Volume 0
295.
number
1991 Fcdcmtion
12.3,
of Europcun
146-148
FEBS
Biochemical
Socictics
0014579319
10533
December
199 I
liS3.50
Inhibition of protein tyrosine phosphatase activity by diarnide is reversed by epidermal growth factor in fibroblasts Hugo P. Monteiro”.
Received
Yuri
I6 Scpiember
!vaschenko,
l99i;
revisal
Roland
Fischer
version rrceiveii
and Arnold
23 Ociobcr
Stern
199:
Diamidc (azodicarbosylic acid bis(dimcthyl;tmidc)) inhibits protein tyrosins phosphatasc activity in librobktsts without altering protein lyrosine kinusc nctivity associated with the cpidcrmal growth factor rcccptor. The loss of protein tyrosinc phosphutusauctivity CitusCd by diamidc is rcvcrscd by 2-mcrcapto~tlmnol or cpidcrmal growth factor. EGF
receptor:
Protein
tyrosine
1. INTRODUCTION Epiclermnl growth factor (EGF) binds to its cell surface receptor containing tyrosine kinase activity. The activation of the tyrosine kinase activity by the binding process triggers a cascade of events that transduces a signal from the cell surface to the nucleus [l]. Receptor tyrosine kinases catalyse the phosphorylation of tyrosine residues either on their own polypeptide chain or on numerous intrinsic substrates in the signal pathway 121.Recent evidence suggests that tyrosine kinase phosphorylation could be regulated by a diverse family of enzymes referred to as protein tyrosine phosphatases (PT-Pax) [3,4], whose catalytic activity is dependent on a specific cysteine residue [5]. PTPase activity is found associated with the plasma membrane and/or in the cytosol [6]. Their specific activities are much greater than protein tyrosine kinases .xrd they therefore have thecapacity to regulate the levei of tyrosine phosphorylation of proteins involved in signal transduction pathways in the cell [7].
pl~osplu~~asc: Oxidation-reduction
G 108. I) wus provided by Dr. Francoisc Bcllot (Row Central Rcscurch. King of Prussiu. PA). Affinity-purilicd antiphosphotyrosine untibodics wcrc u gih of Dr. Ben Margolis (NYU Medical Ccntcr. NY). NIH3T3 cells cxprcssing the human EGF rcccptor (HER I4 cells) [8] wcrc grown in Dulbccco’s modilied Euglc’s medium c’ fetal bovine serum. in IOO-mm (DMEM) supplcmcntcd with lOJo plastic culture dishes. Incubations wcrc done in DMEM conmining 20 mM HEPES und I% bovine serum albumin ut 25°C. After incubation. cells wcrc solubilizcd in lysis buffer A (20 mM HEPES, pH 7.5. I SO mM NaCI. 10% g!yfcrol. I% Triton X-100. I .S mM MgC12. I tnM EGTA. I fig/ml aprotmm, I ~.g/ml leupcptin uud I mM phcnylmcthylsulfonyltluoridc). After 30 min incubation 011 ice. lysmcs wcrc spundown for IO min al 4OC us IZ 000 x g. The supwwtunts wcrc collcctcd and scrvcd as the source of PTPase activity. PTPnsc activity was essayed by mixing 100 ~1 ofcdl Iysate, 7 :!~!t!i H s?ispcnsion conlilinitu3 immunoprccipituted rJP]-lubclcd EGF rcc:ptors, prepared csscntially us dcscribcd by Honcger ct al. [8]. Reactions wcrc curried out for 30 min at 37°C with stirring. The supernatant wits rapidly uspirutcd and the rndiolubclcd immunoprccipitatcs were colkctcd and mixed with 3x conccntrutcd SDS gel-loading butI& and resolved on 7.5% SDS polyacryi~tnridc gel clcctrophorcsis. Itnn~unoblot analysis WBS perrormcd cssc~~~hlly ;IS previously dcscribrd [9] cxcep~ that cells wre solubilizcd in Iysis bulkr A. Binding cxpcriments wcrc pctformcd according to Lax Cl al. [9].
