We want to know how kinases and phosphatases act on the myosin motor in muscle cells in relation to chemical second messengers controlling signaling networks. We have now identified two new kinases in heart muscle that may be key regulators of its myosin motor protein. One kinase, cardiac myosin light chain kinase (cMLCK), is greatly elevated in diseased heart and thus, may be recruited to enhance myosin function to help the heart pump blood. We have established that this kinase is essential for the basal phosphorylation of myosin and maintenance of heart performance. Another newly identified kinase (ZIPK) may be recruited to phosphorylate myosin with stresses acting on the heart during diseases.
We have also established that smooth muscle requires myosin phosphorylation by a smooth muscle specific myosin light chain kinase. This phosphorylation is necessary for control of blood pressure by smooth muscle cells in blood vessels, movement of digested food in the stomach and intestines, maintenance of airways in the lungs, and emptying of the urinary bladder. We have investigated interconnected chemical networks that affect myosin phosphorylation and using genetically modified mice with biophysical, biochemical and physiological measurements. Primary hypotheses are directed to identifying the key signaling proteins essential for smooth muscle contraction that may contribute to the development of different smooth muscle based diseases, including our recent observation that mutations in the human gene of smooth muscle myosin light chain kinase caused aortic aneurysm and dissection.
Activation of many cell surface receptors initiates diverse cellular movements such as cell migration, cell-matrix adhesion and contraction. These movements respond to increased cytosolic Ca2+ concentrations ([Ca2+]i) and activation of a different Ca2+/calmodulin (CaM)-dependent MLCK. MLCK phosphorylates smooth muscle specific myosin RLC, allowing myosin to bind actin filaments for cell shortening and force development. Signaling pathways are proposed for inhibition of myosin light chain phosphatase (MLCP) activity which increases RLC phosphorylation (Ca2+-sensitization). The MLCP regulatory subunit MYPT1 and the inhibitor protein CPI-17 may be phosphorylated by different Ca2+-independent kinases. Based on our recent successes in using molecular transgenic and conditional gene ablation approaches to establish MLCK’s role in Ca2+-dependent signaling in mice, we propose similar approaches to unravel integrative signaling pathways in relation to MLCP and Ca2+-sensitization mechanisms.
Familial thoracic aortic aneurysms and dissections (TAAD) are linked to mutations in smooth muscle myosin heavy chain (MYH11), actin (ACTA2) and MLCK (MYLK). We plan to determine if disease-causing mutations reduce smooth muscle contractile function.
The synergy of the interactions among these different projects and our worldwide collaborators provide unique opportunities for understanding of the molecular mechanisms that influence normal contractile functions in smooth muscles as well as derangements caused by specific diseases.