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ALTERATIONS IN THE SUBCELLULAR DISTRIBUTION OF p21raS-GTPase ACTIVATING PROTEIN IN PROLIFERATING RAT ACINAR CELLS Yoichi Nakagawa,* Karnam R. Purushotham,* Pao-Li Wang, * James E. Fischer,* William A. Dunn,f Charlotte A. Schneyes and Michael G. Humphreys-Beher t* Department
of Oral Biology, P.O. Box 100424 and Department of Anatomy and Cell Biology,* University of Florida, Gainesville, FL 32610$
Department of Physiology and Biophysics, 6 University of Alabama, Birmingham, AL 35294 Received
August
12,
1992
Rat parotid acinarcells undergotransientproliferation in responseto chronic administration of the B-adrenergicagonistisoproterenolor epidermalgrowth factor (EGF). Treatment with these agentscausedan increasein tyrosine phosphorylationof p21ras-GTPaseactivating protein (GAP). This phosphorylation event was accompaniedby a redistribution of the protein from the plasma membraneto internal membranecompartments. Separationof subcellularmembranesrevealed increasedGAP associatedwith a low density population of vesicles concomitant with growth stimulation as well as to the nuclear membrane,but not the nucleoplasm. Upon cessationof hyperplasia induced by isoproterenol, phosphorylated GAP present in the plasma membrane returnedto control cell levels. D 19~ ~~~~~~~~ press,1°C.
Chronic administration of the 13-adrenergic agonist, isoproterenol, (ISO) leadsto rat and murine parotid gland acinar cell proliferation (l-3). This transient increasein cell hyperplasiais not, however, neoplastic,and after 5 to 6 days, cell division declinesand is replacedby continued cell hypertrophy (l-3). The transition from stasisto active cell division in mature differentiated acinar cells appearsto be mediatedin part by the presenceof cell surfacegalactosyltransferase(46). Changesin diet or treatmentof cells in vivo or in vitro with growth factors also causesacinar cell proliferation (7, 8). Cell division, mediatedby EGF, occursthrough the classicalinteraction of this ligand with the EGF-Receptor (EGF-R) in the cell surfacewith the subsequentactivation of intrinsic receptor tyrosine kinase activity. A seriesof cytosolic protein constituents undergo phosphorylation and membrane association, which are involved in the generation of the intracellular signalling pathway for the tyrosine kinases (9-13). The activation of the phosphotyrosinesignalling pathway involved in growth stimulation by isoproterenol appearsto take place by an alternative cell surface activation of the EGF-R through the interaction of galactosyltransferase with the carbohydrate moieties of the EGF-R (9). Both EGF and isoproterenoltreatmentof acinarcells lead to EGF-R autophosphorylationand transientmembrane associationof phospholipaseCy and phosphotidylinositol3-kinase with the EGF-R in the plasma t TO whom correspondenceshouldbe addressed 0006-29 I X/Y? $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reprodrrcfion in any form reserved.
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through the SH2-domains
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of these proteins
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(Puruthotham
ef al.,
manuscript submitted; 14, 15). Immunoprecipitation of detergent solubilized lysates from various transformed
cells in
culture or cell lines overexpressing constituents of the activated EGF-R or PDGF-R complex have demonstrated an association with phospholipase Cy, phosphotidylinositol3-kinase, ~190, p62 and GAP (GTPase activating protein) (10-13). GAP levels in the plasma membrane and its tyrosine phosphorylation are proposed to mediate the activity of p21ra . Cellular p21ra activity is regulated by the level of protein bound GTP (16). The plasma membrane-associated p21ras are biologically inactive when bound to GDP, but are active only when bound to GTP (16- 18). Activated p21ras may then propagate tyrosine kinase stimulated cell growth by modulation of serine/threonine kinase signal transducing molecules (17, 18). The activity of ~21 ras are regulated by two types of mechanisms; enhancement of GDP/GTP exchange and inhibition of GTPase activity (19-22). The weak intrinsic
GTPase activity of p21ras can be greatly accelerated by GAP (23-25).
GAP
promotes the return of p21ras to an inactive GDP-bound state and is thought to be a regulator of p2lras activity and may therefore be a possible downstream target molecule. GAP acts on the effector domain of p2 1ras which is necessary for oncogenic transformation (16-22). To investigate the role of p2lras-GAP regulation in in vivo cell growth, we examined the subcellular distribution and phosphorylation of GAP upon stimulation of proliferation and subsequent withdrawal from hyperplasia in rat parotid acinar cells. Acinar cell proliferation was induced with a chronic treatment of either the O-adrenergic agonist, isoproterenol or EGF.
