Regulation of cAMP-dependent protein kinases: the human protein kinase X (PrKX) reveals the role of the catalytic subunit alphaH-alphaI loop.
cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human protein kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e. RIα or RIβ but not by substrate inhibitors RIIα or RIIβ. Detailed examination of the capacity of five PrKX-like kinases ranging from human to protozoa (Trypanosoma brucei) to form holoenzymes with human R subunits in living cells shows that this preference for pseudosubstrate inhibitors is evolutionarily conserved. To elucidate the molecular basis of this inhibitory pattern, we applied bioluminescence resonance energy transfer and surface plasmon resonance in combination with site-directed mutagenesis. We observed that the conserved αH-αI loop residue Arg-283 in PrKX is crucial for its RI over RII preference, as a R283L mutant was able to form a holoenzyme complex with wild type RII subunits. Changing the corresponding αH-αI loop residue in PKA Cα (L277R), significantly destabilized holoenzyme complexes in vitro, as cAMP-mediated holoenzyme activation was facilitated by a factor of 2-4, and lead to a decreased affinity of the mutant C subunit for R subunits, significantly affecting RII containing holoenzymes.
Diskar
Mandy
M
Department of Biochemistry, University of Kassel, Heinrich-Plett-Strasse 40, 34132 Kassel, Germany.
Zenn
Hans-Michael
HM
Kaupisch
Alexandra
A
Kaufholz
Melanie
M
Brockmeyer
Stefanie
S
Sohmen
Daniel
D
Berrera
Marco
M
Zaccolo
Manuela
M
Boshart
Michael
M
Herberg
Friedrich W
FW
Prinz
Anke
A
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics; D002522:Chlorocebus aethiops; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D014346:Trypanosoma brucei brucei / Q000201:enzymology
D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D029721:Drosophila Proteins; D015800:Protozoan Proteins; D017868:Cyclic AMP-Dependent Protein Kinases
J Leukoc Biol. 2003 Feb;73(2):281-8
12554805
J Phys Chem B. 2007 Feb 15;111(6):1496-501
17243667
J Neurosci. 2008 Oct 22;28(43):10885-92
18945896
J Biol Chem. 2005 Apr 8;280(14):13279-84
15691833
J Biol Chem. 2009 Mar 6;284(10):6241-8
19122195
J Mol Biol. 2007 Nov 23;374(2):487-99
17942118
Biochemistry. 1993 Sep 21;32(37):9532-8
8373760
J Biol Chem. 1972 Jan 10;247(1):36-44
4336043
Genetics. 1995 Dec;141(4):1507-20
8601490
J Am Soc Nephrol. 2005 Dec;16(12):3543-52
16236808
Science. 2007 Oct 12;318(5848):274-9
17932298
BMC Chem Biol. 2009 Feb 12;9:3
19216744
Cell Signal. 2007 Oct;19(10):2024-34
17614255
Genomics. 2000 Mar 1;64(2):187-94
10729225
Cell Signal. 2006 Oct;18(10):1616-25
16524697
Chem Rev. 2001 Aug;101(8):2243-70
11749372
Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9260-5
12082174
J Biol Chem. 1999 Feb 26;274(9):5370-8
10026146
Genome Biol. 2003;4(5):111
12734000
Neuropeptides. 2006 Feb;40(1):23-34
16442618
Genetics. 2009 Jun;182(2):529-39
19364808
J Biol Chem. 1992 Mar 5;267(7):4806-14
1537860
Front Biosci. 2000 Aug 01;5:D678-93
10922298
J Biol Chem. 2010 Feb 5;285(6):3825-3832
19965870
Biophys J. 2006 May 15;90(10):3428-33
16500960
Kidney Int. 2009 Jul;76(1):54-62
19367327
Chem Biol. 2007 Sep;14(9):1031-42
17884635
Adv Protein Chem. 2003;66:27-85
14631816
J Mol Biol. 2009 Nov 13;393(5):1070-82
19748511
J Biol Chem. 1994 Sep 16;269(37):23025-31
8083203
J Mol Biol. 2006 Mar 31;357(3):880-9
16460759
Hum Mol Genet. 1997 Oct;6(11):1985-9
9302280
Biochim Biophys Acta. 2008 Jan;1782(1):1-9
17980165
Biophys J. 2006 Sep 15;91(6):2063-71
16798810
Genetics. 2006 Aug;173(4):1909-17
16751660
Eur J Biochem. 1997 Sep 15;248(3):820-6
9342234
J Histochem Cytochem. 2005 Aug;53(8):1003-9
15879576
Biochemistry. 1996 Mar 5;35(9):2934-42
8608131
Mol Cell Biol. 1987 Apr;7(4):1371-7
3037314
Structure. 2006 Jan;14(1):141-9
16407073
Eur J Cell Biol. 2006 Jul;85(7):663-72
16529845
Cell. 2007 Sep 21;130(6):1032-43
17889648
Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15412-7
9860982
Chembiochem. 2006 Jul;7(7):1007-12
16755626
Biochemistry. 1982 Nov 9;21(23):5794-9
6295440
Annu Rev Biophys Biomol Struct. 1999;28:155-79
10410799