Cellular communication and signal transduction: Using kinase activities to analyze intracellular networks

Cells respond and adapt to the environment using networks of molecular interactions that transduce information between cellular components. Investigating the fundamental features of these intracellular networks is expected to improve the diagnosis and treatment of disease. Advances in our understanding are slow because the complex dynamics of molecular associations are challenging to monitor on a large scale. Significant effort has been required to develop techniques that measure multi-component responses and improve the quality of quantitative measurements in a reliable context. Toward these aims, we developed techniques that systematically interrogate key pathways in clinically relevant molecular signaling networks.;Network response is frequently modulated by the phosphorylation of intracellular proteins by kinases. Kinase activities are used to monitor changes in cell response over time and thereby provide a link between functional response and molecular pathway dynamics. Several hundred kinases participate in the regulation of interdependent pathways and correlations between kinase activities are difficult to evaluate without performing simultaneous measurements. A remarkable example of the complexity of molecular signaling networks is the range of cellular responses that comprise innate immunity. Using two examples of cell response in the innate immune system we developed both targeted and global approaches to characterize dynamic changes in intracellular signaling patterns. Our targeted approach was based on the phophorylation of synthetic peptide kinase substrates, immobilized for simultaneous detection by the Luminex bead array system. Using this approach, the response of multiple tyrosine kinases was profiled during inhibition in a model of chronic myelogenous leukemia. Our global approach was based on enrichment for native phosphorylated substrates in neutrophil cell lysates challenged with lipopolysaccharide to induce the inflammatory response. Enrichment was followed by identification and relative quantitation using mass spectrometry. The application of both techniques required the additional development of quantitative methodology for the accurate determination of changes at the level of individual proteins and functional groups of proteins.;The results provide an initial step for an improved understanding of molecular contributions to biological responses at the cellular level. These novel methods provide a foundation for addressing future research toward targeted signal intervention for the treatment of immune response conditions.