Materials
and Methods
Materials. d, 1 isoproterenol, EGF (mouse), phenylmethylsulfonyl fluoride, sodium pyrophosphate and sodium orthovanadate was purchased from Sigma Chemical Co. Phosphotyrosine antibody complexed to sepharose 4B and [ 1251]-1abe11ed protein A were purchased from Amersham (Arlington Heights, IL). Female Sprague-Dawley rats, weighing between 175-225 g were obtained from the University of Florida breeding colony. Chemicals for sodium dodecyl sulfate (SDS) polyacrylamide gels were purchased from Bio-Rad (Richmond, CA). All other reagents were obtained from commercial sources and were of ultrapure quality. GAP irnmunoprecipitation. Rats were injected twice daily with 0.5 ml saline, 25 mg/kg isoproterenol or 25 @kg EGF. Parotid glands were removed 15 min following the final injection at 3 days. The tissue was homogenized in 10 mM Tris-HCl pH 8.0, containing 1% aprotinin, 0.5 pM phenylmethylsulfonyl fluoride, 100 mM NaF, 10 p,M sodium pyrophosphate, 200 PM sodium orthovanadate and 1.0% NP-40. Following removal of insoluble particulate material, 500 p.g of protein were immunoprecipitated with monoclonal antibody to phosphotyrosine coupled to sepharose-4B (6). The immunoprecipitated protein complexes were separated on 8% SDSpolyacrylamide gels (PAGE) and transferred to nitrocellulose by electrophoresis at 17V overnight (26). The immunoblot was reacted with anti-GAP 677 or 638 antibody (which gave identical results) provided by Dr. J. Gibbs of Merck, Sharpe and Dohme Laboratories, West Point, PA (lo13) followed by alkaline phosphatase-conjugated second antibody and incubation in the presence of BCIP or monoclonal antibody to phosphotyrosine (Amersham) followed by l25I-labelled protein A and autoradiography. The EGF-R, at 170 l&a, was identified with polyclonal antibody as described previously (9, 27, 28). Autoradiography was performed using Kodak XAR-5 film exposed for 24 hr at -80°C. Each group of experimental animals consisted of a pool from six individuals. Immunoprecipitation and immunoblotting was repeated 3 times to verify consistency of the results. Density Gradient Separation of Subcellular Fractions. Total membrane fractions (100,000 xg pellets) were prepared at 4°C following homogenization in the lysate mixture described previously (4-6). A prior low speed centrifugation was performed to remove connective tissue and unlysed cells. Plasma membrane was isolated from total membrane preparations by methodology 1173
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specifically designed for rat parotid acinar cells (29). Sixty micrograms of plasma membrane were subjected to Western blotting using anti-GAP 677 antiserum subsequent to separation by 8% SDS-PAGE. Low and high speed fractions were separated on a linear 1.06-1.25 g/cc sucrose gradient (27, 28). One ml fractions were collected using a Buchler auto densi-flow gradient apparatus. Twenty-five c(g of every other sample were analyzed by separation on an 8% SDS-PAGE prior to transfer to nitrocellulose The same results were obtained on three separate occasions for each gradient separation. Color development, using the alkaline phosphate-conjugated second antibody, was stopped after 10 min by transfer to water. Increased levels of GAP from separate gels was again confirmed by running samples (35 pg protein) from the peak fractions (10-13) on the same gel, transfering the samples to the same nitrocellulose and reacting with the antibody (antiGAP 677) dependent protocol (Table 1). Marker enzymes for Golgi complex (81-4 galactosyltransferase) were assayed as described (4) using ovalbumin as acceptor glycoprotein and [3H] Uridine Diphosphate-Galactose (Amersham) as sugar donor. Gamma glutamyltranspeptidase, a plasma membrane marker suitable for rat parotid gland acinar cells, was assayed by the method of Tate and Meister (30). Nuclei isolation. Rat parotid gland acinar cell nuclei were isolated by the method of Blobel and Potter as described previously (6). In brief, parotid glands were homogenized on ice into 5ml TKM buffer (pH 7.4) [5 mM Tris, 2.5 mM KC1 and 5 mM MgC12] containing 0.25M sucrose. Following passage through cheesecloth, the sucrose concentration was adjusted to 1.62M, layered over a 2.2M sucrose cushion, and centrifuged at 6000 xg for 1 h. The pellet containing nuclei was resuspended in 1.0 ml TKM buffer and sonicated for 30 set with intermittent cooling. The nuclear membrane was recovered from nucleoplasm by centrifugation for 1 hr at 145000 xg.
Results
and Discussion
Following chronic administration of EGF or isoproterenol to rats for 3 days (a time of maximal hyperplasia), the parotid glands were removed, total protein solubilized in buffer containing
1.0% NP-40, and the resulting cell lysates immunoprecipitated
with a monoclonal
antibody to phosphotyrosine (6, 24,25). The proteins present in the immunoprecipitated complex were reacted with antibody to bovine GAP (5) or phosphotyrosine (Fig. 1A and B, respectively). When the nitrocellulose blot of proteins from the immune precipitation were probed with antibody
123 205 116.5
205
6123 -
1165
80
80
49.5
49.5-
-
Figure 1. Immunoprecipitation analysis of phosphorylated pl20-GAP and levels followinggrowth stimulationof rat parotidglandacinarcells. Animalswereinjected for 3 days with saline(Lane 1); 25 mg/mlisoproterenol(Lane2) or 25ug/kgEGF (Lane3). PanelA represents a Westernblotanalysisof theimmunopreeipiated material using anti GAP-677 antibody. Panel B representsan autoradiograph of immunoprecipitated materialreactedwith antiphosphotyrosine antibodyand [tzsI]labelledprotein A. Proteinswere immunoprecipitated with antiphosphotyrosine antibody coupled to sepharose 4B. Prestained molecular weight standards (Bio-Rad) are: myosin, 205,000 Da; B-galactosidase, 116,500 Da; bovine serum albumin, 80,000 Da; ovalbumin, 49,500 Da.
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Densitometer Values for GAP associated with Acinar Cell Membranes following Western blot analysis Time*
Treatment/Blot 72
h
IS0 1 .s t 0.2 0.18+0.03 9.8620.41
Phosphotyrosinet Plasmamembranes Low Density Vesicles High Density Vesicles Cytoplasm§
EGF 1.2 +0.2 0.05+0.04 10.65+0.35 0.279.04 0.719.18
0.30+0.06 0.79+0.15
I44h IS0 N.D.* 0.95*0.05 N.D. N.D.
