BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1457-l 453

Vol. 189, No. 3, 1992 December 30, 1992

INSULIN

RECEPTOR TYROSINE KINASE AUTO-DEPHOSPHORYLATION

DOMAIN

Philip A. Gruppuso*‘, Joan M . Boylan*, Barry A. Levine’ and Leland Ellis@

*Departments of Pediatrics and Biochemistry, Brown University, Providence, Rhode Island 02903 “School of Biochemistry, University of Birmingham, Edghaston, Birmingham B13 211, UK @Institute of Biosciences and Technology, Texas A&M University, Houston, Texas 77030 Received

October

28,

1992

We have observed dephosphorylation of the soluble, 48 kDa insulin receptor tyrosine kinase domain following its tyrosine autophosphorylation. Dephosphorylation was associatedwith generation of inorganic phosphate,thereby making catalysis by reversal of the kinase reaction unlikely. The kinase domain preparations could not be shown to contain detectable, contaminating protein tyrosine phosphataseactivity. In addition, dephosphorylation was insensitive to protein phosphataseinhibitors. However, it was blocked by the kinase inhibitor staurosporine. These results are consistentwith insulin receptor kinase domain auto-dephosphorylationvia catalysisinvolving the kinase itself. These findings raise the possibility of a novel mechanismfor termination of the insulin receptor signal. 0 1992ncademlc Press,1°C. t

The primary event in insulin signal transduction is activation of tyrosine kinase activity intrinsic to the beta-subunit of the insulin receptor (1,2). This results in betasubunit tyrosine phosphorylation which enables the receptor kinase to phosphorylate exogenoussubstrateson tyrosine. It is therefore plausible that termination of the insulin signal m ight involve receptor dephosphorylation. Reversal of tyrosine phosphorylation has been assignedto a diverse family of enzymestermed protein tyrosine phosphatases ( PTPases;reviewed in ref. 3). Candidate PTPasesactive with the insulin receptor have been identified (4-8). However, a PTPaseresponsible for insulin receptor J To whom correspondenceshould be addressedat: Division of Pediatric Endocrinology and Metabolism, Rhode Island Hospital, 593 Eddy St, Providence, RI 02903. Abbreviations: PTPase,protein tyrosine phosphatase;IRKD, insulin receptor tyrosine kinase domain; RCML, reduced, carboxyamidomethylated and malyelated lysozyme; ~Npp,pnitrophenyl phosphate; NMR, nuclear magnetic resonance.

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0006-29 1X/92 $4.00 Copyright 0 1992 by Academic Press, 1~. All rights of reproduction in arw form rcsrrvnl.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMfvlUNlCPrTlONS

dephosphorylation has not been specifically designated In an attempt to clarify this area, we undertook studies in which a 48 kDa, soluble insulin receptor tyrosine kinase domain (IRKD [9]) was autophosphorylated on tyrosine for use as a PTPasesubstrate (67). In the course of these studies we observed dephosphorylation of the substrate (p-tyr-IRKD) in the absenceof added PTPases. The present report describesstudies which followed this seminal finding. Methods

IRKD was autophosphorylated as described previously (10) in the presenceof 20 pg/ml protamine, 4 m M Mg” and 0. I m M ATP. Phosphorylation in various experiments reached a maximum stoichiometry of 3 to 5 moles of phosphate per mole IRKD. Analysis of radiolabeled pucleotides present in reaction m ixtures was accomplished using poly(ethylene)imine thin layer chromatography (11). Direct measurementsof phosphataseactivities were performed as follows Reduced, carboxyamidomethylated and malyelated lysozyme phosphorylatedon tyrosine (P-Tyr-RCML) and the autophosphorylated IRKD were prepared and used in a phosphate release PTPase assay(12). Substrate (P-Tyr) concentrations were 1 uM and 0.1 uM for PTyr-RCML and P-Tyr-IRKD, respectively. Hydrolysis ofp-nitrophenyl phosphate @-Npp) was measuredcolorometrically at a substrate concentration of 10 m M . After incubation at 30°C for 60 m in, the reaction was stopped by addition of Na,CO, and absorbanceof the sample was measuredat 410 nm. A detergent (Triton X-100) extract of rat liver membranes (prepared as in ref. 12) was used as a source of exogenousPTPaseswhich show activity againstthe autophosphorylated insulin receptor (6) and the autophosphorylated IRKD (7). Results