3. RESULTS 2. MATERIALS
AND METI-iODS
HER mivl diamidc and incubated for 30 min at 25OC. PTPase activity was inhibited by I mM diamide as shown on SDS-gel electrophoresis (Fig. I, lane 4). Treatment of cells with 83 nM EGF had no apparent effect on PTPase activity (Fig. 1, lane 3). Pre-incubation of cells with 83 nM EGF followed by incubation with 1 mM diamide (Fig. I, lane 5) or pre-incubation of cells with I mM diamide followed by incubation with 83 nM EGF (Fig. 1, lane 6) showed an increase in PTPase activity when compared to I mM diamide (Fig. 1, lane 4), with the increase greatest in the latter situation (Fig. I, lane 6). The addition of 2-mcrcapto~thanol to lysatcs of cells pretreated with I mM diamide (Fig. 2, lane 5) resulted in a partial i4
EGF from mouse submaxillary glauds wus obtained from Toyobo Co. EIcctrophorcsis rcugcnts L... 1’np* from Bio-R;td. Dinmidc ;tr,d ull other chemicals \‘r’erc of rcagcnt grttdc :rnd wcrc from Sigma. A monoclonul antibody specific for the human EGF rcccptor (Immunoglobin
EGF. cpidcrmttl growth fuctor; PTPasc. protcitl tyrosine pltosphutttsc; DMEM, Dttlbccco’s modilicd Eagle’s medium; SDS. sodiunl dodccyl sulfm. Ahbrc~i~rriorrs:
*fwrrarrcrrr
ddrc.s.s:
Funduclo
Hcmoccntro
dc S. Paulo.
S. Pnulo.
Bruzil. CfJJ’f?~/JUJll/LVlrL!ndrhw: Arnold Slcrn, Dcpurtmcnt of Ph;trmttcology. NW York University Medical Ccntcr. 550 First Avcnuc, NW York, NY 10016, USA. Fux: (I) (212) 263 7133.
146
c~!!s
F~K
treat&
with
I
Volume
295. number
FEBS
1.1.3
1
2
3
4
5
reversion of the inhibitory effect of 1 mM diamide (Fig. 2, lane 3) on PTPase activity. Incubation of 1 mM diamide with cell extracts also showed inhibition of PTPase activity (data not shown). The effect of diamide on autophosphorylation of the EGF-receptor as well as binding of EGF to its receptor was investigated. Diamide at 1 mM did not stimulate autophosphorylation of the EGF-receptor as revealed by immunoblot analysis (Fig. 3. lane 3). The addition of EGF to diamide-treated cells [Fig. 3, lanes 4 and 5) showed tyrosine kinase activity similar to EGF (Fig. 3, lane 2). Furthermore, billding of EGF LO its receptor was no: impaired by treatment of cells with diamide (data not shown). 4. DISCUSSION Diamide can inhibit the activity of PTPases which regulate tyrosine kinasc activity of the EGF receptor. This inhibition probably occurs by the oxidation of sulf-
2
3
4
December
199
I
6
Fig. I. Effect ordiamidc on PTPasc activity in HER 14 cells. HER 14 cells were incubatd Ibr 30 min at 25°C in DMEM conlaining 20 mM HEPES and IQ bovine strum albumiu. PTPasc aclivity was assayed in the supcrnatants as dcscribcd in Mawrials and Xlcthods. (Lane I) phosphorylatcd reccplor. 110 Iysatc added; (lane 2) phosphorylatcd receptor plus Iysatc; (lane 3) as 2 with 83 nM EGF: (lane 4) as 2 with I mM diamidc; (lane 5) us 2 wi\h 83 nM EGF iollowed by I mM diamidc added al 5 min; (lane 6) as 2 with I mM diamidc followed by 83 nM EGF added at 25 min.
1
LETTERS
5
.
Fig. 2. Effect of 2-mcrcep~oc\hunol on PTPasc activity in HER I4 cells. Conditions and assay wcrc the same as in Fig. I. (Lane I) phosphorylutcd rcccp[or, no Iysalc added; (lane 2) phnsphorylakd rcccplor plus lysutc; (law 3) as 2 with I mM diamidc; (lane 4) as 2 with 2 mM Znicrcaptoclhanol; (I;lnc 5) ;1s 2 with 1mM diamidc plus 2 mbl ?- nicrcuploclhanol.
