Calmodulin and Calmodulin-regulated Protein Kinases as Transducers of Intracellular Calcium Signals

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity.

“…We have used the eDNA sequence of nmMLCK described in this report to test, through the use of site-specific mutagenesis, extant models about CaM recognition and relief of autoinhibition of MLCKs. The results, which include the production of a novel conformational suppressor mutation in the CaM binding sequence, are consistent with the hypothesis that the 20-residue CaM binding segment of smMLCK and nmMLCK, originally identified by chemical fragmentation and synthetic peptide analogue studies (48,50), is quantitatively important in MLCK's selective recognition of and response to CaM. Relatedly, the data allow the tentative identification within the MLCK sequence of a CaM regula-tory unit, which includes a new definition of the autoinhibitory region.…”

“…Results from studies that used peptide analogues of residues 1,082=1,101 in the MLCK sequence (48,50), site-specific mutagenesis, and computational chemistry analyses of CaM (14, 80; Table 1I), and enzyme kinetic studies of CaM/MLCK complexes (Table II) raised the possibility of complementary charge features within CaM and MLCK that are important in the formation and functioning of CaM/MLCK signal transduction complexes. The potential importance of protein charge, or electrostatic, properties in biological macromolecular recognition has been recognized for some time (59), and the specific data that showed (14,80) that charge reversal mutations of CaM alter its ability to interact with MLCK are consistent with this possibility.…”

“…Our knowledge of the primary structure of a nmMLCK, and the insight provided by our demonstration that the catalytic and CaM regulatory regions of nmMLCK and smMLCK are identical in amino acid sequence, allowed us to use the information gained from previous studies (19,23,48,50,78) of smMLCK to directly test, by site-specific mutagenesis, models (4, 27, 33, 39, 42-44, 48, 50, 61) of how MLCK structure might correlate with CaM binding and enzyme activation. All of the models have in common the assumption that there are key regions within the MLCK sequence that prevent the manifestation of protein kinase activity due to an intramolecular inhibition, or autoinhibition, and that activation by CaM results from the release of this intramolecular inhibition.…”

A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin–Calmodulin Binding Peptide Interaction

“…CaM is small, highly conserved calcium binding messenger protein (148 residues) that has a key role in intracellular signal transduction, binding and activating enzymes 27 . It is also one of the few examples of a small protein capable of binding to peptides with reasonable affinity (K D ~ 4 nM) in presence of calcium ions which can be completely disrupted by addition of calcium chelators like EDTA or EGTA due to a conformational change of CaM that is triggered on loss of calcium (Supplemental Fig.…”

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

“…Calmodulin, a major calcium target protein, is widely recognized to play a key role in the Ca 2 + signaling pathways in mammals (Hidaka and Ishikawa 1992;Lukas et al 1988). Recently, the involvement of plant CaM in signal transduction in plants has been established from experiments showing that the expression of CaM genes is regulated by environmental stimuli, including light and auxin (Jena et al 1989), wind and touch (Braam and Davis 1990), and heat shock (Braam 1992).…”