Calmodulin (CaM) is a ubiquitous Ca binding protein that has emerged as a universal regulator of the calcium signal. As cytosolic Ca rises, Ca binds to CaM producing structural changes which allow it to bind and activate more than thirty different target proteins. CaM is directly involved in the Ca dependent regulation of smooth muscle contraction, glandular and neurosecretion, DNA replication, gene expression and cell division. The present studies will utilize our new biologically active fluorescent CaM's which selectively report CaM binding to its target proteins. We will determine and relate the Ca dependence of CaM binding to its target proteins in smooth muscle to the Ca dependence of their activation. Using fluorescence stopped-flow techniques we will determine the rates of the Ca dependent association and dissociation of CaM with its target proteins, including myosin light chain kinase (MLCK) and caldesmon. These rates will be compared to the off-rates of calcium from CaM-target protein complexes. These studies will provide temporal information relating Ca transients (such as those muscle) with the rate of CaM-target protein association and disassociation. Most external stimuli influence intracellular events by the activation of cellular kinases and the phosphorylation of specific substrate proteins. Recently, it has been shown that several tyrosine kinases (including, the insulin receptor and the oncogene product, P60-src) are capable of phosphorylating tyrosine residues in Ca binding sites III and IV of CaM. We will examine the effect of this phosphorylation on CaM's calcium (and drug) binding properties and on the Ca dependence of CaM interaction with and activation of its target proteins. If phosphorylated CaM exhibition altered Ca dependent activation of MLCK, it will be examined for its ability to regulate Ca dependent tension in chemically skinned coronary arteries. Because CaM regulates many aspects of cell metabolism and growth its phosphorylation by these tyrosine kinases might play a pivotal role in the mechanism of action of Insulin and in the transformation of cells by oncogenic viruses which code for tyrosine kinases.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Ohio State University
Schools of Medicine
United States
Zip Code
Black, D J; Halling, D Brent; Mandich, David V et al. (2005) Calmodulin interactions with IQ peptides from voltage-dependent calcium channels. Am J Physiol Cell Physiol 288:C669-76
Tang, Wei; Halling, D Brent; Black, D J et al. (2003) Apocalmodulin and Ca2+ calmodulin-binding sites on the CaV1.2 channel. Biophys J 85:1538-47
Van Lierop, Jacquelyn E; Wilson, David P; Davis, Jonathan P et al. (2002) Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40). J Biol Chem 277:6550-8
Davis, Jonathan P; Rall, Jack A; Reiser, Peter J et al. (2002) Engineering competitive magnesium binding into the first EF-hand of skeletal troponin C. J Biol Chem 277:49716-26
Tikunova, Svetlana B; Rall, Jack A; Davis, Jonathan P (2002) Effect of hydrophobic residue substitutions with glutamine on Ca(2+) binding and exchange with the N-domain of troponin C. Biochemistry 41:6697-705
Johnson, J David; Tikunova, Svetlana B (2002) Fluorescence methods for measuring calcium affinity and calcium exchange with proteins. Methods Mol Biol 173:89-102
Tikunova, S B; Black, D J; Johnson, J D et al. (2001) Modifying Mg2+ binding and exchange with the N-terminal of calmodulin. Biochemistry 40:3348-53
Adak, S; Santolini, J; Tikunova, S et al. (2001) Neuronal nitric-oxide synthase mutant (Ser-1412 --> Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis. J Biol Chem 276:1244-52
Black, D J; Tikunova, S B; Johnson, J D et al. (2000) Acid pairs increase the N-terminal Ca2+ affinity of CaM by increasing the rate of Ca2+ association. Biochemistry 39:13831-7
Lee, S H; Johnson, J D; Walsh, M P et al. (2000) Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration. Biochem J 350 Pt 1:299-306

Showing the most recent 10 out of 39 publications