The protein kinase superfamily is one of the largest protein classes in the human genome. Kinases regulate key signaling processes that have been implicated in development and disease, including cancer. Small molecule kinase modulators have become important therapeutic tools and often target catalytic domains that are among the most structurally and functionally conserved regions of these enzymes. While these tools have provided tremendous insight into kinase activity, specificity has been difficult to achieve due to the high degree of structural conservation among kinases. Further, many protein: protein interactions that regulate kinase activity are mediated through -helical interactions that are difficult to disrupt using small molecule approaches. As a means to circumvent these problems, I propose to develop synthetically modified -helices that will be applied as investigative tools to mediate protein: protein interaction events involved in kinase regulation. As a model for understanding kinase regulation, cAMP-dependent Protein Kinase A (PKA) will be used as a paradigm for probing kinase regulation. PKA has broad substrate specificity, so activity is partly regulated by intracellular localization near specific target subsets via interactions with a diverse set of proteins called A Kinase Anchoring proteins (AKAPs). AKAPs interact directly with the regulatory subunits to localize the tetrameric complex. Single nucleotide polymorphism (SNP) analysis has revealed that single amino acid changes in the PKA Binding Region (AKB) of the AKAP protein D-AKAP2 is linked to a variety of disease states, including familial breast cancer. Synthetically stabilized, isoform-specific peptides will be designed to probe AKAP- mediated interactions with the R-subunits of PKA. Cell signaling events, as monitored by protein phosphorylation and expression changes, will be comparatively analyzed by mass spectrometry to uncover isoform-specific kinase activity in non-cancerous and mammary epithelial cancer cell lines. This will be used as a foundation for studying altered kinase signaling in cancer models.
Greater insight into kinase regulation will allow us to understand the implications of kinases on cancer. By taking a chemical biology approach, we can specifically probe a kinase of interest to study its role in cellular signaling in various cancers and compare this to its role in non-disease states. The written critiques and criteria scores of individual reviewers are provided in essentially unedited form in the Critique section below. Please note that these critiques and criteria scores were prepared prior to the meeting and may not have been revised subsequent to any discussions at the review meeting. The Resume and Summary of Discussion section above summarizes the final opinions of the committee.
|Wang, Yuxiao; Ho, Tienhuei G; Bertinetti, Daniela et al. (2014) Isoform-selective disruption of AKAP-localized PKA using hydrocarbon stapled peptides. ACS Chem Biol 9:635-42|