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Dissociation of the hepatic insulin receptor favours its phosphorylation by casein kinase 2
Volume 2t~3, number 1, i~-!8
FEBS 09715
May 199t
|991 Federation of European Biochemlc¢,l lgo¢letJ,~s 0014~'/9~1191~$L~0 ADONIS 0014['19~91004,1),M
Dissociation of the hepatic insulin receptor favours its phosphorylation by casein kinase 2 R a m o n Trujillo. M a r t a Jose, M a r i a Pinna, Elena M o l i n a and Emilio Itarte Depart¢~ment de Bioqubntea i Biologia Mol¢¢tdar. Faeultat de Ci~nci¢~. Unlversitat Aut~t~oma de Baecelona,
E.08193 Bellaterra (Barcelona), Spain Re~ived 7 March 1991 The ap heterodimerie form of untreated hepatic insulin receptor was a substrate for casein kina~: 2, whereas the a~//a heterotetramer was not. On the contrary, autophosphorylation was detected only in the heterotetram©r, Dissociation of the r¢~:eptor by treatment with dithiothreitol decreased its autophosphorylation bat favoured phosphorylation of ill fl.subunh by casein kiuas¢ 2.
Insulin receptor; Casein klnase 2; Protein phosphorylation; Rat liver
1. INTRODUCTION Insulin receptor is a glycoprotein composed of extracellular a,-subunits, which contains the insulin binding site, and transmembrane #-subunits, which possess the insulin stimulated tyrosine kinase activity [!]. In addition to its autophosphorylation on tyrosine, the B-subuait of the insulin receptor has been reported to be a substrate in vitro for serine/threonine kinases such as the cyclic AMP-dependent protein kinase [2], protein kinase C [3], casein kinase 2 [41, and a protein kinase associated to the receptor [5]. Phosphorylation of insulin receptor on serine/threonine residues is well documented to occur in rive [6-8], and causes a reduction in the tyrosine kinase activity of the receptor. The receptor extracted from human placenta and rat hepatocytes or hepatoma cells presents several oligomeric forms which correspond to different aggregation states of the co- and #.subunits [9-11]. Dissociation of the c~zflz heterotetramer into a,fl heterodimer causes a loss of the l!igh affinity insulin binding cc3mponent inherent to the heterotetramer [10]. Furthermore, the a'~z heterotetramer is the form autophosphorylated in rive [1 1], and interaction bet. ween two c~# heterodimers is necessary to show insulindependent autophosphorylation in vitro [12], The aim of the present work was to investigate if the oligomeric state of the insulin receptor also influenced Correspondence address: E, Itarte, Departament de Bioquimica i Biologia Molecular, Facultat de Ci~ncies, Universitat Autbnoma de Barcelona, Campus de Bel!aterra, E-08193 Bellaterra IBarcelona), Spain
A bbreviatio~2s: DTT. dithiothreitol; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate
Published by Elsevier Science Publishers B V.
