Biochimica ct Biopl5"sica Acta. 1136(1992) 151)-154 z: 1992_Elsexier Science Publishers B.V. All rights rcscr.'¢d 0167-4889/92/S05.00
1.50
Guanine nucleotides stimulate carboxyl methylation of kidney
cytosolic proteins Denis Gingras and Richard
B61iveau
Lahoratoire de Membranologic .ttoldculaire. I ~ r t t T n e n t dr" chinue-biochimie. Unit c~iL.: du Qta~occ A ,*.fon/r~a/Momr~al. Q u ~ c ICanada~ and Groupe de Recht'rche en Transport Mt~zbranaire. Unire~ti de Montreal. Montrial Quf,bec ¢Cana~a)
(Recei~.cd4 [X-cember 1991) (Revis4.'dmanuscript reoeixetl20 March Iq92_1
K~y words: Protein cztrbo:q;tnlcll'q,'lllalasferas¢:Guanine nudeotid¢
We studied the effect of guanine nucleotides on the carbo~l methyla:ion catabzed by class ii protein carbox~/lmethyltransferases tPCMT). Additioo of guanosine 5°-O4y-thiohriphosphate (GTPyS) promoted a time- and concentration-dependent enhancemcnt of protein mctbylatio.~ ia tnc cytosolic fraction isolated from kidney cortex. GTPyS affected the kinetics of the methylation reaction, as reflected by alterations of both apparent K~ and V ~ of the metl~'ltransfetase. This effect was specific for guanine nucleotides and was completely abolished by addition of S-adenosyl-t_-homocysteine, a well-known inh~itor of methyltransferasc--catalyzed reactions. No GTP~,S stimulation of melh~,°.~t~ was found in q,losolic extracts from any of the other tissues studied, including brain, testis, spleen, and liver, nor in brush-border membranes isolated from the kidney cortex. The metbylated proteins were highly sensitive to moderately alkaline conditions, suggesting that the methyl esters were formed on t.-isoaspartyl residues and thus mcthylated by a cla.gs II PCMT. Th.csc results suggest that class-II-associated protein mcthylation activity from the soluble iraction of the kidney can be regulated by g,,anine nuclcotid,~.
h droduO.iou Protein carboxylmethyhransferases (PCMT) represent a broad group of enzymes that transfer a methyl group from S-adenosyI-L-methionine to a carboxyl group in proteins [1]. In eucar~'otes, two major classes of PCMT have been studied to date. C!ass II PCMT (E.C. 2.1.1.77) methylate a wide variety of proteins and peptides to form extremely base-labile atethyl esters and possess a high :eactivity towards aspartic residues covalently altered by isomerization reactions [2.3]. These unusual properties have led to the hypothesis that these methyltransferases may be involved in the metabolism of d a m a g e d proteins [4.5]. Another class of ir, ethyltransferases (class l i d catalyze the metbylation Cortespondenc~ to: R. I~liveau. D~partement de chimie-biochimie. Uni','ersit6 dy Qu6bec ~ MontrdaL C.P. 8888. Succursalc "A". Mow tribal. Out,bee IDC 3P8. Canada. Abbreviations: AdoMet. S-adenos¥l-i.-mcthionine: AdoHcy. Sadcnosyl-t-homo~'st¢in~: ApplNHh~.adenyl-5'-yl imidodiphnsphatc; BBM. brush-border mcrqbrant:s; G-protein. guanine-nucleotidebinding prolein; GTPyS. guanosin¢ 5"-O4y-thioRriphosphate: GDP~OS. guannsine 5°-O4/~-thiokliphosphate: PCMT. protein carbox~'lmethyltransfe~ase; 16-BAC. benz~'ldimethyl-n-hexadeq,'lammonium chloride.
