+/-1. 4 sd) pM; Y4-analogue of Trp peptide (Tyr peptide), 7.3 (+/-0.1) nM; and A-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-S-S (RS20-like peptide), 0. 11-0.39 nM. The Ki values were consistent with kinetically determined Kd values of the peptides to calmodulin. Kinetic determination of Kd values required the use of a fluorescently labeled calmodulin, 2-chloro-(epsilon-amino-Lys75)-[6-(4-N, N-diethylamino-phenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-calmodulin).1 Since, as here, Lys75 is a convenient labeling site on calmodulin for the introduction of fluorescent probes, the biological activity of the Lys-modified calmodulins was evaluated. TA-calmodulin and calmodulin selectively modified by 1-N, N-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-C1) at Lys75 (dansyl-calmodulin) were characterized as activators of cyclic AMP phosphodiesterase (PDE) and inhibitors of MLCK. The Km value for dansyl-calmodulin was equal to that of calmodulin, and that of TA-calmodulin was 3.5-fold greater. TA-calmodulin and Lys75-labeled dansyl-calmodulin thus distinguish between PDE and MLCK being agonists to the former and antagonists to the latter.

Ca2+-dependent and -independent mechanisms of calmodulin nuclear translocation

“…1H). The previously described Trp peptide (Török and Trentham, 1994;Török et al, 1998a;Török et al, 1998b) was myristoylated at the N-terminus to make it membrane permeant. The Ca 2+ /CaM-binding properties of mTrp peptide were determined by stopped-flow fluorimetry using TA-CaM.…”

“…A cell permeant CaM-binding peptide based on the acetylated Trp peptide (Ac-RRKWQKTGHAVRAIGRL-CONH 2 ) (Török and CaM nuclear translocation Trentham, 1994;Török et al, 1998a;Török et al, 1998b) was generated by N-terminal myristoylation instead of acetylation. This myristoylated peptide (mTrp) was HPLC-purified to homogeneity and its K d for 2-chloro-(ε-amino-Lys75) [6-4-(N,N-diethhylamino)phenyl]-1,3,5-triazin-4-yl-CaM (TA-CaM) was determined by stopped-flow kinetic experiments in a Hi-Tech PQ/SF-51MX multimixing stoppedflow spectrofluorimeter (Hi-Tech Scientific, UK) in the solution conditions above with the exception of EGTA being replaced by 100 µM CaCl 2 .…”

“…Measurement value was the decrease in adenosine extinction at 265 nm. For detailed methodology see Török et al (Török et al, 1998b).…”

Mechanism of Calmodulin Recognition of the Binding Domain of Isoform 1b of the Plasma Membrane Ca2+-ATPase: Kinetic Pathway and Effects of Methionine Oxidation

“…Scheme 1 was therefore modified to include k 1 : The system of rate equations for Scheme 3 (see eq S13 in the Supporting Information) yielded further conditions for the kinetic rate constants. The fluorescence experiment reported here lacked the time resolution to directly measure k 1 , which has been reported for various enzymes to be on the order of 10 8 M −1 s −1 (14,15,30). Caride and co-workers recently analyzed CaM binding to the CaM binding domain of isoform 4b of PMCA (14,15).…”

KCNE4 Juxtamembrane Region Is Required for Interaction with Calmodulin and for Functional Suppression of KCNQ1

“…1 and 3. In a previous characterization of MLCK-P ("Trp peptide"), the K d value of calmodulin and MLCK-P was calculated to be 6 pM from assay of MLCK inhibition, yielding the conclusion that the peptide inhibits MLCK by trapping calmodulin and leaving free calmodulin concentrations very low (23). Similarly, prior characterization of CaMKII-P ("peptide 290 -309") determined that CaMKII-P inhibits CaMKII and CaM-dependent phosphodiesterase activity by peptide interaction with CaM and not by a direct effect on the enzyme (24).…”