Browsing by Author "Dr. Wayne A. Tompkins, Committee Co-Chair"

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    Protein Kinase A Regulates beta-2 Integrin Avidity Activation and Subsequent Neutrophil Activation via Modulation of Myosin Light Chain Kinase.
    (2006-04-11) Chilcoat, Clayton Douglas; Dr. Robert B. Rose, Committee Member; Dr. Samuel L. Jones, Committee Co-Chair; Dr. Scott M. Laster, Committee Member; Dr. Edward A. Havell, Committee Member; Dr. Wayne A. Tompkins, Committee Co-Chair
    β2 integrins are adhesion molecules on the surface of neutrophils. Avidity activation of β2 integrins includes transportation of pre-formed integrins to the cell surface and a conformational change in the integrin to a high-binding state. Upon binding ligand, β2 integrins initiate a signaling cascade that results in activation of the neutrophil to a pro-inflammatory state, and the inhibition of this signal can prevent further activation of the neutrophil. cAMP and it effector protein kinase A (PKA) exert a generally inhibitory effect upon neutrophil activation. PKA has been shown to inactivate myosin light chain kinase (MLCK). Myosin light chain (MLC) phosphorylation is crucial for actin-myosin complex formation, which is required for stability and contraction of the actin cytoskeleton in neutrophils as well as β2 integrin-dependent adhesion. We hypothesize that the inhibitory effect of PKA upon neutrophils is due to inhibition of β2 integrin avidity activation resulting in the subsequent inhibition of neutrophil activation. Furthermore we hypothesize that the effect of PKA upon β2 integrin avidity activation is mediated through PKA's effect upon MLCK. We demonstrate that inhibition of PKA induces β2 integrin-dependent adhesion and that augmentation of cAMP prevented β2 integrin-dependent adhesion and subsequent respiratory burst activity. Further, we demonstrate via flow cytometric detection of antibodies directed against β2 integrins that pharmacologic inhibition of PKA activity results in overall increased β2 integrin expression on the neutrophil surface, as well as increased expression of the activated form of the integrin. This upregulation and activation of β2 integrins due to inhibition of PKA is abolished by pharmacologic MLCK inhibition. Inhibition of MLCK also blocked β2 integrin-dependent neutrophil adhesion achieved by inhibition of PKA, as well as neutrophil migration along towards a PKA inhibitor. These findings demonstrate that PKA regulation of β2 integrin affinity activation and subsequent neutrophil activation is via an MLCK-dependent pathway.