of the a-cart~'~.~.jl group of C-terminal residues located in a consens::s sequence ( C A A X ) of yeast and mammalian pro:~.ins. The pro!ein substrates of this class include t~c small GTP-binding protein ras [6-8], the nuc:-_ar |amin B [9], the -~- subonit of some trimeric G - p r m e i n s [10L and c G M P phnsphodieslerase [11]. The data obtained so far with these substrates have suggested that metbylation occurs after isoprenylation of a cysteine residue 4 amino acids from the C-terminns and proteolytic removal of the last 3 amino acids [ 12,13]. The nature of the a-carboxyl-terminal-metbylated proteins and the fact that this methylation is in some cases enhanced by guanine nucleotides [14,15], suggest an involvement of this methylation reaction in hormonal transduction. However, no studies have focnsed so far on a similar regulatory role of d a s s - l l catalyzed protein metbylation. In the kidney, the cells lining the proximal tubule are the site of reabsorption of important metabolites filtered by the glomerolns. These cells are characterized by a high degree of asymmetry with segregation of sodium-coupled active-transport systems in the brush-border membrane [16], while hormone receptors and bormone-sensitive adenylyl t3'clases are concentrated at the basolateral pole, in close contact with the circulation [17]. W e have recently reported the pres-
encc of two distinct class !1 PCMT in the kidney cortex, a soluble and a brush-border-membrane-associated PCMT [18]. In the present work, we examined the effects of guanine nucieotides on protein methylation occurring in the cytosolic fraction and in brush-border membranes of the kidney cortex. We demonstrate that guanine nucleotides enhance the activity of the soluble class !1 PCMT.
60 ~
,--r---
Materials and Meflmds All experiments were performed on adult SpragueDawley male raLs weighing 300-350 g (Charles River). Tissues were homogenized in 5 vols. of 250 mM sucrose-5 mM Tris-HCI (pH 7.5) using a Potter-Elvehjem tissue grinder. The homogenates were centrifuged at l l 0 0 0 0 x g for 60 min and the resulting supernatants used as cytosols. Brush-border membranes from the kidney cortex were isolated by a magnesium precipitation method [19]. The medium used for metbylation reactions contained, in a final volume of 30/zl : 100 mM Hepes-Tris buffer (pH 7.5), 2 p.M (2/zCi) [methyl-3H]S-adenosyi L-rnethionine (69-73 Ci/mmol, Dupont-New England Nuclear) and 100 p.g of proteins. The reaction mixture was incubated at 3TC for 60 rain or the times indicated in the figures. For GTP-dcpcndent methylation, GTP,/S (10 to 200 p.M) was added to the medium. Total incorporation of radioactivity into protein methyl esters was measured with a methanol vapor diffusion assay [20]. Kinetic parameters were determined by non-linear regression analysis of the data using a computer graphics program (Kaleidagraph). Methylated proteins were separated by 16-BAC acid electrophoresis using 7.5% acrylamide gels followed by fluorograpby with i M sodium salicylat¢, as described previously [18]. Control experiments have previously shown that the total amount of radioactivity present in the acidic gel is in the form of basc-scusith'c methyl esters. No significant radioactivity is detected following SDS-PAGE at pH 8.8, the methyl estcrs being hydrolyzed at this alkaline pH [18]~
10
t~~" ~
8 0
P
~
Incubationtime(min) Fig. I. Time dependence of endogenouscarboxyl mcthylation of kidney c~o.-,olicproteins.Cytosolicproteins(100 pg) were incubated in 100 mM Hepes/Tris(pH 7.5) with 2 pM [3HlAdoMct(2 ~.Ci) in the absence (Q) or presence of 100 pM App[NH.]p (11). GDP~S (o), GTP',/S(o) or GTP-tS+ 500 pM AdoHcy( A). Methylationwas quantified as described elsewhere[20]. Values are means+_S.D,of three experimentsdone in triplicate. Inset. Initial velocitiesof protein carboxyl mcthylation for contrd (1:3) and GTPTS-trcatcd (e) cytosols. (100 pM) also resulted in a stimulation of the endogenous methylation, but to a smaller extent, while App[NH]p, a non-hydrolysable analog of ATP, was without effect (Fig. 1). Both basal and GTPyS-stimulated methylation activities were almost completely abolished by the addition of 500/zM AdoHcy, a wellknown inhibitor of methyltrausferase-catalyzed reactions. The effect of GTPTS was strongly concentration-dependent. As shown in Fig. 2, addition of GTP~/S to the incubation medium resulted in a progressive stimula-
~0o
:~'~
is0.