N.D.
7 From Figure IA. GAP was identified in Western blots following immune precipitation with antiphosphotyrosine antibody coupled to sepharose (Amersham). * All values were obtained from the same nitrocellulose filter and expressed relative to unstimulated acinar cell values (1 .O units) + standard deviation. Densitometer analysis (Hoeffer GS300 gel scanner) was performed on 3 separate nitrocellulose determinations of GAP protein. Low density vesicles (high speed centrifugation fraction) were pooled fractions lo- I3 (6=1.14g/cc); high density vesicles (low speed centrifugation fraction) were pooled fractions 15-19 (61.2g/cc). $ Not Determined. 0 Cytoplasm is defined as soluble material from the 145,000 xg centrifugation to isolate low density vessicles (27,28).
to GAP, a protein
Mr = 120 kDa was detected in control and experimental samples(10-13). There was a 1.550.2 and 1.2+0.2-fold increasein the level of GAP presentin of approximately
cell lysatesasdeterminedby densitometer,following isoproterenolor EGF treatmentrespectively when compared to control cell lysates (Table 1). When the immunoprecipitated complex was subsequently probed with the antiphosphotyrosine antibody, the presumed GAP at 120 kDa showed an S-fold increase in phosphotyrosine upon stimulation of proliferation with either isoproterenolor EGF (Fig. 1B). Immunoprecipitationof cell lysatesusinga polyclonal antibody to rat EGF-R failed to coprecipitateGAP. An analysis of plasmamembranesshowedthat in parotid acinar cells, GAP activity was unexpectedly lost following growth stimulation by isoproterenol or EGF. A time course for plasma membrane detection is presented in Figure 2A and C. Within 15 min of the first
A 205 116.5 a0
12345
B
123
205 116.5 80
C
12345
205 116.5 60 49.5
49.5
49.5
Figure 2. Decreased levels of GAP associated with the plasma membrane following a time course of isoproterenol or EGF stimulation. Separate groups of animals (n=6) received daily injections of isoproterenol (panel A and B) or EGF (panel C). Parotid glands (panels A and C) were removed 15 min after the injection of saline for 72 h (lane 1) or 15 min following the injection of the initial drug regimen (lane 2); 1 h after a single injection (lane 3); 24 h after a chronic injection (lane 4) and 72 h of the chronic drug regimen (lane 5). In panel B, lane 1 represents control membranes (72 h saline); lane 2, 72 h after the chronic injection of isoproterenol and lane 3, 144 h of chronic injection of isoproterenol.
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of EGF, GAP associated with the plasma membrane began to decline (Fig. 2C).
The same trend was also evident following isoproterenol administration, however, the decline in plasma membrane-associated GAP did not appear to occur until the 24 h time point. Isolation of acinar cells and in vitro culturing
for 24 h produced the same loss of GAP from the plasma
membrane in a time dependent manner upon the inclusion of EGF or isoproterenol into culture media following a15 min exposure (data not shown). Since acinar cells demonstrate a self limited hyperplasia in response to chronic treatment with B-agonist, plasma membranes were isolated at 6 days of a chronic drug regimen. Plasma membrane GAP activity declined at 72 h (maximal hyperplasia) but at 144 h (a time of declining cell proliferation) had returned to unstimulated control levels (Table 1; Fig. 2B). To assess the changes in subcellular distribution
of GAP that were associated with its
activation by tyrosine phosphorylation, membrane preparations were separated by a continuous sucrose gradient following the isolation of a low and high speed microsomal preparation (27,28). The low speed, 12,000 xg preparation contained plasma membrane sheets, lysosomes and mitochondria while the high speed 145,000 xg fraction contained plasma membrane vesicles, Golgi complex, endoplasmic reticulum and endosomes. When the later fraction was applied to a sucrose gradient, plasma membrane derived vesicles (as assayed by the acinar cell marker enzyme y-glutamyltranspeptidase; 29) were clearly separated from the low density vesicles containing and Golgi complex (assayed by l31-4 galactosyltransferase;
4, 5) (Fig. 3A). Western blot of fractions
from the high speed microsome preparation showed an increase in GAP protein associated with the Golgi complex fractions 10-14. Upon treatment with EGF (panel D and F) or isoproterenol (panel C and E), the amount of GAP protein associated with the low density vesicles was significantly increased. GAP associated with low density vesicles fractions increased 2-fold over control levels at 15 min and IO-fold at 72 h (Table 1). Detection of the EGF-R was confined to the high density microsomal fraction (plasma mebrane fraction) in untreated acinar cells but was detected in the low density microsomal fraction following treatment with the growth factor or R-agonist. This observation is consistent with receptor internalization into endosomes (27,28).
The high density
microsomal fraction again showed a decrease in the levels of detectable GAP when separated by sucrose gradient (Table 1). Increased GAP was also found associated with the nuclear envelope. As shown in Fig. 4, GAP activity was increased in the nuclear membrane following either EGF or isoproterenol treatment at 72 h. Densitometer analysis revealed a 2-fold increase in GAP following isoproterenol treatment and a 5-fold increase following EGF treatment of acinar cells. There was no detection of GAP in the nucleoplasm from any of the three groups of animals (data not shown). Thus, the results presented for growth stimulation of rat parotid gland acinar cells showed a different membrane association pattern than has been commonly reported. GAP is typically localized in the cytosol of non-dividing cells (10, 11). Upon EGF or PDGF stimulation or in cells transformed by pp60src, GAP activity associated with the plasma membrane occurs (10-13). In contrast to the cytosolic serine phosphorylated form, membrane GAP shows increased tyrosine phosphoryiation presumably through the kinase activity of the receptor complex. In acinar cells tyrosine phosphorylation may take place by a non-receptor tyrosine kinase activity since GAP could not be immunoprecipitated along with the EGF-R. Since the activation ofp2lras requires the 1176
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!