its phosphorylation on serine/threonine residues by casein kinase 2. 2. EXPERIMENTAL Insulin was from Eli Lilly. ~:Sl-insulin B-26 was from Amersham. Casein kinase 2 was purified from rat liver cytosol [4], Liver m~mbranes were prepared from 5.week-old female rats by differential centrifugatioa, Membrane proteins were extracted by solubilization with 2olo Triton X-100 and subjected to chromatogra.nhy on wheat germ leetin.agaros¢, The retained proteins were eluted by washing the cOlumn with 0,3 M N.acetyl-D-glucosamine as in [4]. Autophosphorylation of insulin receptor and its phosphorylation by casein kinase 2 was carried out as described previously [41. In all cases, samples were analyzed by 0,1°70 SDS/5% PAGE under nonreducing conditions or by 0,1070 SDS/7.5% PAGE under reducing conditions, in some experiments, the samples were also analyzed by non-denaturing polyacrylamide gel electrophoresis using a linear gra. dient of 2-15% acrylamide, according to [13l. The gels were dried, autoradiographed and the intensity of the bands was quantitated by densitometry. Cross.linking experiments with 1,25 nM 12Sl-insulin, in the absence or in the presence of I~M unlabeled insulin, were carried out using 1 mM disuecinimidyl suberate as in [14],
3. RESULTS 3.1. Analysis o f the phosphorylated insulin receptor: oligomeric structure o f insulin receptor species Cross-linking experiments with t25I-insulin of rat liver insulin receptor preparations purified through lectin-agarose showed the existence of two receptor species with apparent Mr of 300 and 200 kDa (Fig. 1A). This pattern is sinaJ!ar to that observed by other groups in rat liver and human placenta [9,101 where they have been shown to correspond to the azd2 and co# species. As expected from previous data [15], the amount of
15
May 1991
FEBS LETTERS
Vohime 283, I'~t:tnber I A
B -300
-200
Filg. I, O l i B o m e r i c
1251- INSULIN
+
+
I
INSULIN
--
,t,
I
---
" ÷
÷ -
+ ÷
CK-2 iNSULIN
-÷
--
÷ -
÷ -
[),..32 p ] G T P D,-32¢1 Arp
t i l r U ¢ l U r e o f Jll,lulin rer~eiHor .spe¢ie.s. ( A } C r o s s , l i n k i n i l
÷
whh
I"~l-in~ulin
[ 1 , 2 I ~ M ) , e i l h ¢ r ili Ille itbleli~;c o r i n t | t c pre.sence ¢ff
I t,M unhlbeled ilt~ulin, w.'l~ carried out tlsin~ I nhM di,~u¢¢ininlJd)'l ~uberate tinder standard ¢ondilionrl, S~nlple;i ~s,ere eleglropJlOre~i~,d, dried lind autoradiol]ruphed, (B) Insulin receptor autopho~phorylatiol~ (lanes I and 2) and phasphorylalion by ¢a~lein kina~e 2 [CR.2) (lane~ ~ nnd 4), The partially purified insulin receptor wa.~ incuba|ed either in the absem:e or in Ihe prexenee of 0,1 j~hl in~iulin prior to ilho~phorylalion. S a m p l ~ were almlyzed by SDS.PAGE under non.redu¢inlg conditions, Tile autoradiot~ram of the l/el is shown, The apparen~ M, of the band,~ are indicated in RD.~I o n tile side of 111¢ attloradiotlram
I-~Sl-insulin bound to the c~/3 species was very small as compared with that observed with the tv~: heterotetramer. The two receptor species showed different abilities to autophosphorylate and to serve as substrates for casein kinase 2 (Fig, tB). Only the larger M~ species showed autophosphorylation whereas phosphorylation by casein kinase 2 seemed to be restricted to the lower Mr species, The different oligomeric structure of the receptor subjected to autophosphorylation or to phosphorylation by casein kinase 2 was also observed
when analyzed by electrophoresis in non-denaturing gels (Fig, 2A), Phosphate incorporation due to autophosphorylation was clearly stimulated by insulin whereas phosphorylation by casein kinase 2 was not. Both the high and low M~ species gave rise to bands that migrate with M~ in the range of 95-103 kDa when ana!yzed in SDS-PAGE under reducing conditions (Fig. 2B). The presence of the 95-103 kDa doublet may arise from different levels of phosphorylation of the g-subunit, as has been reported to occur with insulin receptor from H-35 hepatoma cells [161.
