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Structural and Biochemical Effects of Phosphorylation of Protein Kinase A (PKA).

Jon Steichen

Appointment Period: 2011-2012, Grant Years: [27]

Jon SteichenProtein kinases are one of the largest classes of proteins in the human genome (~2%) and they are widely recognized to be important oncogenes. One of the most common mechanisms for regulating protein kinase activity is through the phosphorylation of a conserved structural element known as the activation loop. Currently there are nine FDA approved protein kinase inhibitors and some of them distinguish between between the phosphorylated and unphosphorylated form of the kinase. In addition to the activation loop many kinases are regulated by their own C-terminal tail interacting with the kinase core and this interaction can also be regulated by phosphorylation. In the laboratory of Dr. Susan Taylor I study the structural and biochemical effects of phosphorylation of protein kinase A (PKA) on its activation loop and its C-terminal tail (turn motif) in hopes of better understanding generally how these phosphorylation events regulate kinase activity. I have used x-ray crystallography to characterize the structural changes associated with both activation loop as well as turn motif phosphorylation, and hydrogen/deuterium exchange to characterize the dynamic effects of activation loop phosphorylation in solution. Additionally, I am characterizing the kinetic effects as well as the effects on binding to regulatory proteins. In a similar project I am characterizing the interaction between the phosphoinositide-dependent protein kinase (PDK1) and PKA. PDK1 is known to phosphorylate the activation loop of numerous protein kinases and mutations of PDK1 have been correlated with colorectal cancer. To characterize this interaction I am using mutational analysis of both PDK1 and PKA to define the binding interface between the two proteins.

Publications (resulting from this training)

Yang J, Wu J, Steichen JM, Kornev AP, Deal MS, Li S, Sankaran B, Woods VL Jr, Taylor SS. A conserved glu-arg salt bridge connects coevolved motifs that define the eukaryotic protein kinase fold. J Mol Biol. (2012) 415:666-79. PMID: 22138346.

Steichen JM, Kuchinskas M, Keshwani MM, Yang J, Adams JA, Taylor SS. Structural basis for the regulation of protein kinase A by activation loop phosphorylation. J Biol Chem. 2012 Feb 10. [Epub ahead of print] PubMed PMID: 22334660.

Other publications (prior to Training Grant appointment):

Steichen JM, Iyer GH, Li S, Saldanha SA, Deal MS, Woods VL Jr, Taylor SS. Global consequences of activation loop phosphorylation on protein kinase A. J Biol Chem. 2010 Feb 5;285(6):3825-32. Epub 2009 Dec 4. PubMed PMID: 19965870; PMC2823524.

Steichen JM, Petty RV, Sharkey TD. Domain characterization of a 4-alpha-glucanotransferase essential for maltose metabolism in photosynthetic leaves. J Biol Chem. 2008 Jul 25;283(30):20797-804. Epub 2008 May 22. PubMed PMID: 18499663.

Lu Y, Steichen JM, Yao J, Sharkey TD. The role of cytosolic alpha-glucan phosphorylase in maltose metabolism and the comparison of amylomaltase in Arabidopsis and Escherichia coli. Plant Physiol. 2006 Nov;142(3):878-89. Epub 2006 Sep 15. PubMed PMID: 16980562; PMC1630732.

Lu Y, Steichen JM, Weise SE, Sharkey TD. Cellular and organ level localization of maltose in maltose-excess Arabidopsis mutants. Planta. 2006 Sep;224(4):935-43. Epub 2006 Apr 5. PubMed PMID: 16596410.