Fig. 3. El’fcct of diamide on autophosphorylalion of the EGF-rcccplor in HER I4 cells. Con$tions wcrc the same as in Fig. I. Immunoprccipiiation of the EGF-rcccplor by mAb I08 was followed by an immunoblot analysis oTthc prccipilalcd receptor with anti-phospholyrosinc antibodies. (Lane I) control; (lane 2) 83 nM EGF; (lane 3) I mM diamidc; (lane 4) 83 nX1 EGF Tollowcd by I mM diamidc added al 5 min: (lane 5) I mM diamidc followed by 83 nM EGF added al 25 min.
hydryl groups on the enzyme and is consistent with the evidence that its activity is dependent on specific cysteine residues in their catalytic domains [j]. The finding that 2-mercaptoethanol reverses the inhibition by diamide provides evidence in favor of oxidized sullhydryl groups as being responsible for the loss of PTPasc activity, and suggests that oxidation-reduction processes may be of importance in the regulation of protein phosphorylation in receptor signal pathways. An oxidationreduction ljroccss was rcccntly demonstra!ed for maintaining the activity of a protein tyrosine kinasc in the endoplasmic reticulum of a B lymphocyte cell line [IO]. The attenuation of the diamide-inhibited PTPase activity by EGF indicates that activation of the EGF receptor could have consequences on PTPase activity. The underlying mechanism is not apparent, but EGF must inlluence processes that regulate the reduction of critical sulfhydryl groups affecting PTPase activity. Consistent with this idea is the observation of increased production of reducing equivalents by EGF activation of the hexose monophosphate shunt in rat kidney cells [l I]. That binding of EGF to its receptor was not affected by diamide suggests that the sulfhydryl oxidant modulates PTPase activity with some specificity. An increase in EGF receptor phosphorylation was not observed in cells treated with diamide and EGF. This indicates that turnover of tyrosine kinasc phosphorylation is not a rate-limiting step in the signal transduction pathway of EGF. A similar pattern was observed for the insulin receptor, where phenylarsine oxide, a dithiol complexing agent that inhibits PTPase, did not enhance phosphorylation of the tyrosine kinasc activity of the b-subunit of the insulin receptor, but activated tyrosine phosphorylation of a I5 kDa (~~15) protein in adipocytes [12]. 147
Volume
295,
number
123
FEBS LETTERS
Decen?ber I99 I
Ackr~os,lr,~~ertre,~rs:Supported by National Institutes or Heahh Grant ES.03425. We thank Dr. Joseph Schlcssinger for support of Y.I. and R.F. and for careful reading of the manuscript.
[7] Fischer, E.H., Charbonncau, H. and Tanks, N.K. (1991) Science 253, 40!-406. [8] Honcgger, A.M.: Dull, J.S.. Felder. S., van Obbcrghcn, E., Bcllot, F., Szapary, D., Schmidl, h., Ullrich. A. and Schlessingcr, J. (1987) Cell 51, 199-209.
REFERENCES
[9] Lax, I., Johnson, A., Hawk, R., Sap. J., Bellor, F., Winkler, hl.. Ullrich, A., Vennslrom, B., Schlcssingcr. J. and Givol, D. (1988) Mol. Cell. Biol. 8, 1970-1978.
[I] Ullrich, A. and Schlcssingcr. J. (1990) Cell 61. 203-212. [I?] hlargolis, B., Rhee. Xi.. Feldcr, S.. Mervic, M.. Lyail, R., Levitski, A., Ullrich. A., Silberslein, A. and Schlessingcr, J. (19S9) Cell 57. IIOI-1107. [3] Lau, K.-H.W., Farley, J.R. and Baylink. D.J. (1989) Biochem. J. 257, 23-36. [4] Aiexander. D.R. (19SO) New Biologist 1, 1039-1062. [5] Tonks, X.K., Diltz, C.D. and Fischer, E.M. (1988) J. Biol. Chcm. 263, 6722-6730. [6] Nelson, R.L. and Branton. P.E. (1984) Mol. Cell Biol. 4. 10031016.
145
[iO] Bauskin, A.R., Alkalay. 1. and Ben-Neriah. Y. (1991) Cell 66, 685-696. [I I] Stanton, R.C. and Sciftcr, J.L. (1988) Am. J. Physiol. 254, C267c271. [12] Bernier, M., Laird, D.M. nnd Lane, M.E. Acad. Sci. USA 84, 1844-1848.
(1987) Proc. Nail.
