Searching the Human Trancriptome for Novel MAP-Kinase Substrates Utilizing a Docking Motif & Live Cell Imaging of the Tumor Suppressor P53 Oligomer Formation in Response to Ribotoxic Stress

When: Friday, July 20, 2012 at 12:00
Where: DA 114
Speaker: Elizabeth Ann Gordon
Organization: UC Irvine
Sponsor: Biophysical Group Meeting

In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases. The Bardwell lab has developed a hybrid computational search algorithm that combines machine learning and expert knowledge to to search the human transcriptome for novel mitogen activated protein kinase docking sites.  Predictions are tested by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and docking site mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). We have also identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, and a functionally uncharacterized protein SMTNL2.

 

Inactivation of the tumor suppressor protein p53 often leads to uncontrolled cell growth and cancer. p53 is mutated in about 50% of all cancers.   Mutations in the c-Jun n-terminal kinase (JNK) phosphoacceptor site of p53 render it incapable of responding to stress and are found frequently mutated in human tumors.  p53 forms multimers in response to JNK activation in vivo and the induction of dimer, tetramer and higher order oligomers can be observed in live cells utilizing p53-eGFP.  Utilizing the T81A JNK kinase insensitive mutant of p53-eGFP, tetramer and higher order oligomer were observed prior to induction of JNK activity and did not change over time.  Additionally, in the breast cancer cell line MDA-MB-231 wild-type p53-eGFP does not respond properly to JNK activation, forming dimer yet failing to form tetramer or higher order oligomer, suggesting that the mutant form of p53-R280K expressed by these cells binds to and blocks the formation of higher order WT oligomers.