Förster resonance energy transfer measurements of cofactor-dependent effects on protein arginine N-methyltransferase homodimerization.
Protein arginine N-methyltransferase (PRMT) dimerization is required for methyl group transfer from the cofactor S-adenosyl-L-methionine (AdoMet) to arginine residues in protein substrates, forming S-adenosyl-L-homocysteine (AdoHcy) and methylarginine residues. In this study, we use Förster resonance energy transfer (FRET) to determine dissociation constant (K(D)) values for dimerization of PRMT1 and PRMT6. By attaching monomeric Cerulean and Citrine fluorescent proteins to their N-termini, fluorescent PRMTs are formed that exhibit similar enzyme kinetics to unconjugated PRMTs. These fluorescent proteins are used in FRET-based binding studies in a multi-well format. In the presence of AdoMet, fluorescent PRMT1 and PRMT6 exhibit 4- and 6-fold lower dimerization K(D) values, respectively, than in the presence of AdoHcy, suggesting that AdoMet promotes PRMT homodimerization in contrast to AdoHcy. We also find that the dimerization K(D) values for PRMT1 in the presence of AdoMet or AdoHcy are, respectively, 6- and 10-fold lower than the corresponding values for PRMT6. Considering that the affinity of PRMT6 for AdoHcy is 10-fold higher than for AdoMet, PRMT6 function may be subject to cofactor-dependent regulation in cells where the methylation potential (i.e., ratio of AdoMet to AdoHcy) is low. Since PRMT1 affinity for AdoMet and AdoHcy is similar, however, a low methylation potential may not affect PRMT1 function.
Thomas
Dylan
D
Division of Biomolecular and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.
Lakowski
Ted M
TM
Pak
Magnolia L
ML
Kim
Jenny J
JJ
Frankel
Adam
A
D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D011484:Protein-Arginine N-Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000378:metabolism
D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; C099988:PRMT1 protein, human; C447833:PRMT6 protein, human; D011484:Protein-Arginine N-Methyltransferases
J Biol Chem. 2008 Apr 11;283(15):10015-25
18263580
Exp Cell Res. 2004 Apr 1;294(2):325-34
15023523
J Biol Chem. 2002 Feb 1;277(5):3537-43
11724789
J Biol Chem. 2005 Feb 4;280(5):3656-64
15494416
J Am Soc Mass Spectrom. 2009 Mar;20(3):507-19
19110445
J Biol Chem. 1996 Jun 21;271(25):15034-44
8663146
J Biol Chem. 2000 Mar 17;275(11):7723-30
10713084
EMBO J. 2007 Oct 17;26(20):4402-12
17882261
J Biol Chem. 2004 May 28;279(22):22902-7
15044439
Mol Cell Biol. 2000 Jul;20(13):4859-69
10848611
Mol Endocrinol. 2009 Apr;23(4):425-33
19164444
Biophys J. 1999 May;76(5):2587-99
10233073
Annu Rev Biochem. 1998;67:509-44
9759496
J Biol Chem. 1999 Oct 29;274(44):31531-42
10531356
EMBO J. 2000 Jul 17;19(14):3509-19
10899106
Nat Struct Biol. 2000 Dec;7(12):1165-71
11101900
Biochemistry. 2007 Nov 20;46(46):13370-81
17960915
Science. 1999 Jun 25;284(5423):2174-7
10381882
Structure. 2003 May;11(5):509-20
12737817
Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12318-23
17640894
Nat Methods. 2005 Dec;2(12):905-9
16299475
Biochemistry. 2001 May 15;40(19):5747-56
11341840
Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11996-2001
11050231
J Cell Sci. 2009 Mar 1;122(Pt 5):667-77
19208762
EMBO J. 2007 Oct 17;26(20):4391-401
17882262
Biochemistry. 1994 May 10;33(18):5539-44
8180176
Mol Cell. 2009 Jan 16;33(1):1-13
19150423
Anal Biochem. 2002 Jun 15;305(2):195-205
12054448
J Biol Chem. 2001 Aug 31;276(35):32971-6
11413150
Nat Biotechnol. 2004 Apr;22(4):445-9
14990965
J Biol Chem. 1998 Jul 3;273(27):16935-45
9642256
J Biol Chem. 2007 Nov 9;282(45):33009-21
17848568
Biochem J. 2009 Jun 26;421(2):253-61
19405910