B
A
t
300
oK-2
'NSULIN I-)>-3~ P'I G T P - r- l ' - ~ P ] ATP i
-
103 95
1
2
3
4
2
3
4
-
-
+
÷
-
•
+
+
"t" -'P-
-÷ --
4" ÷ =
+ ,P
* -
+ +
Figl 2, Analysis of phosphorylated insulin receptor under non-denaturing conditions, (A) Insulin receptor was autophosphorylated either in the aDs÷nee or in the presence of 75 nM insulin, or phosphorylated by casein kinase 2 (CK-2) using either 0.025 mM [7-'~zP]ATP or 0.025 mM [7-'~2P]GTP and analyzed in non
16
May 1991
FEB$ LETTERS
Volume 2~t), number I
incorporation decreased as the concentration of DTT in the phosphorylation assay increased. In contrast, tt~e presence of DTT increased considerably the phosphorylation of the receptor by casein kinase 2, but this increase was more prominent at DTT concentrations that give rise to the appearance of free#.subunlts, In addition to the 95-1021 kDa band, treatment with DTT prior to incubation with casein kinase 2 led to a marked increase in the phospl~orylation of a !75-300 kDa band, This band was not observed in samples that were first phosphorylated with casein kinase 2 and then treated with similar amounts o f DTT
3.2. lrtfluen¢'e of D T T on insulin receptor phosphor/I. orion Reduction with D°TT causes the cleavat/,e or' the heterotetrameric form o f insulin receptor into , ~ dimers 1101. Since the form o f the receptor which i~ preferentially pllosphorylat~d by casein kinase 2 corresponded to the ¢~# dimer, we assayed the influence of prior treatment of insulin receptor preparations with DTT on its phosphor~/lation by this klnase. As shown in Fig, 3A, DTT-treatment led to the appearance of autophosphory|ated c¢# heterodimers and free #-subunlts, However, the level of total ~=P-
A
-300
-200
103
3 95
I'D T T~ cK-=
(mMI
.
.
.
O
0.1
.
.
l:p-32p'Ior+,
-
Ep.3gp3
4,
A TP
1
4,
10
O
0.1
I
10
+
+
+
+
+
+
+
-
-
+
+
4'
.
.
.
.
B
103 "1 95
[DTT~ (raM) CK-2 [ y_32p] GTP
[ v-"2P3 ArP INSULIN App. M r
(kDa)
0 --
10 -
+ --~l" 300
+ -
,t95 ~
0 ,4. 't"
10 ÷ +.
10 4+
-- 4' -- ÷ 200 300
-
4. 95 e
Fig, 3, Effect of DTT on insulin receptor pl~osphorylation, (A) Insulin receptor was treated with different concentrations of DT~i" in the presence of 75 nM insulin prior to phosphorylation by casein kinase 2 (CK-2) and analysis by SDS-PAGE under non.reducing conditions, (B) The bands from a gel similar to that shown in (A) were extracted, treated with 6'-mereaptoethanol and analyzed in SDS-PAGE undel' reducing conditions. The app, Mr indicated at the bottom o f the figure refers to that determined for each band under the conditions shown in (A). The 95-103 kDa doublet was referred as 95*, The apparent M, of the bands are indicated in kDa on the side of the autoradiogram.
17
Volume 283. number I
FEBS LETTERS
(data not shown). This rules out that its presence is due
to any effect of DTT facilitating its entrance in the SDS-PAGE gels. The apparent M~ of the high M~ band phosphorylated by casein kinase 2 did not v a r y when analyzed in SDS-PAGE (Fig, 3B),
under reducing conditions
May 199I
Acknowlcd~tcmcmx: Tl~h work w~ ~upporled tn p~tr~ t~y Gnm|~ $C1,02.~:t,C from Ih¢ F.EC and PB 1~6.0374from I)GiC'YT (Sp~io), REFERENCES i l l R ~ n . O,M, (i9~7) Science 237, i4~2~14~I~. 121 Roch, R.R. and I|¢~utl~in. J. (19~7} Diab~le~ ]6. 123=126, 131 Bolhtu, el.E,, Roll~, R,A,. BeltudohL J., ModdpRo~en, D, and Ko~hland. D,I~, (It~}~6) Pro¢. N~II, Acad, Sel, USA a3. ~M22=$#24,
4. D I S C U S S I O N
14l Granitic, J,, P~¢c~, M, and I~ane, E. (19#8) FI~BS LeH. 2.12,
The data obtained on the phosphorylation o f insulin receptor by casein kinase 2 suggest that the phosphorylatlon sites for this kinase are hindered in the ~'..#2 heterotetramer and become accessible upon its dissociation into ~# heterodimer, and especially in the free #-subunits, This is in contrast to the autophosphorylation o f the receptor which took place m a i n l y in ~he < ~ 2 heterotctramer, as already reported
l$) Lewis, R.F~., Perry Wu, G,, Ma~Domtld. R,C. and Czech. P,M. (1990) J. Btol, Chem, 265, 947=9~4, [6l Kasulla, M., Zlck, Y,, Blith, D,L., Karlsson, F,A., H~ring, H,U, and Kahn, C,R, (1982) J. Biol, Chem. 257, 9891~9894. [?) Parqh D.T,. Slmrma, B,R,, Sh~tfer. J.A,. Whi|e, M F. and Ketttn, R, (1985) J. Biol, Cl~cm. 260, 7131~7136. [8] Takayama, S,, While. M.F, and Knhn. C,R. (1988} J. Biol. Chem, 263, 3440=3447,
by other groups [i 1,12,15]. This fact is interesting since the c~,0 heterodimer has been shown to represent an inactive state of the receptor, whereas the heterotetramer would correspond to the active receptor [15]. The possible physiological meaning of this different susceptibility to autophosphorylation and to phosphorylation by casein kinase 2 remains to be elucidated. NevertheLess, our data indicate that the inactivation of insulin receptor by dissociation into o~.~ heterodimers is accompanied by strong conformational changes in the #-subunit and reinforce the idea that changes in the oligomeric state o f the insulin receptor have a strong influence on its properties.