295.
number
1991 Fcdcmtion
12.3,
of Europcun
146-148
FEBS
Biochemical
Socictics
0014579319
10533
December
199 I
liS3.50
Inhibition of protein tyrosine phosphatase activity by diarnide is reversed by epidermal growth factor in fibroblasts Hugo P. Monteiro”.
Received
Yuri
I6 Scpiember
!vaschenko,
l99i;
revisal
Roland
Fischer
version rrceiveii
and Arnold
23 Ociobcr
Stern
199:
Diamidc (azodicarbosylic acid bis(dimcthyl;tmidc)) inhibits protein tyrosins phosphatasc activity in librobktsts without altering protein lyrosine kinusc nctivity associated with the cpidcrmal growth factor rcccptor. The loss of protein tyrosinc phosphutusauctivity CitusCd by diamidc is rcvcrscd by 2-mcrcapto~tlmnol or cpidcrmal growth factor. EGF
receptor:
Protein
tyrosine
1. INTRODUCTION Epiclermnl growth factor (EGF) binds to its cell surface receptor containing tyrosine kinase activity. The activation of the tyrosine kinase activity by the binding process triggers a cascade of events that transduces a signal from the cell surface to the nucleus [l]. Receptor tyrosine kinases catalyse the phosphorylation of tyrosine residues either on their own polypeptide chain or on numerous intrinsic substrates in the signal pathway 121.Recent evidence suggests that tyrosine kinase phosphorylation could be regulated by a diverse family of enzymes referred to as protein tyrosine phosphatases (PT-Pax) [3,4], whose catalytic activity is dependent on a specific cysteine residue [5]. PTPase activity is found associated with the plasma membrane and/or in the cytosol [6]. Their specific activities are much greater than protein tyrosine kinases .xrd they therefore have thecapacity to regulate the levei of tyrosine phosphorylation of proteins involved in signal transduction pathways in the cell [7].
pl~osplu~~asc: Oxidation-reduction
G 108. I) wus provided by Dr. Francoisc Bcllot (Row Central Rcscurch. King of Prussiu. PA). Affinity-purilicd antiphosphotyrosine untibodics wcrc u gih of Dr. Ben Margolis (NYU Medical Ccntcr. NY). NIH3T3 cells cxprcssing the human EGF rcccptor (HER I4 cells) [8] wcrc grown in Dulbccco’s modilied Euglc’s medium c’ fetal bovine serum. in IOO-mm (DMEM) supplcmcntcd with lOJo plastic culture dishes. Incubations wcrc done in DMEM conmining 20 mM HEPES und I% bovine serum albumin ut 25°C. After incubation. cells wcrc solubilizcd in lysis buffer A (20 mM HEPES, pH 7.5. I SO mM NaCI. 10% g!yfcrol. I% Triton X-100. I .S mM MgC12. I tnM EGTA. I fig/ml aprotmm, I ~.g/ml leupcptin uud I mM phcnylmcthylsulfonyltluoridc). After 30 min incubation 011 ice. lysmcs wcrc spundown for IO min al 4OC us IZ 000 x g. The supwwtunts wcrc collcctcd and scrvcd as the source of PTPase activity. PTPnsc activity was essayed by mixing 100 ~1 ofcdl Iysate, 7 :!~!t!i H s?ispcnsion conlilinitu3 immunoprccipituted rJP]-lubclcd EGF rcc:ptors, prepared csscntially us dcscribcd by Honcger ct al. [8]. Reactions wcrc curried out for 30 min at 37°C with stirring. The supernatant wits rapidly uspirutcd and the rndiolubclcd immunoprccipitatcs were colkctcd and mixed with 3x conccntrutcd SDS gel-loading butI& and resolved on 7.5% SDS polyacryi~tnridc gel clcctrophorcsis. Itnn~unoblot analysis WBS perrormcd cssc~~~hlly ;IS previously dcscribrd [9] cxcep~ that cells wre solubilizcd in Iysis bulkr A. Binding cxpcriments wcrc pctformcd according to Lax Cl al. [9].
