General Biology Seminar

Protein phosphorylation plays roles in the majority of cellular processes, ranging from transcription to metabolism, cell division and cell movement. Given the fundamental importance of protein phosphorylation, it is not surprising that aberrations in phosphorylation occurring as a result of mutations or by epigenetic means play a major role in human diseases, such as cancer. There are more than 30 examples of protein kinases (PKs) where mutations play well-defined roles in human cancer. However, recent cancer kinome sequencing studies have uncovered potential driver mutations in a large number of additional PK genes that have not previously been implicated in cancer. We have devised a rank-ordering system to define the PK mutations most likely to play a role in cancer. We have begun to investigate several of these for their involvement in carcinogenesis, including cancer-associated mutations in DAPK3 and two PKC isoforms, and found that they decrease kinase activity, suggesting these kinases normally play tumor suppressor roles and are mutationally inactivated in some cancers. In response to DNA double-strand breaks (DSBs), cells sense the DNA ends at the break and then activate the protein kinase ATM to initiate a signaling cascade, known as the checkpoint response, that is driven by phosphorylation and ubiquitylation. We have found that the tumor suppressor protein CtIP is essential downstream of ATM for the resection of 5¡¦ ends of DSBs, which generates a 3¡¦ single stranded region that is needed for activation of the ATR kinase and subsequent repair of the break by homologous recombination. Interestingly, prior DSB damage in a cell precludes response to a later DSB damage signal by blocking CtIP recruitment to the damage site. RNF4 is a highly conserved eukaryotic RING domain E3 ubiquitin ligase that recognizes and ubiquitylates SUMOylated proteins through its SUMO-interacting motifs (SIM). Depletion of RNF4 impairs the cell¡¦s response to DNA damage and results in genomic instability. We have solved the structure of the RNF4 RING domain, and shown that it is a symmetrical dimer; the integrity of the RNF4 RING dimer is critical for its activity as a SUMO-targeted ubiquitin ligase (STUbL) in vitro and for its function in maintaining genomic integrity in fission yeast cells. We have also identified Arkadia/RNF111 as a second type of STUbL, which is involved in promoting signaling through the TGF Ò signaling pathway.