Date of Award

2007

Degree Name

Doctor of Philosophy

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Michael Moore

Second Advisor

Larry Grover

Third Advisor

Elsa Mangiarua

Abstract

Myosin light chain kinase (MLCK) is an enzyme that phosphorylates the serine-19 residue on myosin regulatory light chains (MLCs) which serves to activate the Mg2+-ATPase of myosin. This catalytic activity is thought to be the primary role of MLCK; however, it has recently been suggested that MLCK’s actin binding and bundling properties may also be of importance in smooth muscle contraction. In the absence of calcium and calmodulin (CaM), MLCK will bundle actin filaments with its N-terminus. During calcium influx and subsequent CaM activation, MLCK binding to actin decreases resulting in unbundling of actin filaments and allows myosin and actin to slide past each other for force development. Despite these signals, some contractile agonists develop high levels of force in the relative absence of increased levels of intracellular calcium or MLC phosphorylation. One agonist that falls into this category is phorbol 12,13-dibutyrate (PDBu). PDBu activates the protein kinase C (PKCα) pathway which inhibits myosin light chain phosphatase (MLCP) and allows the MLCs to stay in a phosphorylated state. PKCα can also phosphorylate the kinase domain of MLCK and inhibit activation via CaM. This pathway suggests that MLCK and its ability to bind to actin filaments may still be intact in PDBu-stimulated smooth muscle. Therefore, the present studies look at the interaction between MLCK and α- and β-actin, the two predominant isoforms found in vascular smooth muscle, during PDBu-induced contraction of A7r5 smooth muscle cells in culture and highly differentiated vascular smooth muscle freshly excised from the rat.

Subject(s)

Smooth muscle - Contraction - Research.

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