0.1 ;g 56 z--0 4
8
12
16
Fraction
205 116.5
8
20
24
28
0.0 y
number
----
80 49.5
_‘
205
-
116.5
-
80
-
49.5
-
mr-.,a
Figure 3. Distribution of GAP on sucrose gradients of the Golgi enriched high speed microsomal fraction. Subcellular fractions were prepared from total cell lysates by centrifugation at 15,000 xg to obtain a plasma membrane, mitrochondria and lysosomeenriched fraction (low speed) and centrifugation at 145,000 xg of the supernatant fraction of the low speed preparation to obtain a Colgi complex, endoplasic reticulum and endosome enriched pool (high speed). Animals (pooled, n=6) in each group received either chronic injection of saline (panel B) isoproterenol for 1.5 min prior to killing (panel C); 15 min EGF injection prior to killing (panel D); 72 h of isoproterenol treatment (panel E); 72 h of EGF treatment (panel F). The low density vesicle fraction (10-13) is indicated by arrow.
participation
of bound GTP, it would appearthat GAP activation and translocation to the plasma
membraneis a late event in the progressionfrom stasisto active cell proliferation. The intracellular recycling of internalized ligand-receptor complexes in endosomesmay therefore play a role in mediating the subsequentphosphorlyation of GAP by virtue of this mechanismof protein sorting. PlasmamembraneGAP could be internalized with the EGF-R upon growth stimulation. This, however, would have to occur following the initial phosphorylation of phospholipaseCr and phosphatidylinositol 3-kinase.,since activated EGF-R from rat acinar cell surface fractions have been shownto co-immunoprecipitatethesetwo proteinsearly in acinarcell growth stimulation 1177
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2051165 604435kDa Figure 4. Distribution of GAP following isolation of the nuclear membrane. Parotid gland cell nuclei were isolated as described previously (6). Ten pg of the nuclear envelope were separated by 8% SDS polyacrylamide gel and stained for protein by Coomassie Brilliant blue R-250 (Dane1 A) or transferred to nitrocellulose and reacted with antibody to GAP and 125;:labelled protein A (panel B). Molecular weight standards are as in Figure 1. Lane 1, control rat parotid gland nuclear membrane; lane 2, ISO-treated acinar cell nuclear membrane; lane 3, EGF-treated parotid gland acinar cell nuclear membrane.
(9; unpublishedresults). Activation of p21ras could therefore proceed by stimulation of the RasGuanine nucleotide exchange factor (19, 22, 31). As acinar cell hyperplasia is down-regulated, reappearanceof GAP at the cell surfacecould act upon p21‘as-GTP to stimulate GTP hydrolysis and thus inactivate p21rassignal transduction. Alternatively, GAP alone may be taken up into endosomes,independentof EGF-R and recycled to the plasmamembrane. Indeed we have found GAP in low density vesiclesof unstimulatedcontrols (Fig. 3B). In this mechanism,GAP might be storedin endosomesor the Golgi complex asseenin certain receptor densitizationmechanisms (32). Tyrosine phosphorylation could occur in a membranecompartmentindependentof receptor kinaseactivity. GAP itself may have other effector functions besidesinteraction with p21rasduring the early transition to growth. Growth factor stimulation or transformation by tyrosine kinase genes, also leads to the phosphorylation of p62 and ~190 (10-13). Recently, p62 and ~190, which interact with GAP, have been purified and cDNA clones isolated. Analysis of p62 revealed extensive sequencesimilarity to a putative hnRNA protein, GRP33. p62 has the ability to bind single strandedDNA and RNA and appearsto be localized, in part, to the cell nucleus(33). ~190 is localized within the cytoplasm, and nucleus, and its sequenceincludes predicted protein homology to the transcriptionalrepressorof glucorticoid receptor (GRF-l),BDR, n-chimaerin and the GTPase superfamily (34). These two reports speculatethat GAP can transducesignalsfrom p21rasthrough GAP-protein complexesat sitesdistal to the plasmamembrane.Thus the early loss of GAP from the plasmamembrane,and from the cytoplasm, in stimulated acinar cells may be directly related to theseother cellular functions requiredfor the transitionfrom quiescenceto active proliferation asobservedhereby the localization of increasedGAP to the nuclearmembrane.
Acknowledgments This work was supported by NIH grants DE 08778 and DE 00291 (MHB); DE 02110 (CAS); and DK 33326 (WAD). The authors thank Dr. JacksonGibbs for his generousprovision 1178
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of antibody to bovine GAP 677 and 638. Acknowledgment is given to Ms. Marilyn Lietz for preparationof the manuscript.
