Accessible Requires Authentication Published by De Gruyter August 8, 2005

Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA

Claudia Hahnefeld, Daniela Moll, Maik Goette and Friedrich W. Herberg
From the journal

Abstract

cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.

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References

Anand, G.S., Hughes, C.A., Jones, J.M., Taylor, S.S., and Komives, E.A. (2002). Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)α subunit of protein kinase A. J. Mol. Biol.323, 377–386. Search in Google Scholar

Bossemeyer, D., Engh, R.A., Kinzel, V., Ponstingl, H., and Huber, R. (1993). Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 20 Å structure of the complex with Mn2+ adenylyl imidodiphosphate and inhibitor peptide PKI(5–24). EMBO J.12, 849–859. Search in Google Scholar

Buechler, J.A. and Taylor, S.S. (1990). Differential labeling of the catalytic subunit of cAMP-dependent protein kinase with a water-soluble carbodiimide: identification of carboxyl groups protected by MgATP and inhibitor peptides. Biochemistry29, 1937–1943. Search in Google Scholar

Canaves, J.M. and Taylor, S.S. (2002). Classification and phylogenetic analysis of the cAMP-dependent protein kinase regulatory subunit family. J. Mol. Evol.54, 17–29. Search in Google Scholar

Cook, P.F., Neville, M.E. Jr., Vrana, K.E., Hartl, F.T., and Roskoski, R. Jr. (1982). Adenosine cyclic 3′,5′-monophosphate dependent protein kinase: kinetic mechanism for the bovine skeletal muscle catalytic subunit. Biochemistry21, 5794–5799. Search in Google Scholar

de Rooij, J., Zwartkruis, F.J., Verheijen, M.H., Cool, R.H., Nijman, S.M., Wittinghofer, A., and Bos, J.L. (1998). Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature396, 474–477. Search in Google Scholar

de Rooij, J., Rehmann, H., van Triest, M., Cool, R.H., Wittinghofer, A., and Bos, J.L. (2000). Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs. J. Biol. Chem.275, 20829–20836. Search in Google Scholar

Diller, T.C., Madhusudan, Xuong, N.H., and Taylor, S.S. (2001). Molecular basis for regulatory subunit diversity in cAMP-dependent protein kinase: crystal structure of the type II β regulatory subunit. Structure (Camb.)9, 73–82. Search in Google Scholar

Gibson, R.M., Ji-Buechler, Y., and Taylor, S.S. (1997). Interaction of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Electrostatic sites on the type Iα regulatory subunit. J. Biol. Chem.272, 16343–16350. Search in Google Scholar

Hahnefeld, C., Drewianka, S., and Herberg, F.W. (2004). Determination of kinetic data using surface plasmon resonance biosensors. Methods Mol. Med.94, 299–320. Search in Google Scholar

Herberg, F.W. and Zimmermann, B. (1999). Analysis of protein kinase interactions using biomolecular interaction analysis. In: Protein Phosphorylation – A Practical Approach, Vol. 2, D.G. Hardie, ed. (Oxford, UK: Oxford University Press), pp. 335–371. Search in Google Scholar

Herberg, F.W., Bell, S.M., and Taylor, S.S. (1993). Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states. Protein Eng.6, 771–777. Search in Google Scholar

Herberg, F.W., Dostmann, W.R., Zorn, M., Davis, S.J., and Taylor, S.S. (1994). Crosstalk between domains in the regulatory subunit of cAMP-dependent protein kinase: influence of amino terminus on cAMP binding and holoenzyme formation. Biochemistry33, 7485–7494. Search in Google Scholar

Herberg, F.W., Taylor, S.S., and Dostmann, W.R. (1996). Active site mutations define the pathway for the cooperative activation of cAMP-dependent protein kinase. Biochemistry35, 2934–2942. Search in Google Scholar

Houge, G., Steinberg, R.A., Ogreid, D., and Doskeland, S.O. (1990). The rate of recombination of the subunits (RI and C) of cAMP-dependent protein kinase depends on whether one or two cAMP molecules are bound per RI monomer. J. Biol. Chem.265, 19507–19516. Search in Google Scholar

Huang, L.J. and Taylor, S.S. (1998). Dissecting cAMP binding domain A in the RIα subunit of cAMP-dependent protein kinase. Distinct subsites for recognition of cAMP and the catalytic subunit. J. Biol. Chem.273, 26739–26746. Search in Google Scholar

Humphrey, W., Dalke, A., and Schulten, K. (1996). VMD: visual molecular dynamics. J. Mol. Graph.14, 27–38. Search in Google Scholar

Kaupp, U.B. and Seifert, R. (2002). Cyclic nucleotide-gated ion channels. Physiol. Rev.82, 769–824. Search in Google Scholar

Kawasaki, H., Springett, G.M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D.E., and Graybiel, A.M. (1998). A family of cAMP-binding proteins that directly activate Rap1. Science282, 2275–2279. Search in Google Scholar

Kim, C., Xuong, N.-H., and Taylor, S.S. (2005). Crystal structure of a complex between the catalytic and regulatory (RIα) subunits of PKA. Science307, 690–696. Search in Google Scholar

Knighton, D.R., Zheng, J.H., Ten Eyck, L.F., Xuong, N.H., Taylor, S.S., and Sowadski, J.M. (1991). Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science253, 414–420. Search in Google Scholar

Krebs, E.G. and Beavo, J.A. (1979). Phosphorylation-dephosphorylation of enzymes. Annu. Rev. Biochem.48, 923–959. Search in Google Scholar

Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685. Search in Google Scholar

