Deciphering mechanisms of substrate targeting by protein kinases
The completion of the human genome a decade ago revealed more than 500 genes encoding protein kinases, and mass spectrometry based phosphoproteomics efforts have now cataloged over 100,000 sites of phosphorylation in mammalian cells. These studies have clearly outpaced our ability to understand signaling networks through analysis of individual kinases and their substrates: for the vast majority of the phosphorylation sites, the responsible kinase and functional significance are simply not known. My group studies basic mechanisms used by kinases to target specific substrates within the cell, with the idea of applying this knowledge to identify new kinase-substrate pairs on a proteomic scale. Kinases interact with their substrates through short sequence motifs found both at the site of phosphorylation and at distal sites. We develop methodology that allows for the rapid identification of these protein kinase recognition motifs. Using synthetic peptide arrays, we have recently conducted a biochemical screen to identify phosphorylation site motifs recognized by the entire set of kinases from budding yeast, and a similar screen of the human kinome is in progress. Information from these screens is used to map cellular phosphorylation networks and to relate mechanisms of substrate targeting to specific structural features of kinases.
Development of “orthogonal” kinase-substrate signaling systems
Investigations into the function of a protein kinase typically involve modulating its activity with small molecule inhibitors or genetic disruption, perturbations that necessarily impact the phosphorylation of the full set of substrates of the kinase. Understanding the functional impact of signaling through specific individual substrates of a kinase is more challenging, yet is essential to understanding mechanisms by which kinases effect changes in cell physiology. To develop systems that allow individual phosphorylation events to be studied, we are generating protein kinase mutants that change its phosphorylation site sequence motif. Such kinase mutants are effectively inactive in that they cannot recognize, and thus do not phosphorylate, their endogenous substrates. However, introduction of a complementary mutation into a substrate restores its ability to be phosphorylated by the mutant kinase. This process generates an “orthogonal” signaling system that allows us to assess the consequence of phosphorylating only a single substrate in cells.
Mutagenic rewiring of phosphorylation networks in cancer
Mutation and amplification of protein kinases and their upstream regulators are among the most common events driving cancer initiation and progression. We are specifically interested in recurrent cancer-associated mutations that drive mitogen-activated protein kinase (MAPK) signaling pathways. We are carrying out biochemical and cellular investigations into mutations in both kinases and phosphatases to better understand mechanisms of activation/inactivation and their downstream consequences. Intriguingly, we have found in some cases that cancer-associated mutations serve to change the substrate specificity of a protein kinase, suggesting that simultaneous gain and loss of specific substrates may in some cases constitute driver events in cancer. We use our knowledge of sequence motifs involved in substrate recognition to identify substrates of oncogenic kinases important for control of cell growth and survival. These studies provide insight into mechanisms underlying tumorigenesis and help to guide the development of kinase-targeting drugs.