This proposal is to study the role of metablastin, a highly conserved cytosolic protein involved in signal transduction, in the control of mammalian cell division. Metablastin is expressed in many immature cells during development and in most cancer cells. It is strongly induced in lymphocytes after exposure to mitogens and in hepatocytes during liver regeneration. Metablastin undergoes serine phosphorylation in response to a variety of extracellular factors and during mitosis, at sites that serve as substrates for both cyclin-dependent (CDKs) and mitogen- activated protein kinases (MAPKs/ERKs) in vitro. Conditional expression of a phosphorylation site mutant of metablastin in human leukemia cells was recently shown to result in cell cycle arrest at the G2/M interface, suggesting that metablastin plays a role in the control of the G2/M transition. Guided by structural features of metablastin, we have isolated a human metablastin-interacting protein, Mip1, using a yeast two-hybrid interaction screen. Mip1 is novel but may be related to the dbl proto-oncogene family of guanine nucleotide exchange factors for certain Ras-related GTP-binding proteins. The hypothesis we wish to test is that the interaction of metablastin with Mip1 is part of a cell cycle control switch that is operated through phosphorylation of metablastin by the cyclin-dependent protein kinase p34cdc2. The first specific aim is to characterize Mip1. We will isolate and sequence full-length human and murine cDNAs encoding Mip1. Putative functional motifs will then be tested in vitro using recombinant Mip1. Once a functional activity has been identified, we will test if it is altered in the presence of phosphorylated or non-phosphorylated metablastin. We will raise antibodies to Mip1 and study the expression of the protein and its mRNA in murine tissues during development. To explore the biological function of Mip1 and the functional significance of its interaction with metablastin, we will carry out microinjection studies using HeLa cells. In these studies, we will compare the ability of phosphorylation site mutants of metablastin and of Mip1 antibodies to induce cell cycle arrest. To further explore if Mip1 is a critical cellular target of metablastin, we will test if co-injection of Mip1 will reverse the inhibitory effect of metablastin mutants on the cell cycle. We will also express full-length and truncated forms of Mip1 in NIH-3T3 cells in order to test the oncogenic potential of Mip1. In addition, we will use the yeast two-hybrid system to search for interacting proteins that might be potential downstream targets or upstream regulators of Mip1. The second specific aim is to study the interaction of metablastin and Mip1 in vitro and in vivo and to explore if site-specific phosphorylation of metablastin alters this interaction. A more long-range aim is to determine, using the yeast two-hybrid interaction screen, if there is a family of metablastin-interacting proteins. The proposed studies promise to shed light on a novel mechanism in the control cell replication, perturbations of which may play a role in oncogenesis.