Leon, D.A., Canaves, J.M., and Taylor, S.S. (2000). Probing the multidomain structure of the type I regulatory subunit of cAMP-dependent protein kinase using mutational analysis: role and environment of endogenous tryptophans. Biochemistry39, 5662–5671. Search in Google Scholar

Mazzolini, M., Punta, M., and Torre, V. (2002). Movement of the C-helix during the gating of cyclic nucleotide-gated channels. Biophys. J.83, 3283–3295. Search in Google Scholar

McRee, D.E. (1999). XtalView/Xfit – A versatile program for manipulating atomic coordinates and electron density.J. Struct. Biol.125, 156–165. Search in Google Scholar

Morgenstern, B. (1999). DIALIGN 2: improvement of the segment-to-segment approach to multiple sequence alignment. Bioinformatics15, 211–218. Search in Google Scholar

Neitzel, J.J., Dostmann, W.R., and Taylor, S.S. (1991). Role of MgATP in the activation and reassociation of cAMP-dependent protein kinase I: consequences of replacing the essential arginine in cAMP binding site A. Biochemistry30, 733–739. Search in Google Scholar

Ogreid, D. and Doskeland, S.O. (1981). The kinetics of the interaction between cyclic AMP and the regulatory moiety of protein kinase II. Evidence for interaction between the binding sites for cyclic AMP. FEBS Lett.129, 282–286. Search in Google Scholar

Ogreid, D., Ekanger, R., Suva, R.H., Miller, J.P., and Doskeland, S.O. (1989). Comparison of the two classes of binding sites (A and B) of type I and type II cyclic-AMP-dependent protein kinases by using cyclic nucleotide analogs. Eur. J. Biochem.181, 19–31. Search in Google Scholar

Rehmann, H., Prakash, B., Wolf, E., Rueppel, A., De Rooij, J., Bos, J.L., and Wittinghofer, A. (2003). Structure and regulation of the cAMP-binding domains of Epac2. Nat. Struct. Biol.10, 26–32. Search in Google Scholar

Ringheim, G.E. and Taylor, S.S. (1990). Dissecting the domain structure of the regulatory subunit of cAMP-dependent protein kinase I and elucidating the role of MgATP. J. Biol. Chem.265, 4800–4808. Search in Google Scholar

Shabb, J.B. and Corbin, J.D. (1992). Cyclic nucleotide-binding domains in proteins having diverse functions. J. Biol. Chem.267, 5723–5726. Search in Google Scholar

Slice, L.W. and Taylor, S.S. (1989). Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli. J. Biol. Chem.264, 20940–20946. Search in Google Scholar

Sorol, M.R., Pastori, R.L., Muro, A., Moreno, S., and Rossi, S. (2000). Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A. Arch. Biochem. Biophys.382, 173–181. Search in Google Scholar

Stenberg, E., Persson, B., Roos, H., and Urbaniczky, C. (1991). Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins. J. Colloid Interface Sci.143, 513–526. Search in Google Scholar

Stergiopoulos, S.G. and Stratakis, C.A. (2003). Human tumors associated with Carney complex and germline PRKAR1A mutations: a protein kinase A disease! FEBS Lett.546, 59–64. Search in Google Scholar

Su, Y., Dostmann, W., Herberg, F.W., Durick, K., Xuong, N.H., Ten Eyck, L., Taylor, S.S., and Varughese, K.I. (1995). Regulatory subunit of protein kinase A: structure of a deletion mutant with cAMP binding domains. Science269, 807–813. Search in Google Scholar

Symcox, M.M., Cauthron, R.D., Ogreid, D., and Steinberg, R.A. (1994). Arg-242 is necessary for allosteric coupling of cyclic AMP-binding sites A and B of RI subunit of cyclic AMP-dependent protein kinase. J. Biol. Chem.269, 23025–23031. Search in Google Scholar

Tasken, K. and Aandahl, E.M. (2004). Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol. Rev.84, 137–167. Search in Google Scholar

Taylor, S.S., Buechler, J.A., and Yonemoto, W. (1990). cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu. Rev. Biochem.59, 971–1005. Search in Google Scholar

Wainger, B.J., DeGennaro, M., Santoro, B., Siegelbaum, S.A., and Tibbs, G.R. (2001). Molecular mechanism of cAMP modulation of HCN pacemaker channels. Nature411, 805–810. Search in Google Scholar

Weber, I.T. and Steitz, T.A. (1987). Structure of a complex of catabolite gene activator protein and cyclic AMP refined at 2.5 Å resolution. J. Mol. Biol.198, 311–326. Search in Google Scholar

Weber, I.T., Steitz, T.A., Bubis, J., and Taylor, S.S. (1987). Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase. Biochemistry26, 343–351. Search in Google Scholar

Wu, J., Brown, S., Xuong, N.H., and Taylor, S.S. (2004a). RIα subunit of PKA; a cAMP-free structure reveals a hydrophobic capping mechanism for docking cAMP into site B. Structure (Camb.)12, 1056–1064. Search in Google Scholar

Wu, J., Jones, J.M., Xuong, N.H., Eyck, L.F., and Taylor, S.S. (2004b). Crystal structures of RIα subunit of cyclic adenosine 5′-monophosphate (cAMP)-dependent protein kinase complexed with (Rp)-adenosine 3′,5′-cyclic monophosphothioate and (Sp)-adenosine 3′,5′-cyclic monophosphothioate, the phosphothioate analogues of cAMP. Biochemistry43, 6620–6629. Search in Google Scholar

Zimmermann, B., Hahnefeld, C., and Herberg, F.W. (2002). Applications of biomolecular interaction analysis in drug development. TARGETS1, 66–73. Search in Google Scholar

Published Online: 2005-08-08
Published in Print: 2005-07-01

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