18
130= 134.
[9] Max.~agu~,J, and Czech. M,P. (1980) Di,'tbeees29, 945-947, [IO] BOni.Sehnetxler,M,, Scott, W,. Wautlh, S,M, ~DiBella, E. and Pilch P,F, (1087) J, Biol, Chem. 262, 8395~8401. (11] Le Marchand.Bru~tel, Y,. Ballotli, R,, Grc~,meaux,T,. T~mi. J.F,, Brandenburih D. anti Van ObherBIlen. E, (1989) J, Biol. Chem, 264, 21316=2132l. (12] Boni.Schnetzler, M.. Kali~ian, A,, DelVecchio. R, and Pilch, P. (1988) J, Biol, Chem. 263, 6822-6828, [13] Kubar. J, and Van Obbergh~n, E. (1989) Bio¢ttemistry 28, 1086-1093, [14] Pilch, P.F. ;,nd Czech, M,P. (19~0) J Biol. Chem, 255,
1722-1731. (15) FI6rke, R,R,. Klein, H,W. and Reinaucr. H. (1990) Eur. J, Btochem, 191, 4?3-482, [16] Tashiro.Hashimo|o, Y,, Tobe, K,, Koshio, O., lzumi, T., Tak:lku, F,, Akanuma, Y, and Kasuga, M, 0989) J, Biol. Chem, 264, 6879-6885,
FEBS 09715
May 199t
|991 Federation of European Biochemlc¢,l lgo¢letJ,~s 0014~'/9~1191~$L~0 ADONIS 0014['19~91004,1),M
Dissociation of the hepatic insulin receptor favours its phosphorylation by casein kinase 2 R a m o n Trujillo. M a r t a Jose, M a r i a Pinna, Elena M o l i n a and Emilio Itarte Depart¢~ment de Bioqubntea i Biologia Mol¢¢tdar. Faeultat de Ci~nci¢~. Unlversitat Aut~t~oma de Baecelona,
E.08193 Bellaterra (Barcelona), Spain Re~ived 7 March 1991 The ap heterodimerie form of untreated hepatic insulin receptor was a substrate for casein kina~: 2, whereas the a~//a heterotetramer was not. On the contrary, autophosphorylation was detected only in the heterotetram©r, Dissociation of the r¢~:eptor by treatment with dithiothreitol decreased its autophosphorylation bat favoured phosphorylation of ill fl.subunh by casein kiuas¢ 2.