Results
Time-, concentration-, and AdoMet-dependence GTPyS-stimulated protein methflation
of
Incubation of the soluble fraction isolated from kidney cortex with the radioactive methyl donor [3H]AdoMet resulted in a time-dependent methylation of endogenous proteins (Fig. i). The addition of 100 lzM GTPyS, a non-hydrolysable analog of GTP, increased markedly both the rate and extent of methylation, with an average 3-fold stimulation of the initial rate of incorporation of labelled methyl groups (33 vs. 10 pmol/mg per h) (Fig. i, inset). Addition of GDP/3S
o'
. ~ . , , ~. 50
i00
. , tso
2oo
Nucleotides (I.tM) Fig Z Concentration depcndcocc and specificity of nuclontides on carboxyl methylation ol kidney-corlex cylosolie proteins. Cytosolic proteins (I00 p.g) were incubated in I00 mM Hepes/Tris (~'H 7.5) with 2 p M [~H]AdoMet (2 pCi) in the presence of increasing concentrations of App[Nll]p ([3), GDP~S (o) or GTPTS (o) for 60 rain at 37*(::. Control value refers to methyhtion measured in the absence of nucleotides (9.8 pmol/mg per h). Means _+S.D. of three experiments done in triplicate are shown.
152 TABLE I Effect of GIPyS on rite etulogou,ui u#uhle PC.'tfT activity of t~ti~nL~ tusues
.
i
Preparatton of the soluble e~lraCts rfld determination of I ~ , l T act~.'ilywere I~rformed as ~k'-~'ribet:in "Materials and Methods'. A reprc~cnZatk¢ cxperlmenl :~ilh lrip.t-~:alcdeterminations is sh~rwn.
20b 150 100
Seluble cslracl
O
I~.?,fF a,_-lk-il) Ipmol/mg per h~
control
5O 0
. 0
5
10
15 20 AdoMet (pM)
25
,
30
Fig. 3. Effca of GTI'?S on the AdoMet dependence ot kidno'
1.50
Guanine nucleotides stimulate carboxyl methylation of kidney
cytosolic proteins Denis Gingras and Richard
B61iveau
Lahoratoire de Membranologic .ttoldculaire. I ~ r t t T n e n t dr" chinue-biochimie. Unit c~iL.: du Qta~occ A ,*.fon/r~a/Momr~al. Q u ~ c ICanada~ and Groupe de Recht'rche en Transport Mt~zbranaire. Unire~ti de Montreal. Montrial Quf,bec ¢Cana~a)
(Recei~.cd4 [X-cember 1991) (Revis4.'dmanuscript reoeixetl20 March Iq92_1
K~y words: Protein cztrbo:q;tnlcll'q,'lllalasferas¢:Guanine nudeotid¢
We studied the effect of guanine nucleotides on the carbo~l methyla:ion catabzed by class ii protein carbox~/lmethyltransferases tPCMT). Additioo of guanosine 5°-O4y-thiohriphosphate (GTPyS) promoted a time- and concentration-dependent enhancemcnt of protein mctbylatio.~ ia tnc cytosolic fraction isolated from kidney cortex. GTPyS affected the kinetics of the methylation reaction, as reflected by alterations of both apparent K~ and V ~ of the metl~'ltransfetase. This effect was specific for guanine nucleotides and was completely abolished by addition of S-adenosyl-t_-homocysteine, a well-known inh~itor of methyltransferasc--catalyzed reactions. No GTP~,S stimulation of melh~,°.~t~ was found in q,losolic extracts from any of the other tissues studied, including brain, testis, spleen, and liver, nor in brush-border membranes isolated from the kidney cortex. The metbylated proteins were highly sensitive to moderately alkaline conditions, suggesting that the methyl esters were formed on t.-isoaspartyl residues and thus mcthylated by a cla.gs II PCMT. Th.csc results suggest that class-II-associated protein mcthylation activity from the soluble iraction of the kidney can be regulated by g,,anine nuclcotid,~.