3. RESULTS 2. MATERIALS
AND METI-iODS
HER mivl diamidc and incubated for 30 min at 25OC. PTPase activity was inhibited by I mM diamide as shown on SDS-gel electrophoresis (Fig. I, lane 4). Treatment of cells with 83 nM EGF had no apparent effect on PTPase activity (Fig. 1, lane 3). Pre-incubation of cells with 83 nM EGF followed by incubation with 1 mM diamide (Fig. I, lane 5) or pre-incubation of cells with I mM diamide followed by incubation with 83 nM EGF (Fig. 1, lane 6) showed an increase in PTPase activity when compared to I mM diamide (Fig. 1, lane 4), with the increase greatest in the latter situation (Fig. I, lane 6). The addition of 2-mcrcapto~thanol to lysatcs of cells pretreated with I mM diamide (Fig. 2, lane 5) resulted in a partial i4
EGF from mouse submaxillary glauds wus obtained from Toyobo Co. EIcctrophorcsis rcugcnts L... 1’np* from Bio-R;td. Dinmidc ;tr,d ull other chemicals \‘r’erc of rcagcnt grttdc :rnd wcrc from Sigma. A monoclonul antibody specific for the human EGF rcccptor (Immunoglobin
EGF. cpidcrmttl growth fuctor; PTPasc. protcitl tyrosine pltosphutttsc; DMEM, Dttlbccco’s modilicd Eagle’s medium; SDS. sodiunl dodccyl sulfm. Ahbrc~i~rriorrs:
*fwrrarrcrrr
ddrc.s.s:
Funduclo
Hcmoccntro
dc S. Paulo.
S. Pnulo.
Bruzil. CfJJ’f?~/JUJll/LVlrL!ndrhw: Arnold Slcrn, Dcpurtmcnt of Ph;trmttcology. NW York University Medical Ccntcr. 550 First Avcnuc, NW York, NY 10016, USA. Fux: (I) (212) 263 7133.
146
c~!!s
F~K
treat&
with
I
Volume
295. number
FEBS
1.1.3
1
2
3
4
5
reversion of the inhibitory effect of 1 mM diamide (Fig. 2, lane 3) on PTPase activity. Incubation of 1 mM diamide with cell extracts also showed inhibition of PTPase activity (data not shown). The effect of diamide on autophosphorylation of the EGF-receptor as well as binding of EGF to its receptor was investigated. Diamide at 1 mM did not stimulate autophosphorylation of the EGF-receptor as revealed by immunoblot analysis (Fig. 3. lane 3). The addition of EGF to diamide-treated cells [Fig. 3, lanes 4 and 5) showed tyrosine kinase activity similar to EGF (Fig. 3, lane 2). Furthermore, billding of EGF LO its receptor was no: impaired by treatment of cells with diamide (data not shown). 4. DISCUSSION Diamide can inhibit the activity of PTPases which regulate tyrosine kinasc activity of the EGF receptor. This inhibition probably occurs by the oxidation of sulf-
2
3
4
December
199
I
6
Fig. I. Effect ordiamidc on PTPasc activity in HER 14 cells. HER 14 cells were incubatd Ibr 30 min at 25°C in DMEM conlaining 20 mM HEPES and IQ bovine strum albumiu. PTPasc aclivity was assayed in the supcrnatants as dcscribcd in Mawrials and Xlcthods. (Lane I) phosphorylatcd reccplor. 110 Iysatc added; (lane 2) phosphorylatcd receptor plus Iysatc; (lane 3) as 2 with 83 nM EGF: (lane 4) as 2 with I mM diamidc; (lane 5) us 2 wi\h 83 nM EGF iollowed by I mM diamidc added al 5 min; (lane 6) as 2 with I mM diamidc followed by 83 nM EGF added at 25 min.
1
LETTERS
5
.
Fig. 2. Effect of 2-mcrcep~oc\hunol on PTPasc activity in HER I4 cells. Conditions and assay wcrc the same as in Fig. I. (Lane I) phosphorylutcd rcccp[or, no Iysalc added; (lane 2) phnsphorylakd rcccplor plus lysutc; (law 3) as 2 with I mM diamidc; (lane 4) as 2 with 2 mM Znicrcaptoclhanol; (I;lnc 5) ;1s 2 with 1mM diamidc plus 2 mbl ?- nicrcuploclhanol.
