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Overview

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.

Smooth Muscles

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.

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  • Project 1: Is the regulatory subunit of MLCP (MYPT1) necessary for both rapid and sustained RLC phosphorylation and contraction? We will knock out MYPT1 in adult mice containing floxed MYPT1 alleles by tamoxifen-controlled Cre expression specifically in smooth muscle cells for these investigations.
  • Project 2: How does MYPT1 regulate MLCP activity to sustain RLC phosphorylation during Ca2+-sensitization?The two regulatory phosphorylation sites in MYPT1 will be mutated (Thr696Ala and Thr853Ala) individually or together, for biochemical and cell response studies.  We will identify which of the two phosphorylation sites are important for Ca2+-sensitization responses.
  • Project 3: Does Ca2+-independent ZIPK affect Ca2+-sensitizing MYPT1 and CPI-17 phosphorylation? Mice containing floxed genes for ZIPK will be used for gene ablation in adult animals because there are no selective pharmacological inhibitors for this kinase. We will determine if Ca2+-independent kinase ZIPK phosphorylates MYPT1 and CPI-17 as an integral component of the Ca2+-sensitization process.

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.

  • Project 4: Do MYH11 mutations that cause TAAD impair contractile output of smooth muscle cells? We will analyze aortic smooth muscle tissues from genetically modified mice for age-dependent adaptive changes in vasomotor performance.
  • Project 5: Does heterozygous loss of MLCK activity impair contractile output because of attenuated RLC phosphorylation in smooth muscle cells and lead to development of TAAD in the ascending aorta?
  • Project 6: Do ACTA2 mutations that cause TAAD impair contractile output of smooth muscles? We will analyze the adhesion and contractile signaling modules to determine if specific actin mutations promote selective dysfunction in one module or both.
  • Project 7: Do ACTA2 mutations perturb myofibroblast contractions? MRTF-A will be used to induce myofibroblast phenotype expressing high amounts of smooth muscle α-actin in human dermal fibroblasts from patients harboring mutations in ACTA2.

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.

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