References :: 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
Brown-Grant, K. (1961) Nature 191, 10761078. Schneyer, C. A. (1962) Am.J. Physiol. 203, 232-236. Selye, H., Cantin, M. and R. Veilleux, R. (1961) Growth 25, 243-248. Humphreys-Beher, M. G., Schneyer, C. A., Kidd, V. J. and Mat-chase,R. B. (1987) L Biol. Chem. 262, 11706-l 1713. Marchase, R. B., Kidd, V. J., Rivera, A. A. and M. G. Humphreys-Beher, M.G. (1988) J. Cell. Biochem, 36,453-465. Purushotham,K. R., Zelles, T. and Humphreys-Beher, M. G. (1991) Molec. Cell, Biochem 102, 19-34. Schneyer; C. A. and Humphreys-Beher,M. G. (1988) ,I. Oral Pathol. 17, 250-256. Humphreys-Beher, M. G., Zelles, T., Maeda, N., Purushotham,K. R. and Schneyer, C. A. (1990) Molec. Cell. Biochem, 95, l-l 1. Purushotham,K. R., Dunn, W. A., Schneyer, C.A. and Humphreys-Beher, M. G. (1992) Biochem. J. 284,767-776. Molley, C.J., Bottaro, D.R., Fleming, T.R., Marshall, M.S., Gibbs, J.B. and Aaronson, S.A. (1989) Nature 342, 7 11-714. Ellis, C., Moran, M., McCormick, F. and Pawson,T. (1990) Nature 343, 377-381. Ria, F., Chan, B.M.C., Scherer, M.T., Smith, J.A. and Gefter, M.L. (1990) Nature 343, 381-383. Bouton, A. H., Kanner, S.B., Vines, R.R., Wang, H.C.R., Gibbs, J.B. and Pawson, J.T. (1991) Molec. Cell. Biol. 11, 945953. Koch, C. A., Anderson, D., Moran, M. F., Ellis, C. and Parsons,T (1991) Science 252, 668-674. Anderson, D., Koch, C.A., Grey, L., Ellis, C., Moran, M.F. and Pawson,T. (1990) Science 250,979. Gibbs, J. B., Schaber, M. D., Marshall, M. S., Scolnick, E. M. and Sigal, I. S. (1987) L Biol. 262, 1042610429. Field, J., Broek, D., Kataoka , T. and Wigler, M. (1987) Molec. Cell. Biol. 7, 21282133. Trahey, M. and McCormick, F.(1987) Science238,542-545. Satoh, T., Nakamura, S. and Kaziro, Y. (1987) Molec. Cell. Biol. 7, 4553-4556. Wood, K. W., Sarnecki, C., Roberts, T. M. and Blenis, J. (1992) C&l 68, 1041-1050. Sassone-Corsi,P., Der, C.J. and Verma, I.M. (1989) Molec. Cell. B~ol. 9, 3174-3183. Kamata, T. and Feramisco,J. R. (1984) Nature 310, 147-150. West, M., Kung, H. and Kamata, T. (1990) FEBS Lett, 259, 245-248. Wolfman, A. and Macara, I. G. (1990) Science 248,67-69. Downward, J., Riehl, E., Wu, L. and Weinberg, R. A. (1990) Proc. USA 87, 5998-6002. Towbin, H., Stachalin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci USA 76, 4350. Dunn , W. A. and Hubbard, A. L. (1984) J. Cell. Biol. 98, 2148-2159. Dunn, W. A., Connolly, T. P. and Hubbard, A. L.(1986) J. cell. Biol. 102, 24-36. Arvan, P. and Castle, J. D. (1982) J. Ceil. Biol. 95, 8. Tate, S. S. and Meister, A. (1974) ,I. Biol. Chem. 249,7593. Li, B.-Q., Kaplan, H.-F. and Katama, T. (1992) Science 256, 1456-1459. Lefkowitz, R. J., Stadel, J.M. and Caron, M.G. (1983) Ann. Rev. Biochem, 52, 159186. Wong, G., Mtiller, O., Clark, R., Conroy, L., Moran, M. F., Polakis, P. and McCormick, F. (1992) &Ll69,55 l-558. Settleman, J., Narashimhan,V.. Foster, L. C., and Weinberg, R.A. (1992) &!!69,539549.
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ALTERATIONS IN THE SUBCELLULAR DISTRIBUTION OF p21raS-GTPase ACTIVATING PROTEIN IN PROLIFERATING RAT ACINAR CELLS Yoichi Nakagawa,* Karnam R. Purushotham,* Pao-Li Wang, * James E. Fischer,* William A. Dunn,f Charlotte A. Schneyes and Michael G. Humphreys-Beher t* Department
of Oral Biology, P.O. Box 100424 and Department of Anatomy and Cell Biology,* University of Florida, Gainesville, FL 32610$
Department of Physiology and Biophysics, 6 University of Alabama, Birmingham, AL 35294 Received
August
12,
1992
Rat parotid acinarcells undergotransientproliferation in responseto chronic administration of the B-adrenergicagonistisoproterenolor epidermalgrowth factor (EGF). Treatment with these agentscausedan increasein tyrosine phosphorylationof p21ras-GTPaseactivating protein (GAP). This phosphorylation event was accompaniedby a redistribution of the protein from the plasma membraneto internal membranecompartments. Separationof subcellularmembranesrevealed increasedGAP associatedwith a low density population of vesicles concomitant with growth stimulation as well as to the nuclear membrane,but not the nucleoplasm. Upon cessationof hyperplasia induced by isoproterenol, phosphorylated GAP present in the plasma membrane returnedto control cell levels. D 19~ ~~~~~~~~ press,1°C.
