title: Implementing fluorescence anisotropy screening and crystallographic analysis to define PKA isoform-selective activation by cAMP analogs.
abstract: Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates many proteins, most notably cAMP-dependent protein kinase (PKA). PKA holoenzymes (comprised of two catalytic (C) and two regulatory (R) subunits) regulate a wide variety of cellular processes, and its functional diversity is amplified by the presence of four R-subunit isoforms, RIα, RIβ, RIIα, and RIIβ. Although these isoforms all respond to cAMP, they are functionally nonredundant and exhibit different biochemical properties. In order to understand the functional differences between these isoforms, we screened cAMP derivatives for their ability to selectively activate RI and RII PKA holoenzymes using a fluorescence anisotropy assay. Our results indicate that RIα holoenzymes are selectively activated by C8-substituted analogs and RIIβ holoenzymes by N6-substituted analogs, where HE33 is the most prominent RII activator. We also solved the crystal structures of both RIα and RIIβ bound to HE33. The RIIβ structure shows the bulky aliphatic substituent of HE33 is fully encompassed by a pocket comprising of hydrophobic residues. RIα lacks this hydrophobic lining in Domain A, and the side chains are displaced to accommodate the HE33 dipropyl groups. Comparison between cAMP-bound structures reveals that RIIβ, but not RIα, contains a cavity near the N6 site. This study suggests that the selective activation of RII over RI isoforms by N6 analogs is driven by the spatial and chemical constraints of Domain A and paves the way for the development of potent noncyclic nucleotide activators to specifically target PKA iso-holoenyzmes.
authors: Department of Chemistry and Biochemistry, ‡Department of Pharmacology and Howard Hughes Medical Institute, §Moores Cancer Center, University of California , San Diego, La Jolla, California 92037-0654, United States.
authors: Simon H J
authors: SH
authors: Brown
authors: Cecilia Y
authors: CY
authors: Cheng
authors: S Adrian
authors: SA
authors: Saldanha
authors: Jian
authors: J
authors: Wu
authors: Howard B
authors: HB
authors: Cottam
authors: Banumathi
authors: B
authors: Sankaran
authors: Susan S
authors: SS
authors: Taylor
mesh_terms: D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D005454:Fluorescence Polarization; D008958:Models, Molecular; D020033:Protein Isoforms; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D020033:Protein Isoforms; D017868:Cyclic AMP-Dependent Protein Kinases
references: J Clin Invest. 1994 Jul;94(1):422-30
references: 8040283
references: Curr Drug Targets. 2005 Sep;6(6):655-64
references: 16178799
references: Ann N Y Acad Sci. 2002 Jun;968:37-48
references: 12119266
references: BMC Med Genomics. 2008 Sep 26;1:43
references: 18822129
references: Structure. 2004 Jun;12(6):1057-65
references: 15274925
references: Anal Chem. 2006 Dec 15;78(24):8265-72
references: 17165815
references: Acta Crystallogr D Biol Crystallogr. 2001 Jan;57(Pt 1):122-33
references: 11134934
references: Science. 1995 Aug 11;269(5225):807-13
references: 7638597
references: Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10037-41
references: 11517324
references: Eur J Biochem. 1989 Apr 15;181(1):19-31
references: 2540965
references: J Biol Chem. 1975 Oct 10;250(19):7788-94
references: 240840
references: Acta Crystallogr D Biol Crystallogr. 2004 Mar;60(Pt 3):432-8
references: 14993666
references: Biochemistry. 1996 Mar 5;35(9):2934-42
references: 8608131
references: Cell. 2007 Sep 21;130(6):1032-43
references: 17889648
references: Nat Methods. 2008 Apr;5(4):277-8
references: 18376388
references: Biochemistry. 2004 Jun 1;43(21):6620-9
references: 15157095
references: Science. 2007 Oct 12;318(5848):274-9
references: 17932298
references: Cell Signal. 2007 Oct;19(10):2024-34
references: 17614255
references: Acta Crystallogr D Biol Crystallogr. 1994 Sep 1;50(Pt 5):760-3
references: 15299374
references: Nat Rev Mol Cell Biol. 2004 Dec;5(12):959-70
references: 15573134
references: Structure. 2001 Jan 10;9(1):73-82
references: 11342137
references: J Immunol. 2001 Jun 15;166(12):7600-5
references: 11390516
references: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references: 15299926
references: Methods Enzymol. 1997;276:307-26
references: 27754618
references: Br J Cancer. 1999 Feb;79(5-6):933-9
references: 10070893
references: Oncogene. 2004 Nov 18;23(54):8847-56
references: 15480415
references: Protein Eng. 1993 Sep;6(7):771-7
references: 8248101
references: J Biol Chem. 2004 Feb 20;279(8):7029-36
references: 14625280
references: J Mol Biol. 2009 Nov 13;393(5):1070-82
references: 19748511
references: J Mol Biol. 2007 Nov 23;374(2):487-99
references: 17942118
references: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references: 15572765
references: Chem Pharm Bull (Tokyo). 1990 Nov;38(11):3147-54
references: 1964880
references: Cancer Res. 2008 May 1;68(9):3133-41
references: 18451138
references: Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1948-54
references: 12393927
references: J Synchrotron Radiat. 2004 Jan 1;11(Pt 1):53-5
references: 14646133
references: Cancer Res. 1990 Nov 15;50(22):7093-100
references: 2224844
references: Biochemistry. 1982 Nov 9;21(23):5794-9
references: 6295440