Cells activate complex signaling pathways in response to stresses and injuries such as DNA damage, hypoxia, or infections. These pathways control cell fate by activating protein serine/threonine kinases that phosphorylate critical downstream targets. Mutations in signaling pathways that normally respond to either DNA damage or to disruption of the mitotic spindle, for example, play critical roles in the genesis of most human cancers. The goal of our research is to understand how protein phosphorylation controls progression through the cell cycle at the molecular level and how defects in phosphorylation bypass normal cell cycle checkpoints and lead to human cancer. Our lab uses a broad proteomics approach to decode how these cell signaling pathways are "wired" using bioinformatics, combinatorial chemistry, cell biology, physical biochemistry, structural biology and molecular genetics.
A major clue to understanding cell signaling was the discovery that many molecules contain discrete modular signaling domains such as SH2, SH3 and PDZ domains, arranged in a combinatorial fashion. SH2 and PTB domains bind to phosphotyrosine-containing sequence motifs, but the fundamental mechanisms underlying phosphoserine/phosphothreonine signaling have remained poorly understood. More recently, a subset of new modules, including 14-3-3 proteins, FHA domains and WW domains, were found to bind specifically to short phosphoserine or phosphothreonine-containing amino acid sequences within proteins. These pSer/pThr-binding modules integrate signals from upstream protein Ser/Thr kinases to control the actions of downstream effector molecules, controlling cell cycle checkpoints and activating patterns of gene transcription. We are using phosphoserine- and phosphothreonine-oriented peptide libraries to elucidate the specific sequence motifs recognized by each of these modules and have developed bioinformatics algorithms that use this information to identify likely interacting proteins within the mouse, human and yeast proteomes. In parallel with this combinatorial chemistry/computational approach, we are using high- density protein arrays and expression libraries to identify specific interacting proteins that play critical roles in establishing cell cycle checkpoints that respond to DNA damage or disruption of the mitotic spindle. Our lab is also developing a novel library-against-library screening approach which should reveal additional phosphoserine/threonine-binding modules that have not yet been identified.
In addition, we have a long-standing history in studying phosphorylation-mediated signal transduction, particularly in cell cycle regulation and the DNA damage response. We have identified protein modules that recognize phospho-serine/phospho-threonine motifs and elucidated the specific sequence of the motifs recognized by these modules. Furthermore, we identified the kinase MK2 as a critical node in DNA-damage checkpoint signaling in tumor cells that lack a functional p53-response. At the moment, we are studying how cell cycle kinases like Aurora-A, Plk1 and the Nek-family of kinases regulate mitosis, using a combination of structural biology, biochemistry, and molecular biology. In addition, we try to identify new kinase-substrate relationships using high-throughput peptide library screens, combined with computational biology and phosphoproteomics. Lastly, we aim to take our findings into the clinic and identify novel strategies to inhibit kinase-mediated signal transduction to treat cancer and inflammation. We have previously shown that time-staggered treatment of EGFR inhibitors with doxorubicin efficiently kills cancer cells, and we are currently focusing on the combination of cell-cycle kinase inhibitors with a variety of other targeted or non-targeted therapies. We identified several combination therapies that result in strong synergistic killing of cancer cells, and use cell-line screens together with computational analysis of existing databases to delineate the mechanism of action, and find potential biomarkers.
p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38 MAPK/MK2 pathway for survival after DNA damage (Reinhardt et al, Cancer Cell, 2007)