Our initial observation was that prolonged incubation aimed at maximizing IRKD autophosphorylation actually resulted in decreasedIRKD phosphorylation. A representative time course is shown in Figure 1. We have shown previously that autophosphorylation of the soluble IRKD can be monitored directly by nuclear magnetic resonance(NMR [13]). Under the conditions used for the present experiments, in which ATP was present in excess,autophosphorylation of all five of the known, typical tyrosine phosphorylation sites reached a maximum within approximately 5 m in. Upon further incubation, a different set of temporal changesin the spectrum of the enzyme was observed to evolve more slowly. These changes(Figure 2) corresponded to an increasein the signal intensity of unmodified tyrosine residues whose phosphorylation was detected in the early, faster time course. The phosphorylation/dephosphorylation monitored by NMR was consistentwith that shown in Figure 1. Comparison of the NMR autophosphorylation profiles of the 48 kDa IRKD with that of the 38 kDa which lacks the C-terminal region (13) indicated that dephosphorylation of the carboxy tail tyrosine (residues 1328and 1334in the intact receptor ]I]) occurred in the latter stagesof incubation. 1458

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cpm x 12.5 -

10.0 -

7.5 -

5.0 -

2.5-

0' w

01

60

lncubatlon

Time,

mln

FlGU RF 1. Time cour.w of insulin receptor tyrosine kinase domain (IRKD) phosphotylution and dephosphorylation. Time versus “P content is shown in the graph. I RKD (5 pg) was incubated in 45 pl for 15 min at 30°C with 50 m M HEPES,pH 7.6,0.3 m M EDTA, 4 m M M&II, 2Oug/ml protamine and 0. I mg/ml bovine serum albumin. At time 0, WATP (5 pl, 100 uCi) was added to a final concentration of 0.1 mM. At the designated times, 4 ul of the reaction mixture was removed and added to gel electrophoresis sample buffer. The phosphorylated IRKD was detected as a 48 kDa protein (arrow) following electrophoresis in IO % polyactylamide gels in the presence of dodecyl sulfate. Bands were excised and ‘*P content was measured using Cherenkov counting. ‘*P content of the 48 kDa band is shown in the graph as a function of incubation time. FIGURE 2. ‘H NMR spectra of the aromatic region of the IR KD post autopllospliorylatinn. IRKD (18 mg/ml) was autophosphorylated in the presence of4 m M ATP and 8 m M ‘Mg”. Spectra A to E were obtained (as described previously 1lo]) at 3.5,5,7, 15.5 and 23 minutes, respectively. after obtaining maximal autophosphorylation (I I). Spectra were recorded at 500 MHz using 64 scans (spectra A-C) and 5 12 scans (spectra D. E) at an ambient probe temperature of 300°K. Results show the reversal of changes observed during autophosphorylation (I 3) with a progressive decrease in intensity of tyrosine phosphate and a concomitant increase in unmodified tyrosine signal intensity.

IRKD

dephosphorylation

could be stopped

by addition

of EDTA

in amounts

sufficient to completely chelate the Mg” present during the autophosphorylation reaction (Figure 3). We confirmed our own prior observation (10) that loss of phosphate from the IRKD was accelerated by the addition of ADP (Figure 3A). In contrast, AMP was ineffective.

The effect of ADP raised the possibility that IRKD dephosphorylation

was occurring via reversal of the kinase reaction. To assess the pathway for IRKD dephosphorylation

thin layer chromatography was used to analyze reaction mixtures at

several time points during experiments analogous to that shown in Figure 1. Results (not shown) demonstrated that dephosphorylation was observed beginning at the point at which the ATP had been completely depleted. More importantly, dephosphorylation was associated with the appearance of inorganic phosphate. In separate studies using ‘“C-ADP, no metabolism of ADP was detected. Long-term incubations analyzed by NMR (not shown) contirmed appearance of inorganic phosphate as the reaction product associated with IRKD dephosphorylation These findings favored catalysis of IRKD 1459