Chronic administration of the 13-adrenergic agonist, isoproterenol, (ISO) leadsto rat and murine parotid gland acinar cell proliferation (l-3). This transient increasein cell hyperplasiais not, however, neoplastic,and after 5 to 6 days, cell division declinesand is replacedby continued cell hypertrophy (l-3). The transition from stasisto active cell division in mature differentiated acinar cells appearsto be mediatedin part by the presenceof cell surfacegalactosyltransferase(46). Changesin diet or treatmentof cells in vivo or in vitro with growth factors also causesacinar cell proliferation (7, 8). Cell division, mediatedby EGF, occursthrough the classicalinteraction of this ligand with the EGF-Receptor (EGF-R) in the cell surfacewith the subsequentactivation of intrinsic receptor tyrosine kinase activity. A seriesof cytosolic protein constituents undergo phosphorylation and membrane association, which are involved in the generation of the intracellular signalling pathway for the tyrosine kinases (9-13). The activation of the phosphotyrosinesignalling pathway involved in growth stimulation by isoproterenol appearsto take place by an alternative cell surface activation of the EGF-R through the interaction of galactosyltransferase with the carbohydrate moieties of the EGF-R (9). Both EGF and isoproterenoltreatmentof acinarcells lead to EGF-R autophosphorylationand transientmembrane associationof phospholipaseCy and phosphotidylinositol3-kinase with the EGF-R in the plasma t TO whom correspondenceshouldbe addressed 0006-29 I X/Y? $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reprodrrcfion in any form reserved.
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of these proteins
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(Puruthotham
ef al.,
manuscript submitted; 14, 15). Immunoprecipitation of detergent solubilized lysates from various transformed
cells in
culture or cell lines overexpressing constituents of the activated EGF-R or PDGF-R complex have demonstrated an association with phospholipase Cy, phosphotidylinositol3-kinase, ~190, p62 and GAP (GTPase activating protein) (10-13). GAP levels in the plasma membrane and its tyrosine phosphorylation are proposed to mediate the activity of p21ra . Cellular p21ra activity is regulated by the level of protein bound GTP (16). The plasma membrane-associated p21ras are biologically inactive when bound to GDP, but are active only when bound to GTP (16- 18). Activated p21ras may then propagate tyrosine kinase stimulated cell growth by modulation of serine/threonine kinase signal transducing molecules (17, 18). The activity of ~21 ras are regulated by two types of mechanisms; enhancement of GDP/GTP exchange and inhibition of GTPase activity (19-22). The weak intrinsic
GTPase activity of p21ras can be greatly accelerated by GAP (23-25).
GAP
promotes the return of p21ras to an inactive GDP-bound state and is thought to be a regulator of p2lras activity and may therefore be a possible downstream target molecule. GAP acts on the effector domain of p2 1ras which is necessary for oncogenic transformation (16-22). To investigate the role of p2lras-GAP regulation in in vivo cell growth, we examined the subcellular distribution and phosphorylation of GAP upon stimulation of proliferation and subsequent withdrawal from hyperplasia in rat parotid acinar cells. Acinar cell proliferation was induced with a chronic treatment of either the O-adrenergic agonist, isoproterenol or EGF.
Materials
and Methods
Materials. d, 1 isoproterenol, EGF (mouse), phenylmethylsulfonyl fluoride, sodium pyrophosphate and sodium orthovanadate was purchased from Sigma Chemical Co. Phosphotyrosine antibody complexed to sepharose 4B and [ 1251]-1abe11ed protein A were purchased from Amersham (Arlington Heights, IL). Female Sprague-Dawley rats, weighing between 175-225 g were obtained from the University of Florida breeding colony. Chemicals for sodium dodecyl sulfate (SDS) polyacrylamide gels were purchased from Bio-Rad (Richmond, CA). All other reagents were obtained from commercial sources and were of ultrapure quality. GAP irnmunoprecipitation. Rats were injected twice daily with 0.5 ml saline, 25 mg/kg isoproterenol or 25 @kg EGF. Parotid glands were removed 15 min following the final injection at 3 days. The tissue was homogenized in 10 mM Tris-HCl pH 8.0, containing 1% aprotinin, 0.5 pM phenylmethylsulfonyl fluoride, 100 mM NaF, 10 p,M sodium pyrophosphate, 200 PM sodium orthovanadate and 1.0% NP-40. Following removal of insoluble particulate material, 500 p.g of protein were immunoprecipitated with monoclonal antibody to phosphotyrosine coupled to sepharose-4B (6). The immunoprecipitated protein complexes were separated on 8% SDSpolyacrylamide gels (PAGE) and transferred to nitrocellulose by electrophoresis at 17V overnight (26). The immunoblot was reacted with anti-GAP 677 or 638 antibody (which gave identical results) provided by Dr. J. Gibbs of Merck, Sharpe and Dohme Laboratories, West Point, PA (lo13) followed by alkaline phosphatase-conjugated second antibody and incubation in the presence of BCIP or monoclonal antibody to phosphotyrosine (Amersham) followed by l25I-labelled protein A and autoradiography. The EGF-R, at 170 l&a, was identified with polyclonal antibody as described previously (9, 27, 28). Autoradiography was performed using Kodak XAR-5 film exposed for 24 hr at -80°C. Each group of experimental animals consisted of a pool from six individuals. Immunoprecipitation and immunoblotting was repeated 3 times to verify consistency of the results. Density Gradient Separation of Subcellular Fractions. Total membrane fractions (100,000 xg pellets) were prepared at 4°C following homogenization in the lysate mixture described previously (4-6). A prior low speed centrifugation was performed to remove connective tissue and unlysed cells. Plasma membrane was isolated from total membrane preparations by methodology 1173
