Characterization of thePITALRE/CDK9 and PISSLREcdc2-related kinases

The proteins involved in determining when and how fast a eukaryotic cell can progress towards cell division have been an intensive topic of study for the last two decades. A subset of these proteins controlling the cell cycle are kinases that phosphorylate critical substrates during progression or arrest of the cell cycle. The Saccharomyces cerevisiae cdc28 protein was found to be a kinase that regulated both the onset of DNA replication and mitosis in this yeast. Mammalian cells possess a homolog of cdc28, termed cdc2, that also allows entry into mitosis. However, mammalian systems have evolved a much more complex system of cell cycle regulation in that several similar yet distinct kinases are present at different points of the cell cycle to ensure efficient control. Two relatives of cdc2, PITALRE and PISSLRE, have been cloned in our lab that are named with respect to their homology to cdc2 in the PSTAIRE amino acid domain. This dissertation provides evidence assigning these two kinases to functions both within and outside of cell cycle control. By using a kinase-inactive mutant strategy, PISSLRE is shown here to be essential for normal cellular growth and localized temporally to the early G2 phase of the cell cycle. The PISSLRE protein is nuclear, 35kDa and associates with three intracellular proteins. Although these functions are similar to mammalian cdc2 characteristics, PISSLRE is not able to complement the loss of the S. cerevisiae cdc2 homolog, cdc28. These data suggest that PISSLRE acts similarly to cdc2 in human cells, however the pathways through which it functions are not the same as cdc2 or at least not conserved in yeast. PITALRE, on the other hand, appears to be an atypical cdc2 relative. Expression of PITALRE in the developing mouse is localized to areas that have terminally differentiated and PITALRE kinase activity increases dramatically during in vitro differentiation of certain cell types. Another cdc2 relative, CDKS, has been found to possess much of the same qualities. PITALRE also associates with the TNF signalling protein TRAF2. Interaction occurs in an area of TRAF2 that is required for binding to other kinases and for efficient activation of the NF-$\kappa$B transcription factor. Binding between the two proteins in vivo takes place in terminally differentiated muscle cells, wherein PITALRE partially translocates to the cytoplasm. TRAF2 is highly upregulated during muscle differentiation in vitro and is expressed in areas of skeletal muscle in the developing mouse. A kinase inactive mutant of PITALRE blocks TNF- and TRAF2-mediated NF-$\kappa$B activation and is potentiated only by a mutant of kinase active PITALRE that lacks a nuclear localization sequence. Therefore, I propose here that PITALRE possesses both nuclear and cytoplasmic functions upon onset of differentiation, and that these are not necessarily cooperative