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I 10000

I

I 0

5000

32P-Content

in IRKD After

cpm

20 mln lncubatlon

Control 5 m M EDTA 0.5 m M vanadate DMSO control 10 nM Calyculin 10 nM Okadaic

A Acid 10000

0-

32P-Content

in IAKD After

0 32P-Content

cpm

60 min Incubation

5000 in IRKD After

10000

cpm

60 min Incubation

f:iCi II RE 3. Modulation of IRKD dephosphorylation by nucleotides, protein phosphatase inhibitors and protein kinase inhibitors. For panels A through C, the initial incubation was the same as for Figure 1. Following autophosphorylation for 5 min at 3o”C, 5 ~1 of the reaction mixture was removed to tubes containing 1~11of the designated reagents to obtain the final concentrations shown. Dephosphorylation was allowed to proceed for the times given below each panel. Samples were processedfor polyacrylamide gel electrophoresis and the “P content of the 48 kDa IRKD band was determined Panel A: The effect of AMP and ADP on IRKD dephosphorylation was determined following 5 min of autophosphorylation and a subsequent 20 min dephosphorylation period. Panel B: The ability of EDTA, vanadate, and serine/threonine phosphatase inhibitors to retard IRKD dephosphorylation over a 60 min incubation period was studied. Dimethylsulfoxide (0.06 %), the diluent for calyculin A and okadaic acid, was used in a replicate control experiment. Panel C: The ability of staurosporine to inhibit IRKD dephosphorylation over 60 min was compared to that of EDTA. Identical results were obtained in replicate experiments.

dephosphorylation by a hydrolysis reaction with ADP acting as an allosteric activator, rather than as a substrate in the dephosphorylation reaction. These findings were consistent with the presenceof contaminating phosphataseenzymesin the reaction m ixture. To study the possibility that IRKD dephosphorylation was catalyzed by a contaminating phosphatase, we attempted to detect phosphatases in the reaction mixture by direct measurement Comparisonswere made with a rat liver membrane detergent

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TABLE 1. IRKD and phosphatase liver membrane detergent SUBSTRATES

Detergent Extract IRKD Preparation P-Tyr-IRKD calculated

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substrate dephosphorylation by a rat extract versus the IRKD preparation

P-Tyr-RCML (pmol/min/ml)

P-NPP (pmol/min/ml)

2583

267

none detected dephosphorylation from data similar

BIOPHYSICAL

none detected in to

the absence of those obtained

P-Tyr-IRKD (pmol/min/ml) 30 288 added PTPases in Figure 1.

was

extract, a source of PTPases which can dephosphorylate the native insulin receptor and the autophosphorylated IRKD (67). Results (Table 1) showed that the IRKD reaction mixture contained no detectable phosphatase activity with P-Tyr-RCML, a specific PTPase substrate, orp-Npp, a substrate for both protein phosphatases and non-protein phosphatases such as alkaline phosphatase (14). In contrast, the rat liver membrane extract could dephosphorylate both substrates. In addition, the membrane extract catalyzed IRKD dephosphorylation at a lower rate than the IRKD preparation did in the absence of added phosphatases. In separate experiments, we fount that rat liver membrane PTPases were inhibited approximately 50% by I mM ADP (not shown). Prior studies have also demonstrated modest inhibition or minimal effect of ADP on the activity of purified PTPases (3) while PTPase activation by ADP has not been observed. Thus, the accelerating effect of ADP provided further evidence against catalysis by a PTPase. Experiments using phosphatase inhibitors (Figure 36) also yielded results inconsistent with IRKD dephosphorylation by protein phosphatases. Comparisons were made using 5 mM EDTA which halted IRKD dephosphorylation. Vanadate, which inhibits a broad spectrum of PTPases, did not diminish IRKD dephosphorylation (Figure 3B). Neither did the serine/threonine protein phosphatase inhibitors, calyculin A or okadaic acid (Figure 3B). Further supporting evidence against the presence of contaminating phosphatases in the IRKD preparation has been obtained previously in studies on the phosphorylation of a multiple tyrosine-containing peptide substrate by the IRKD (15). In these experiments, no reversal of peptide substrate phosphorylation seen

was

We interpreted our data as indicating catalysis of IRKD dephosphorylation by the IRKD itself. Additional support for this hypothesis came from experiments in which the protein kinase inhibitor staurosporine, in amounts sufficient to inhibit IRKD autophosphorylation, was shown to inhibit IRKD dephosphorylation (Figure 3C). Discussion Our data are consistent with the interpretation that IRKD dephosphorylation is catalyzed by the IRKD itself via a reaction which is dependent on kinase activity. Other 1461