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specifically designed for rat parotid acinar cells (29). Sixty micrograms of plasma membrane were subjected to Western blotting using anti-GAP 677 antiserum subsequent to separation by 8% SDS-PAGE. Low and high speed fractions were separated on a linear 1.06-1.25 g/cc sucrose gradient (27, 28). One ml fractions were collected using a Buchler auto densi-flow gradient apparatus. Twenty-five c(g of every other sample were analyzed by separation on an 8% SDS-PAGE prior to transfer to nitrocellulose The same results were obtained on three separate occasions for each gradient separation. Color development, using the alkaline phosphate-conjugated second antibody, was stopped after 10 min by transfer to water. Increased levels of GAP from separate gels was again confirmed by running samples (35 pg protein) from the peak fractions (10-13) on the same gel, transfering the samples to the same nitrocellulose and reacting with the antibody (antiGAP 677) dependent protocol (Table 1). Marker enzymes for Golgi complex (81-4 galactosyltransferase) were assayed as described (4) using ovalbumin as acceptor glycoprotein and [3H] Uridine Diphosphate-Galactose (Amersham) as sugar donor. Gamma glutamyltranspeptidase, a plasma membrane marker suitable for rat parotid gland acinar cells, was assayed by the method of Tate and Meister (30). Nuclei isolation. Rat parotid gland acinar cell nuclei were isolated by the method of Blobel and Potter as described previously (6). In brief, parotid glands were homogenized on ice into 5ml TKM buffer (pH 7.4) [5 mM Tris, 2.5 mM KC1 and 5 mM MgC12] containing 0.25M sucrose. Following passage through cheesecloth, the sucrose concentration was adjusted to 1.62M, layered over a 2.2M sucrose cushion, and centrifuged at 6000 xg for 1 h. The pellet containing nuclei was resuspended in 1.0 ml TKM buffer and sonicated for 30 set with intermittent cooling. The nuclear membrane was recovered from nucleoplasm by centrifugation for 1 hr at 145000 xg.
Results
and Discussion
Following chronic administration of EGF or isoproterenol to rats for 3 days (a time of maximal hyperplasia), the parotid glands were removed, total protein solubilized in buffer containing
1.0% NP-40, and the resulting cell lysates immunoprecipitated
with a monoclonal
antibody to phosphotyrosine (6, 24,25). The proteins present in the immunoprecipitated complex were reacted with antibody to bovine GAP (5) or phosphotyrosine (Fig. 1A and B, respectively). When the nitrocellulose blot of proteins from the immune precipitation were probed with antibody
123 205 116.5
205
6123 -
1165
80
80
49.5
49.5-
-
Figure 1. Immunoprecipitation analysis of phosphorylated pl20-GAP and levels followinggrowth stimulationof rat parotidglandacinarcells. Animalswereinjected for 3 days with saline(Lane 1); 25 mg/mlisoproterenol(Lane2) or 25ug/kgEGF (Lane3). PanelA represents a Westernblotanalysisof theimmunopreeipiated material using anti GAP-677 antibody. Panel B representsan autoradiograph of immunoprecipitated materialreactedwith antiphosphotyrosine antibodyand [tzsI]labelledprotein A. Proteinswere immunoprecipitated with antiphosphotyrosine antibody coupled to sepharose 4B. Prestained molecular weight standards (Bio-Rad) are: myosin, 205,000 Da; B-galactosidase, 116,500 Da; bovine serum albumin, 80,000 Da; ovalbumin, 49,500 Da.
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Densitometer Values for GAP associated with Acinar Cell Membranes following Western blot analysis Time*
Treatment/Blot 72
h
IS0 1 .s t 0.2 0.18+0.03 9.8620.41
Phosphotyrosinet Plasmamembranes Low Density Vesicles High Density Vesicles Cytoplasm§
EGF 1.2 +0.2 0.05+0.04 10.65+0.35 0.279.04 0.719.18
0.30+0.06 0.79+0.15
I44h IS0 N.D.* 0.95*0.05 N.D. N.D.
N.D.
7 From Figure IA. GAP was identified in Western blots following immune precipitation with antiphosphotyrosine antibody coupled to sepharose (Amersham). * All values were obtained from the same nitrocellulose filter and expressed relative to unstimulated acinar cell values (1 .O units) + standard deviation. Densitometer analysis (Hoeffer GS300 gel scanner) was performed on 3 separate nitrocellulose determinations of GAP protein. Low density vesicles (high speed centrifugation fraction) were pooled fractions lo- I3 (6=1.14g/cc); high density vesicles (low speed centrifugation fraction) were pooled fractions 15-19 (61.2g/cc). $ Not Determined. 0 Cytoplasm is defined as soluble material from the 145,000 xg centrifugation to isolate low density vessicles (27,28).
