Protein phosphorylation is a dynamic and reversible event essential to the proper functioning of virtually every physiological process, from muscle contraction to cell division. Because protein phosphorylation is a regulatory event, it follows that the protein kinases that catalyze phosphorylation, as well as protein phosphatases that catalyze the reverse reaction, should themselves be subject to regulation. The long term goal of the proposed research is to understand the molecular basis by which protein kinases are regulated in physiological systems. The improper regulation of protein kinases has been implicated in many human diseases, including diabetes and cancer. The proposed research focuses on the regulation of the protein kinase SPRK, a serine/threonine kinase that belongs to an emerging family of protein kinases that are being called mixed-lineage kinases. Several potential mechanisms by which SPRK activity may be regulated, including binding of activators or inhibitors, phosphorylation, and subcellular localization, will be examined. SPRK's contains several domains that may mediate protein-protein interactions, including an SH3 domain, leucine/isoleucine zipper motifs, a sequence for binding to Rho-family GTPases, and a large proline-rich tail. SPRK is the first demonstrated example of a protein kinase that both contains an SH3 domain and exhibits serine/threonine kinase activity. Distantly related to Raf, SPRK, has emerged as a candidate activator of the Jun N-terminal kinase (JNK) pathway. In engineered cell lines, SPRK is rapidly phosphorylated in response to serum or platelet-derived growth factor (PDGF). Besides growth factors, SPRK activity and subcellular localization may be regulated by small GTPases of the Rho subfamily. A combination of biochemical, molecular biological, and cell biological approaches is proposed to investigate the molecular mechanisms by which SPRK is regulated. Specifically, Dr. Gallo and her colleagues will: 1) investigate the mechanism by which Cdc42 activates SPRK, 2) assess the effects of SPRK's putative protein-protein interaction domains on regulating SPRK activity, 3) determine the consequence SPRK phosphorylation in response to PDGF and other extracellular factors, as well as decipher the signaling pathway from the PDGF receptor to SPRK, and 4 )map the precise sites of Cdc42- and PDGF- induced phosphorylation of SPRK. These studies will provide important insight into the molecular mechanisms of SPRK regulation, and of the regulation of protein kinases in general. Furthermore we will learn more about the role of SPRK in cell proliferation and other physiological processes. Certain protein kinases are expressed at abnormally high levels or with abnormally high activity in cancer and other diseases. Understanding how protein kinases are regulated in both normal and cancer cells opens the door to the development of new protein-kinase inhibitors (or inhibitors) that may serve as useful therapeutics.