h droduO.iou Protein carboxylmethyhransferases (PCMT) represent a broad group of enzymes that transfer a methyl group from S-adenosyI-L-methionine to a carboxyl group in proteins [1]. In eucar~'otes, two major classes of PCMT have been studied to date. C!ass II PCMT (E.C. 2.1.1.77) methylate a wide variety of proteins and peptides to form extremely base-labile atethyl esters and possess a high :eactivity towards aspartic residues covalently altered by isomerization reactions [2.3]. These unusual properties have led to the hypothesis that these methyltransferases may be involved in the metabolism of d a m a g e d proteins [4.5]. Another class of ir, ethyltransferases (class l i d catalyze the metbylation Cortespondenc~ to: R. I~liveau. D~partement de chimie-biochimie. Uni','ersit6 dy Qu6bec ~ MontrdaL C.P. 8888. Succursalc "A". Mow tribal. Out,bee IDC 3P8. Canada. Abbreviations: AdoMet. S-adenos¥l-i.-mcthionine: AdoHcy. Sadcnosyl-t-homo~'st¢in~: ApplNHh~.adenyl-5'-yl imidodiphnsphatc; BBM. brush-border mcrqbrant:s; G-protein. guanine-nucleotidebinding prolein; GTPyS. guanosin¢ 5"-O4y-thioRriphosphate: GDP~OS. guannsine 5°-O4/~-thiokliphosphate: PCMT. protein carbox~'lmethyltransfe~ase; 16-BAC. benz~'ldimethyl-n-hexadeq,'lammonium chloride.
of the a-cart~'~.~.jl group of C-terminal residues located in a consens::s sequence ( C A A X ) of yeast and mammalian pro:~.ins. The pro!ein substrates of this class include t~c small GTP-binding protein ras [6-8], the nuc:-_ar |amin B [9], the -~- subonit of some trimeric G - p r m e i n s [10L and c G M P phnsphodieslerase [11]. The data obtained so far with these substrates have suggested that metbylation occurs after isoprenylation of a cysteine residue 4 amino acids from the C-terminns and proteolytic removal of the last 3 amino acids [ 12,13]. The nature of the a-carboxyl-terminal-metbylated proteins and the fact that this methylation is in some cases enhanced by guanine nucleotides [14,15], suggest an involvement of this methylation reaction in hormonal transduction. However, no studies have focnsed so far on a similar regulatory role of d a s s - l l catalyzed protein metbylation. In the kidney, the cells lining the proximal tubule are the site of reabsorption of important metabolites filtered by the glomerolns. These cells are characterized by a high degree of asymmetry with segregation of sodium-coupled active-transport systems in the brush-border membrane [16], while hormone receptors and bormone-sensitive adenylyl t3'clases are concentrated at the basolateral pole, in close contact with the circulation [17]. W e have recently reported the pres-
encc of two distinct class !1 PCMT in the kidney cortex, a soluble and a brush-border-membrane-associated PCMT [18]. In the present work, we examined the effects of guanine nucieotides on protein methylation occurring in the cytosolic fraction and in brush-border membranes of the kidney cortex. We demonstrate that guanine nucleotides enhance the activity of the soluble class !1 PCMT.