Fig. 3. El’fcct of diamide on autophosphorylalion of the EGF-rcccplor in HER I4 cells. Con$tions wcrc the same as in Fig. I. Immunoprccipiiation of the EGF-rcccplor by mAb I08 was followed by an immunoblot analysis oTthc prccipilalcd receptor with anti-phospholyrosinc antibodies. (Lane I) control; (lane 2) 83 nM EGF; (lane 3) I mM diamidc; (lane 4) 83 nX1 EGF Tollowcd by I mM diamidc added al 5 min: (lane 5) I mM diamidc followed by 83 nM EGF added al 25 min.
hydryl groups on the enzyme and is consistent with the evidence that its activity is dependent on specific cysteine residues in their catalytic domains [j]. The finding that 2-mercaptoethanol reverses the inhibition by diamide provides evidence in favor of oxidized sullhydryl groups as being responsible for the loss of PTPasc activity, and suggests that oxidation-reduction processes may be of importance in the regulation of protein phosphorylation in receptor signal pathways. An oxidationreduction ljroccss was rcccntly demonstra!ed for maintaining the activity of a protein tyrosine kinasc in the endoplasmic reticulum of a B lymphocyte cell line [IO]. The attenuation of the diamide-inhibited PTPase activity by EGF indicates that activation of the EGF receptor could have consequences on PTPase activity. The underlying mechanism is not apparent, but EGF must inlluence processes that regulate the reduction of critical sulfhydryl groups affecting PTPase activity. Consistent with this idea is the observation of increased production of reducing equivalents by EGF activation of the hexose monophosphate shunt in rat kidney cells [l I]. That binding of EGF to its receptor was not affected by diamide suggests that the sulfhydryl oxidant modulates PTPase activity with some specificity. An increase in EGF receptor phosphorylation was not observed in cells treated with diamide and EGF. This indicates that turnover of tyrosine kinasc phosphorylation is not a rate-limiting step in the signal transduction pathway of EGF. A similar pattern was observed for the insulin receptor, where phenylarsine oxide, a dithiol complexing agent that inhibits PTPase, did not enhance phosphorylation of the tyrosine kinasc activity of the b-subunit of the insulin receptor, but activated tyrosine phosphorylation of a I5 kDa (~~15) protein in adipocytes [12]. 147
Volume
295,
number
123
FEBS LETTERS
Decen?ber I99 I
Ackr~os,lr,~~ertre,~rs:Supported by National Institutes or Heahh Grant ES.03425. We thank Dr. Joseph Schlcssinger for support of Y.I. and R.F. and for careful reading of the manuscript.
[7] Fischer, E.H., Charbonncau, H. and Tanks, N.K. (1991) Science 253, 40!-406. [8] Honcgger, A.M.: Dull, J.S.. Felder. S., van Obbcrghcn, E., Bcllot, F., Szapary, D., Schmidl, h., Ullrich. A. and Schlessingcr, J. (1987) Cell 51, 199-209.
REFERENCES
[9] Lax, I., Johnson, A., Hawk, R., Sap. J., Bellor, F., Winkler, hl.. Ullrich, A., Vennslrom, B., Schlcssingcr. J. and Givol, D. (1988) Mol. Cell. Biol. 8, 1970-1978.
[I] Ullrich, A. and Schlcssingcr. J. (1990) Cell 61. 203-212. [I?] hlargolis, B., Rhee. Xi.. Feldcr, S.. Mervic, M.. Lyail, R., Levitski, A., Ullrich. A., Silberslein, A. and Schlessingcr, J. (19S9) Cell 57. IIOI-1107. [3] Lau, K.-H.W., Farley, J.R. and Baylink. D.J. (1989) Biochem. J. 257, 23-36. [4] Aiexander. D.R. (19SO) New Biologist 1, 1039-1062. [5] Tonks, X.K., Diltz, C.D. and Fischer, E.M. (1988) J. Biol. Chcm. 263, 6722-6730. [6] Nelson, R.L. and Branton. P.E. (1984) Mol. Cell Biol. 4. 10031016.
145
[iO] Bauskin, A.R., Alkalay. 1. and Ben-Neriah. Y. (1991) Cell 66, 685-696. [I I] Stanton, R.C. and Sciftcr, J.L. (1988) Am. J. Physiol. 254, C267c271. [12] Bernier, M., Laird, D.M. nnd Lane, M.E. Acad. Sci. USA 84, 1844-1848.
(1987) Proc. Nail.