Insulin receptor; Casein klnase 2; Protein phosphorylation; Rat liver
1. INTRODUCTION Insulin receptor is a glycoprotein composed of extracellular a,-subunits, which contains the insulin binding site, and transmembrane #-subunits, which possess the insulin stimulated tyrosine kinase activity [!]. In addition to its autophosphorylation on tyrosine, the B-subuait of the insulin receptor has been reported to be a substrate in vitro for serine/threonine kinases such as the cyclic AMP-dependent protein kinase [2], protein kinase C [3], casein kinase 2 [41, and a protein kinase associated to the receptor [5]. Phosphorylation of insulin receptor on serine/threonine residues is well documented to occur in rive [6-8], and causes a reduction in the tyrosine kinase activity of the receptor. The receptor extracted from human placenta and rat hepatocytes or hepatoma cells presents several oligomeric forms which correspond to different aggregation states of the co- and #.subunits [9-11]. Dissociation of the c~zflz heterotetramer into a,fl heterodimer causes a loss of the l!igh affinity insulin binding cc3mponent inherent to the heterotetramer [10]. Furthermore, the a'~z heterotetramer is the form autophosphorylated in rive [1 1], and interaction bet. ween two c~# heterodimers is necessary to show insulindependent autophosphorylation in vitro [12], The aim of the present work was to investigate if the oligomeric state of the insulin receptor also influenced Correspondence address: E, Itarte, Departament de Bioquimica i Biologia Molecular, Facultat de Ci~ncies, Universitat Autbnoma de Barcelona, Campus de Bel!aterra, E-08193 Bellaterra IBarcelona), Spain
A bbreviatio~2s: DTT. dithiothreitol; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate
Published by Elsevier Science Publishers B V.
its phosphorylation on serine/threonine residues by casein kinase 2. 2. EXPERIMENTAL Insulin was from Eli Lilly. ~:Sl-insulin B-26 was from Amersham. Casein kinase 2 was purified from rat liver cytosol [4], Liver m~mbranes were prepared from 5.week-old female rats by differential centrifugatioa, Membrane proteins were extracted by solubilization with 2olo Triton X-100 and subjected to chromatogra.nhy on wheat germ leetin.agaros¢, The retained proteins were eluted by washing the cOlumn with 0,3 M N.acetyl-D-glucosamine as in [4]. Autophosphorylation of insulin receptor and its phosphorylation by casein kinase 2 was carried out as described previously [41. In all cases, samples were analyzed by 0,1°70 SDS/5% PAGE under nonreducing conditions or by 0,1070 SDS/7.5% PAGE under reducing conditions, in some experiments, the samples were also analyzed by non-denaturing polyacrylamide gel electrophoresis using a linear gra. dient of 2-15% acrylamide, according to [13l. The gels were dried, autoradiographed and the intensity of the bands was quantitated by densitometry. Cross.linking experiments with 1,25 nM 12Sl-insulin, in the absence or in the presence of I~M unlabeled insulin, were carried out using 1 mM disuecinimidyl suberate as in [14],
3. RESULTS 3.1. Analysis o f the phosphorylated insulin receptor: oligomeric structure o f insulin receptor species Cross-linking experiments with t25I-insulin of rat liver insulin receptor preparations purified through lectin-agarose showed the existence of two receptor species with apparent Mr of 300 and 200 kDa (Fig. 1A). This pattern is sinaJ!ar to that observed by other groups in rat liver and human placenta [9,101 where they have been shown to correspond to the azd2 and co# species. As expected from previous data [15], the amount of
15
May 1991
FEBS LETTERS
Vohime 283, I'~t:tnber I A
B -300
-200
Filg. I, O l i B o m e r i c
1251- INSULIN
+
+
I
INSULIN
--
,t,
I
---
" ÷
÷ -
+ ÷
CK-2 iNSULIN
-÷
--
÷ -
÷ -
[),..32 p ] G T P D,-32¢1 Arp
t i l r U ¢ l U r e o f Jll,lulin rer~eiHor .spe¢ie.s. ( A } C r o s s , l i n k i n i l
÷
whh
I"~l-in~ulin
[ 1 , 2 I ~ M ) , e i l h ¢ r ili Ille itbleli~;c o r i n t | t c pre.sence ¢ff
I t,M unhlbeled ilt~ulin, w.'l~ carried out tlsin~ I nhM di,~u¢¢ininlJd)'l ~uberate tinder standard ¢ondilionrl, S~nlple;i ~s,ere eleglropJlOre~i~,d, dried lind autoradiol]ruphed, (B) Insulin receptor autopho~phorylatiol~ (lanes I and 2) and phasphorylalion by ¢a~lein kina~e 2 [CR.2) (lane~ ~ nnd 4), The partially purified insulin receptor wa.~ incuba|ed either in the absem:e or in Ihe prexenee of 0,1 j~hl in~iulin prior to ilho~phorylalion. S a m p l ~ were almlyzed by SDS.PAGE under non.redu¢inlg conditions, Tile autoradiot~ram of the l/el is shown, The apparen~ M, of the band,~ are indicated in RD.~I o n tile side of 111¢ attloradiotlram
I-~Sl-insulin bound to the c~/3 species was very small as compared with that observed with the tv~: heterotetramer. The two receptor species showed different abilities to autophosphorylate and to serve as substrates for casein kinase 2 (Fig, tB). Only the larger M~ species showed autophosphorylation whereas phosphorylation by casein kinase 2 seemed to be restricted to the lower Mr species, The different oligomeric structure of the receptor subjected to autophosphorylation or to phosphorylation by casein kinase 2 was also observed
when analyzed by electrophoresis in non-denaturing gels (Fig, 2A), Phosphate incorporation due to autophosphorylation was clearly stimulated by insulin whereas phosphorylation by casein kinase 2 was not. Both the high and low M~ species gave rise to bands that migrate with M~ in the range of 95-103 kDa when ana!yzed in SDS-PAGE under reducing conditions (Fig. 2B). The presence of the 95-103 kDa doublet may arise from different levels of phosphorylation of the g-subunit, as has been reported to occur with insulin receptor from H-35 hepatoma cells [161.
