The overall objectives of this project are to gain insight into the mechanism by which the newly identified microtubule associated serine/threonine kinase-like molecule, MASTL regulates terminal differentiation of megakaryocytes, as well as to provide the applicant, Dr. Jan Johnson with the scientific tools and career development necessary for a successful career in Basic Science Research. During the next five years, Dr. Johnson will follow a research career development plan consisting of a program of educational sessions and a laboratory-based research prject under the sponsorship of Drs. Diana Gilligan and Neil Josephson and will have the consulting guidance of Drs. Jonathan Drachman, Kenneth Kaushansky, and Barry Paw. The research plan is to define the molecular signaling mechanisms of the specific serine/theronine kinase MASTL during hematopoeisis in order to better understand the differentiation of megakaryocytes. Megakaryocytes arise from a common hematopoeitic stem cell, commit to a megakaryocytic lineage, mature in the bone marrow of mammals and finally produce platelets for circulation. A tightly regulated maturation of megakaryocytes is maintained to control the numbers of circulating platelets in an organism. A single point mutation was identified in the MASTL kinase in a family suffering with an inherited autosomal dominant thrombocytopenia. This observation has provided evidence for the involvement of this novel kinase in the megakaryocytopoeisis process. The specific aims of this research proposal are: 1) Characterize the MASTL protein expression pattern, kinase activity, and modifications in the megakaryocytic cells as they progress through maturation, 2) Determine the role of MASTL in hematopoeisis model systems, and 3) Identify MASTL kinase substrates in megakaryocyte development. Although these specific aims represent an ambitious goal, the variety of experimental methods and approaches will help develop the breadth of Dr. Johnson's scientific experience as well as generate important data regarding the little understood process of terminal megakaryocyte differentiation which may lead to novel approaches for treating both induced as well as inherited thrombocytopenia.