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explanations, such as loss of phosphatefrom the autophosphorylated1RKD via proteolysis, activity of contaminating phosphatasesor reversal of kinase activity, are not consistent with our data. Given the stoichiometry of phosphorylation which would require phosphorylation of tyrosine residues internal to the IRKD, the data in Figures 1 and 2 are not consistent with loss of phosphate from the IRKD via proteolysis. A direct approach to detecting activity of contaminating phosphatasesdetected no such activity. Precedentsexist for reversal of tyrosine kinase activity. Autophosphorylation of the src tyrosine kinase can be reversed upon addition of excessADP via reversal of the kinase reaction (16). Pike et al. (17) reported similar results with the native human insulin receptor. However, our results in Figures 1 and 2 are difficult to explain based on reversal of the kinase reaction. Were this the case, bidirectional catalysis of IRKD phosphorylation /dephosphorylation would lead to a steady state with constant levels of ATP, ADP and phospho-IRKD being attained at equilibrium. We interpret our findings as indicating that reversal of IRKD phosphorylation is catalyzed by the kinase domain itself. Such a mechanismis consistent with the finding that addition of EDTA, which terminates kinase activity via chelation of Mg”. stops IRKD dephosphorylation. However, the most compelling support for this conclusion comes from the observation that dephosphorylation was inhibited by the kinase inhibitor, staurosporine. The present findings raise the possibility of a novel mechanism for termination of signal transduction via a receptor tyrosine kinase. A corollary of our findings is that insulin receptor tyrosine phosphorylation may be in a state of constant turnover which does not require exogenousPTPases. It is possible that phosphate stabilization in vivo is achieved by the binding to autophosphorylation sites of specific target proteins (e.g.,via sx homology domains). Alternatively, the insulin signal could be terminated upon inactivation of the kinase (by dissociation of ligand, for example) which would lead to loss of phosphate from the beta-subunit via mechanismsintrinsic to the beta-subunit. While such a mechanism would not preclude involvement of PTPasesin regulation of insulin signal transduction, it may indicate that termination of the insulin signal involves mechanismsintrinsic to the receptor kinase itself. Acknowledgments We thank Dr. David Brautigan for his helpful discussions. This work was supported by grants HD24455 (to P.G.) and DK40511 (to L.E.) and by a NATO Research Grant (to B.L.). We also acknowledge the contribution to these studies from the Biological NMR Unit at Birmingham University. References I. Rosen, O.M. (1987) Science237, 1452-1458. 2. Roth, R.A., Zhang, B., Chin, J.E., and Kovacina, K. (1992) J. Cell. Biochem. 48, 12-18. 1462

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3. Fischer, E.H., Charbonneau, H., and To&s, N.K. (1991) Science 253,401-406 4. King, M.J., and Sale, G.J. (1990) Biochem J. 266,251-259. 5. Roome, J., O’Hare, T., Pilch, P.F., and Brautigan, D.L. (1988) Biochem. J. 256,493-500. 6. Gruppuso, P.k, Boylan, J.M., Smiley, B.L., Fallon, R.J., and Brautigan, D.L. (1991) Biochem. .I. 274,361-367. 7. Boylan, J.M., Brautigan, D.L., Madden, J., Raven, T., Ellis, L., and Gruppuso, P.A. ( 1992) J. Clin. Invest 90,174-179. 8. Hashimoto, N.: Feener, E.P., Zhang, W.-R., and Goldstein, B.J. (1992) J. Biol. Chem. 267, 13811-13818. 9. Ellis, L., Levitan, A., Cobb, M.H., and Ramos, P. (1988) J. Virol. 62. 1634-1639. 10. Cobb, M.H., Sang, B.-C., Gonzalez, R., Goldsmith, E., and Ellis, L. (1989) J. Biol. Chem 264, 18701-18706.

Il. Cashel, M., Lazzaraini, R.A., and Kalbacher, B. (1969) J. Chromatog 40,103-109. 12. Gruppuso, P.A.; Boylan, J.M., Posner, B.I., Faure, R., and Brautigan, D.L. (1990) J. Clin. Invest 85, 1754-1760. 13. Levine, B.A., Tavare, J.M., Alejos, E., Clack, B., Sayed, N.. and Ellis, L. (1991) J. Biol. Chem. 266,13405-13410. 14.Sparks, J.W., and Brautigan, D.L. (1986) Int J. Biochem. 18,497-504. 15. Levine, B.A., Clack, B., and Ellis, L. (1991) J. Biol. Chem. 266,3565-3570. 16. Fukami, Y., and Lipmann, F. (1983) Proc. Natl. Acad. Sci. USA 80,1872-1876. 17. Pike, L.J., Eakes, A.T., and Krebs, E.G. (1986) J. Biol. Chem. 261,3782-3789.

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Insulin receptor tyrosine kinase domain auto-dephosphorylation.

We have observed dephosphorylation of the soluble, 48 kDa insulin receptor tyrosine kinase domain following its tyrosine autophosphorylation. Dephosph...
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