B
A
t
300
oK-2
'NSULIN I-)>-3~ P'I G T P - r- l ' - ~ P ] ATP i
-
103 95
1
2
3
4
2
3
4
-
-
+
÷
-
•
+
+
"t" -'P-
-÷ --
4" ÷ =
+ ,P
* -
+ +
Figl 2, Analysis of phosphorylated insulin receptor under non-denaturing conditions, (A) Insulin receptor was autophosphorylated either in the aDs÷nee or in the presence of 75 nM insulin, or phosphorylated by casein kinase 2 (CK-2) using either 0.025 mM [7-'~zP]ATP or 0.025 mM [7-'~2P]GTP and analyzed in non
16
May 1991
FEB$ LETTERS
Volume 2~t), number I
incorporation decreased as the concentration of DTT in the phosphorylation assay increased. In contrast, tt~e presence of DTT increased considerably the phosphorylation of the receptor by casein kinase 2, but this increase was more prominent at DTT concentrations that give rise to the appearance of free#.subunlts, In addition to the 95-1021 kDa band, treatment with DTT prior to incubation with casein kinase 2 led to a marked increase in the phospl~orylation of a !75-300 kDa band, This band was not observed in samples that were first phosphorylated with casein kinase 2 and then treated with similar amounts o f DTT
3.2. lrtfluen¢'e of D T T on insulin receptor phosphor/I. orion Reduction with D°TT causes the cleavat/,e or' the heterotetrameric form o f insulin receptor into , ~ dimers 1101. Since the form o f the receptor which i~ preferentially pllosphorylat~d by casein kinase 2 corresponded to the ¢~# dimer, we assayed the influence of prior treatment of insulin receptor preparations with DTT on its phosphor~/lation by this klnase. As shown in Fig, 3A, DTT-treatment led to the appearance of autophosphory|ated c¢# heterodimers and free #-subunlts, However, the level of total ~=P-
A
-300
-200
103
3 95
I'D T T~ cK-=
(mMI
.
.
.
O
0.1
.
.
l:p-32p'Ior+,
-
Ep.3gp3
4,
A TP
1
4,
10
O
0.1
I
10
+
+
+
+
+
+
+
-
-
+
+
4'
.
.
.
.
B
103 "1 95
[DTT~ (raM) CK-2 [ y_32p] GTP
[ v-"2P3 ArP INSULIN App. M r
(kDa)
0 --
10 -
+ --~l" 300
+ -
,t95 ~
0 ,4. 't"
10 ÷ +.
10 4+
-- 4' -- ÷ 200 300
-
4. 95 e
Fig, 3, Effect of DTT on insulin receptor pl~osphorylation, (A) Insulin receptor was treated with different concentrations of DT~i" in the presence of 75 nM insulin prior to phosphorylation by casein kinase 2 (CK-2) and analysis by SDS-PAGE under non.reducing conditions, (B) The bands from a gel similar to that shown in (A) were extracted, treated with 6'-mereaptoethanol and analyzed in SDS-PAGE undel' reducing conditions. The app, Mr indicated at the bottom o f the figure refers to that determined for each band under the conditions shown in (A). The 95-103 kDa doublet was referred as 95*, The apparent M, of the bands are indicated in kDa on the side of the autoradiogram.
