Identifizierung von Inhibitoren der Wachstumsarrest induzierenden Funktion von Miz1 und Charakterisierung von 14.3.3eta als Inhibitor der Funktion von Miz1

The Myc interacting transcription factor Miz1 is required for the transcriptional activation of the cyclin dependent kinase inhibitors p15Ink4b and p21Cip1. It is through the activation of these genes that Miz1 induces a cell cycle arrest in the G1-phase of cells. Myc prevents the Miz1 dependent induction of the G1-arrest by forming a complex with Miz1 and thereby repressing the transcriptional activity of Miz1. In the presented dissertation, a retroviral screen was performed, in an attempt to both identify and subsequently characterize novel genes encoding inhibitors of the growth suppressive function of Miz1. To this end, a human cDNA library was retrovirally infected into Rat1 fibroblasts that had been additionally superinfected with Miz1 expressing retroviruses. The titer of the Miz1 expressing retroviruses caused a complete growth arrest in parental Rat1 fibroblasts. To determine the identity of the inserted cDNAs of the isolated clones, both PCR and sequencing procedures were performed. Altogether, a cDNA encoding human c-Myc was isolated seven times whilst a cDNA encoding a fragment of the human gamma actin1 was isolated twice. In all of the analysed cell clones ectopic Miz1 expression was detectable. The isolation of entire gene sequences with an inhibitory function demonstrated that the executed screening assay was technically working. However the identification of the hit cDNAs inhibiting the Miz1 induced growth arrest as being human c-Myc sequences did not precede new findings because of the already known and well studied interaction of c-Myc and Miz1 (Peukert et al., 1997). The second part of the dissertation deals with the characterization of 14-3-3eta as an inhibitor of the function of Miz1. 14-3-3eta is an isoform of the 14-3-3 protein family which contains nine family members in mammals. 14-3-3eta was found as a potential candidate in a separate retroviral screen that was also performed to identify new inhibitors of the growth suppressive properties of Miz1. In this instance, a retroviral cDNA expression library was used to find systematically inhibitors that could overcome the slow growth phenotype of Miz1 expressing Rat1 fibroblasts. Overexpression of 14-3-3eta overcame Miz1 induced growth arrest in the G1 phase of the cell cycle and inhibited transcriptional activation of the Cdk inhibitors p15Ink4b and p21Cip1 by Miz1 similar to c-Myc and TopBP1. Furthermore 14-3-3eta overexpression antagonizes the Zusammenfassung 126 Miz1 induced apoptosis in Rat1 fibroblasts. Sequence analysis of the Miz1 sequence revealed two potential binding motifs for 14-3-3 proteins. They are located close to and within the DNA binding domain of Miz1. In the first motif lies a phosphorylatable threonine at position 291 and in the second motif lies a phosphorylatable serine at position 428. 14-3-3eta binds directly to Miz1 through these two consensus binding sites. It was shown using point mutants of Miz1 which are deficient in 14-3-3eta binding show that binding of 14-3-3eta to this consensus sites is necessary to block Miz1 mediated transcriptional activation of p21Cip1 and p15 Ink4b. Furthermore, phosphorylation of the serine/ threonine residues in these recognition motifs is prerequisite for the interaction of 14-3-3 to Miz1. The inhibition of Miz1 by 14-3-3eta mechanistically functions by preventing the binding of Miz1 to the initiator elements of the target gene promoters. The interaction with 14-3-3eta had no effect on the subcellular localization of Miz1. An assumption for the interaction of 14-3-3 with its ligands is the phosphorylation of the serine/ threonine residues in the binding motifs. The phosphorylation of the serine at position 428 in the second binding motif is carried out by the Akt/ PKB kinase and is essential for the interaction of 14-3-3eta and Miz1 and the subsequent inhibition of the Miz1 function. Gene expression analysis demonstrated that Miz1 has two distinct functions in regulation of gene expression. Firstly, Miz1 is required for DNA damage induced upregulation of a large group of genes. This function is regulated by Myc but not by Akt/ 14-3-3eta. Secondly, Miz1 represses the expression of a large group of genes in response to DNA damage in an Akt/ 14-3-3eta, but not a Myc regulated manner. Akt dependent phosphorylation of Miz1 and the following binding of 14-3-3eta may thus regulate growth arrest in response to DNA damage.