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Structures of PKA RIIβ Holoenzymes Define Isoform Diversity.

Simon Brown

Appointment Period: 2006-2008, Grant Years: [22,23]

Simon BrownChanges, expression and activity of the signaling enzyme Protein Kinase A have been implicated in various forms of cancer, including both breast and prostate cancer. One approach to selectively modulate PKA signaling is to develop small molecules that exploit the functional and structural variations found between PKA isoforms. An assay system was developed to screen compounds in a dose dependant manner for their ability to affect changes in PKA activity. The incorporation of multiple isoforms of PKA into the assay system has allowed a library of compounds to be directly screened for multiple isoform selectivity of activation. An X-ray crystal structure of the PKA RIIβ regulatory domain bound to an isoform selective compound found in this screen revealed the determinants for compound binding and selectivity. The combination of structural and selectivity information will be used to design additional compounds with higher selectivity for future in-vivo therapeutic testing. Additionally, by solving X-ray crystal structures of the PKA catalytic domain bound to the RIIβ regulatory domain we have discovered the atomic basis for inter-domain interactions and investigated conformational changes of the enzyme that occur after small molecule binding. Comparison of the new RIIβ crystal structures to RIα and RIIα structures has defined the specific characteristics of isoform diversity and how this diversity affects regulation of PKA function. The most important contribution to cancer research is the development of the PKA system as a model for kinase targeting, primarily by innovating a way to use small molecules to affect kinase regulatory systems in an isoform specific manner.

Wu J, Brown S, Xuong NH, Taylor SS. RIalpha subunit of PKA: a cAMP-free structure reveals a hydrophobic capping mechanism for docking cAMP into site B. Structure. (2004) 12:1057-65. PMID: 15274925.

Vigil D, Blumenthal DK, Brown S, Taylor SS, Trewhella J. Differential effects of substrate on type I and type II PKA holoenzyme dissociation. Biochemistry. (2004) 43:5629-36. PMID: 15134437.

Vigil D, Blumenthal DK, Heller WT, Brown S, Canaves JM, Taylor SS, Trewhella J. Conformational differences among solution structures of the type Ialpha, IIalpha and IIbeta protein kinase A regulatory subunit homodimers: role of the linker regions. J Mol Biol. (2004) 337:1183-94. PMID: 15046986.

Heller WT, Vigil D, Brown S, Blumenthal DK, Taylor SS, Trewhella J. C subunits binding to the protein kinase A RI alpha dimer induce a large conformational change. J Biol Chem. (2004) 279:19084-90. PMID: 14985329.

Esposito V, Sjoberg T, Das R, Brown S, Taylor SS, Melacini G. NMR assignment of the cAMP-binding domain A of the PKA regulatory subunit. J Biomol NMR. (2006) 36 Suppl 1:64. PMID: 17016670.

Anand GS, Hotchko M, Brown SH, Ten Eyck LF, Komives EA, Taylor SS. R-subunit isoform specificity in protein kinase A: distinct features of protein interfaces in PKA types I and II by amide H/2H exchange mass spectrometry. J Mol Biol. (2007) 374:487-99. PMID: 17942118.

Wu J, Brown SH, von Daake S, Taylor SS. PKA type IIalpha holoenzyme reveals a combinatorial strategy for isoform diversity. Science. (2007) 318:274-9. PMID: 17932298.

Taylor SS, Kim C, Cheng CY, Brown SH, Wu J, Kannan N. Signaling through cAMP and cAMP-dependent protein kinase: diverse strategies for drug design. Biochim Biophys Acta. (2008) 1784:16-26. PMID: 17996741; PMCID: PMC2561045.

Brown SH, Wu J, Kim C, Alberto K, Taylor SS. Novel isoform-specific interfaces revealed by PKA RIIbeta holoenzyme structures. J Mol Biol. (2009) 393:1070-82. PMID: 19748511.

Brown SHJ, Kim CW, Taylor SS. AMP-PNP Traps PKA RIIbeta Holoenzyme in a Transition State Complex. (2012) In preparation.

Brown SHJ, Saldanha AS, Cottam H, Taylor SS. Protein Kinase A Isozyme Assays Reveal True Selectivity of Commonly Used cAMP Derivatives. (2012) In preparation.