17
Volume 283. number I
FEBS LETTERS
(data not shown). This rules out that its presence is due
to any effect of DTT facilitating its entrance in the SDS-PAGE gels. The apparent M~ of the high M~ band phosphorylated by casein kinase 2 did not v a r y when analyzed in SDS-PAGE (Fig, 3B),
under reducing conditions
May 199I
Acknowlcd~tcmcmx: Tl~h work w~ ~upporled tn p~tr~ t~y Gnm|~ $C1,02.~:t,C from Ih¢ F.EC and PB 1~6.0374from I)GiC'YT (Sp~io), REFERENCES i l l R ~ n . O,M, (i9~7) Science 237, i4~2~14~I~. 121 Roch, R.R. and I|¢~utl~in. J. (19~7} Diab~le~ ]6. 123=126, 131 Bolhtu, el.E,, Roll~, R,A,. BeltudohL J., ModdpRo~en, D, and Ko~hland. D,I~, (It~}~6) Pro¢. N~II, Acad, Sel, USA a3. ~M22=$#24,
4. D I S C U S S I O N
14l Granitic, J,, P~¢c~, M, and I~ane, E. (19#8) FI~BS LeH. 2.12,
The data obtained on the phosphorylation o f insulin receptor by casein kinase 2 suggest that the phosphorylatlon sites for this kinase are hindered in the ~'..#2 heterotetramer and become accessible upon its dissociation into ~# heterodimer, and especially in the free #-subunits, This is in contrast to the autophosphorylation o f the receptor which took place m a i n l y in ~he < ~ 2 heterotctramer, as already reported
l$) Lewis, R.F~., Perry Wu, G,, Ma~Domtld. R,C. and Czech. P,M. (1990) J. Btol, Chem, 265, 947=9~4, [6l Kasulla, M., Zlck, Y,, Blith, D,L., Karlsson, F,A., H~ring, H,U, and Kahn, C,R, (1982) J. Biol, Chem. 257, 9891~9894. [?) Parqh D.T,. Slmrma, B,R,, Sh~tfer. J.A,. Whi|e, M F. and Ketttn, R, (1985) J. Biol, Cl~cm. 260, 7131~7136. [8] Takayama, S,, While. M.F, and Knhn. C,R. (1988} J. Biol. Chem, 263, 3440=3447,
by other groups [i 1,12,15]. This fact is interesting since the c~,0 heterodimer has been shown to represent an inactive state of the receptor, whereas the heterotetramer would correspond to the active receptor [15]. The possible physiological meaning of this different susceptibility to autophosphorylation and to phosphorylation by casein kinase 2 remains to be elucidated. NevertheLess, our data indicate that the inactivation of insulin receptor by dissociation into o~.~ heterodimers is accompanied by strong conformational changes in the #-subunit and reinforce the idea that changes in the oligomeric state o f the insulin receptor have a strong influence on its properties.
18
130= 134.
[9] Max.~agu~,J, and Czech. M,P. (1980) Di,'tbeees29, 945-947, [IO] BOni.Sehnetxler,M,, Scott, W,. Wautlh, S,M, ~DiBella, E. and Pilch P,F, (1087) J, Biol, Chem. 262, 8395~8401. (11] Le Marchand.Bru~tel, Y,. Ballotli, R,, Grc~,meaux,T,. T~mi. J.F,, Brandenburih D. anti Van ObherBIlen. E, (1989) J, Biol. Chem, 264, 21316=2132l. (12] Boni.Schnetzler, M.. Kali~ian, A,, DelVecchio. R, and Pilch, P. (1988) J, Biol, Chem. 263, 6822-6828, [13] Kubar. J, and Van Obbergh~n, E. (1989) Bio¢ttemistry 28, 1086-1093, [14] Pilch, P.F. ;,nd Czech, M,P. (19~0) J Biol. Chem, 255,
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