The broad, long term objective of this proposal is to elucidate the exact sequence of molecular events that are involved in the stimulation of phagocytic leukocytes. Knowledge gained from these studies will be highly relevant to an understanding of host-defense mechanisms. Moreover, studies described here may ultimately suggest novel strategies for enhancing antimicrobial mechanisms during infectious disease. Stimulated neutrophils undergo a variety of changes that include chemotaxis, lysosomal enzyme release and the production of superoxide. Superoxide is a key component of the oxygen-dependent, antimicrobial arsenal of these cells. These cellular responses frequently involve phosphorylation of the myristolyated alanine-rich C kinase substrate (MARCKS-protein) and the 47 kDa subunit of the NADPH-oxidase system (p47-phox) on multiple sites. These proteins are involved in rearrangements of the actin cytoskeleton and superoxide generation, respectively. The investigator has recently observed that neutrophils contain four novel and uncharacterized protein kinases with molecular masses of 69, 63, 49 and 40 kDa that are rapidly activated upon stimulation of these cells with a chemotactic peptide. These novel kinases can catalyze the phosphorylation of peptides which contain the phosphorylation sites of p47-phox and MARCKS. Most importantly, the stimulation of these novel protein kinases appears to be dependent upon the activation of phosphatidylinositol 3- kinase (PI 3-K) and perhaps the production of C-3-phosphorylated phosphoinositides (PPIs). Thus, he may have uncovered a second messenger role for D-3-PPIs in neutrophils and the downstream targets of PI 3-K! Specifically, this project focuses on four unexplored areas in the signal transduction pathways of neutrophils. These are: (1) purifying and characterizing the 63 kDa protein kinase as a paradigm for this family of novel kinases, (2) establishing the structure-function relationships of this enzyme, (3) uncovering the regulatory mechanisms that modulate this kinase (e.g., illuminating the upstream events), and (4) establishing the exact role of this enzyme in neutrophil stimulation (e.g., uncovering the physiological substrates). Techniques of biochemistry (enzymology), molecular biology (cloning and sequencing) and cell biology (e.g., immunolocalization studies) will be employed. Classical and more modern procedures (affinity columns, HPLC systems) will be used to purify the enzyme. Characterization studies will include sequencing this protein and determining its structural motifs along with establishing the substrate specificity, cofactor requirements and the minimal consensus sequence recognized by the kinase. Particular attention will be paid to uncovering the exact role of PI 3-K in the activation of the 63 kDa kinase. Post-translational modifications (e.g., phosphorylation) will be sought. The ultimate goal is to forge a solid link between the regulatory properties of the isolated 63 kDa kinase and the control of the relevant stimulus-response phenomena in intact cells.
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