60 ~
,--r---
Materials and Meflmds All experiments were performed on adult SpragueDawley male raLs weighing 300-350 g (Charles River). Tissues were homogenized in 5 vols. of 250 mM sucrose-5 mM Tris-HCI (pH 7.5) using a Potter-Elvehjem tissue grinder. The homogenates were centrifuged at l l 0 0 0 0 x g for 60 min and the resulting supernatants used as cytosols. Brush-border membranes from the kidney cortex were isolated by a magnesium precipitation method [19]. The medium used for metbylation reactions contained, in a final volume of 30/zl : 100 mM Hepes-Tris buffer (pH 7.5), 2 p.M (2/zCi) [methyl-3H]S-adenosyi L-rnethionine (69-73 Ci/mmol, Dupont-New England Nuclear) and 100 p.g of proteins. The reaction mixture was incubated at 3TC for 60 rain or the times indicated in the figures. For GTP-dcpcndent methylation, GTP,/S (10 to 200 p.M) was added to the medium. Total incorporation of radioactivity into protein methyl esters was measured with a methanol vapor diffusion assay [20]. Kinetic parameters were determined by non-linear regression analysis of the data using a computer graphics program (Kaleidagraph). Methylated proteins were separated by 16-BAC acid electrophoresis using 7.5% acrylamide gels followed by fluorograpby with i M sodium salicylat¢, as described previously [18]. Control experiments have previously shown that the total amount of radioactivity present in the acidic gel is in the form of basc-scusith'c methyl esters. No significant radioactivity is detected following SDS-PAGE at pH 8.8, the methyl estcrs being hydrolyzed at this alkaline pH [18]~
10
t~~" ~
8 0
P
~
Incubationtime(min) Fig. I. Time dependence of endogenouscarboxyl mcthylation of kidney c~o.-,olicproteins.Cytosolicproteins(100 pg) were incubated in 100 mM Hepes/Tris(pH 7.5) with 2 pM [3HlAdoMct(2 ~.Ci) in the absence (Q) or presence of 100 pM App[NH.]p (11). GDP~S (o), GTP',/S(o) or GTP-tS+ 500 pM AdoHcy( A). Methylationwas quantified as described elsewhere[20]. Values are means+_S.D,of three experimentsdone in triplicate. Inset. Initial velocitiesof protein carboxyl mcthylation for contrd (1:3) and GTPTS-trcatcd (e) cytosols. (100 pM) also resulted in a stimulation of the endogenous methylation, but to a smaller extent, while App[NH]p, a non-hydrolysable analog of ATP, was without effect (Fig. 1). Both basal and GTPyS-stimulated methylation activities were almost completely abolished by the addition of 500/zM AdoHcy, a wellknown inhibitor of methyltrausferase-catalyzed reactions. The effect of GTPTS was strongly concentration-dependent. As shown in Fig. 2, addition of GTP~/S to the incubation medium resulted in a progressive stimula-
~0o
:~'~
is0.
Results
Time-, concentration-, and AdoMet-dependence GTPyS-stimulated protein methflation
of
Incubation of the soluble fraction isolated from kidney cortex with the radioactive methyl donor [3H]AdoMet resulted in a time-dependent methylation of endogenous proteins (Fig. i). The addition of 100 lzM GTPyS, a non-hydrolysable analog of GTP, increased markedly both the rate and extent of methylation, with an average 3-fold stimulation of the initial rate of incorporation of labelled methyl groups (33 vs. 10 pmol/mg per h) (Fig. i, inset). Addition of GDP/3S
o'
. ~ . , , ~. 50
i00
. , tso
2oo
Nucleotides (I.tM) Fig Z Concentration depcndcocc and specificity of nuclontides on carboxyl methylation ol kidney-corlex cylosolie proteins. Cytosolic proteins (I00 p.g) were incubated in I00 mM Hepes/Tris (~'H 7.5) with 2 p M [~H]AdoMet (2 pCi) in the presence of increasing concentrations of App[Nll]p ([3), GDP~S (o) or GTPTS (o) for 60 rain at 37*(::. Control value refers to methyhtion measured in the absence of nucleotides (9.8 pmol/mg per h). Means _+S.D. of three experiments done in triplicate are shown.
152 TABLE I Effect of GIPyS on rite etulogou,ui u#uhle PC.'tfT activity of t~ti~nL~ tusues
.
i
Preparatton of the soluble e~lraCts rfld determination of I ~ , l T act~.'ilywere I~rformed as ~k'-~'ribet:in "Materials and Methods'. A reprc~cnZatk¢ cxperlmenl :~ilh lrip.t-~:alcdeterminations is sh~rwn.
20b 150 100
Seluble cslracl
O
I~.?,fF a,_-lk-il) Ipmol/mg per h~
control
5O 0
. 0
5
10
15 20 AdoMet (pM)
25
,
30
Fig. 3. Effca of GTI'?S on the AdoMet dependence ot kidno'