to GAP, a protein
Mr = 120 kDa was detected in control and experimental samples(10-13). There was a 1.550.2 and 1.2+0.2-fold increasein the level of GAP presentin of approximately
cell lysatesasdeterminedby densitometer,following isoproterenolor EGF treatmentrespectively when compared to control cell lysates (Table 1). When the immunoprecipitated complex was subsequently probed with the antiphosphotyrosine antibody, the presumed GAP at 120 kDa showed an S-fold increase in phosphotyrosine upon stimulation of proliferation with either isoproterenolor EGF (Fig. 1B). Immunoprecipitationof cell lysatesusinga polyclonal antibody to rat EGF-R failed to coprecipitateGAP. An analysis of plasmamembranesshowedthat in parotid acinar cells, GAP activity was unexpectedly lost following growth stimulation by isoproterenol or EGF. A time course for plasma membrane detection is presented in Figure 2A and C. Within 15 min of the first
A 205 116.5 a0
12345
B
123
205 116.5 80
C
12345
205 116.5 60 49.5
49.5
49.5
Figure 2. Decreased levels of GAP associated with the plasma membrane following a time course of isoproterenol or EGF stimulation. Separate groups of animals (n=6) received daily injections of isoproterenol (panel A and B) or EGF (panel C). Parotid glands (panels A and C) were removed 15 min after the injection of saline for 72 h (lane 1) or 15 min following the injection of the initial drug regimen (lane 2); 1 h after a single injection (lane 3); 24 h after a chronic injection (lane 4) and 72 h of the chronic drug regimen (lane 5). In panel B, lane 1 represents control membranes (72 h saline); lane 2, 72 h after the chronic injection of isoproterenol and lane 3, 144 h of chronic injection of isoproterenol.
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of EGF, GAP associated with the plasma membrane began to decline (Fig. 2C).
The same trend was also evident following isoproterenol administration, however, the decline in plasma membrane-associated GAP did not appear to occur until the 24 h time point. Isolation of acinar cells and in vitro culturing
for 24 h produced the same loss of GAP from the plasma
membrane in a time dependent manner upon the inclusion of EGF or isoproterenol into culture media following a15 min exposure (data not shown). Since acinar cells demonstrate a self limited hyperplasia in response to chronic treatment with B-agonist, plasma membranes were isolated at 6 days of a chronic drug regimen. Plasma membrane GAP activity declined at 72 h (maximal hyperplasia) but at 144 h (a time of declining cell proliferation) had returned to unstimulated control levels (Table 1; Fig. 2B). To assess the changes in subcellular distribution
of GAP that were associated with its
activation by tyrosine phosphorylation, membrane preparations were separated by a continuous sucrose gradient following the isolation of a low and high speed microsomal preparation (27,28). The low speed, 12,000 xg preparation contained plasma membrane sheets, lysosomes and mitochondria while the high speed 145,000 xg fraction contained plasma membrane vesicles, Golgi complex, endoplasmic reticulum and endosomes. When the later fraction was applied to a sucrose gradient, plasma membrane derived vesicles (as assayed by the acinar cell marker enzyme y-glutamyltranspeptidase; 29) were clearly separated from the low density vesicles containing and Golgi complex (assayed by l31-4 galactosyltransferase;
4, 5) (Fig. 3A). Western blot of fractions
from the high speed microsome preparation showed an increase in GAP protein associated with the Golgi complex fractions 10-14. Upon treatment with EGF (panel D and F) or isoproterenol (panel C and E), the amount of GAP protein associated with the low density vesicles was significantly increased. GAP associated with low density vesicles fractions increased 2-fold over control levels at 15 min and IO-fold at 72 h (Table 1). Detection of the EGF-R was confined to the high density microsomal fraction (plasma mebrane fraction) in untreated acinar cells but was detected in the low density microsomal fraction following treatment with the growth factor or R-agonist. This observation is consistent with receptor internalization into endosomes (27,28).
The high density
microsomal fraction again showed a decrease in the levels of detectable GAP when separated by sucrose gradient (Table 1). Increased GAP was also found associated with the nuclear envelope. As shown in Fig. 4, GAP activity was increased in the nuclear membrane following either EGF or isoproterenol treatment at 72 h. Densitometer analysis revealed a 2-fold increase in GAP following isoproterenol treatment and a 5-fold increase following EGF treatment of acinar cells. There was no detection of GAP in the nucleoplasm from any of the three groups of animals (data not shown). Thus, the results presented for growth stimulation of rat parotid gland acinar cells showed a different membrane association pattern than has been commonly reported. GAP is typically localized in the cytosol of non-dividing cells (10, 11). Upon EGF or PDGF stimulation or in cells transformed by pp60src, GAP activity associated with the plasma membrane occurs (10-13). In contrast to the cytosolic serine phosphorylated form, membrane GAP shows increased tyrosine phosphoryiation presumably through the kinase activity of the receptor complex. In acinar cells tyrosine phosphorylation may take place by a non-receptor tyrosine kinase activity since GAP could not be immunoprecipitated along with the EGF-R. Since the activation ofp2lras requires the 1176
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!
0.1 ;g 56 z--0 4
8
12
16
Fraction
205 116.5
8
20
24
28
0.0 y
number
----
80 49.5
_‘
205
-
116.5
-
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-
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-
mr-.,a
Figure 3. Distribution of GAP on sucrose gradients of the Golgi enriched high speed microsomal fraction. Subcellular fractions were prepared from total cell lysates by centrifugation at 15,000 xg to obtain a plasma membrane, mitrochondria and lysosomeenriched fraction (low speed) and centrifugation at 145,000 xg of the supernatant fraction of the low speed preparation to obtain a Colgi complex, endoplasic reticulum and endosome enriched pool (high speed). Animals (pooled, n=6) in each group received either chronic injection of saline (panel B) isoproterenol for 1.5 min prior to killing (panel C); 15 min EGF injection prior to killing (panel D); 72 h of isoproterenol treatment (panel E); 72 h of EGF treatment (panel F). The low density vesicle fraction (10-13) is indicated by arrow.
participation
of bound GTP, it would appearthat GAP activation and translocation to the plasma
membraneis a late event in the progressionfrom stasisto active cell proliferation. The intracellular recycling of internalized ligand-receptor complexes in endosomesmay therefore play a role in mediating the subsequentphosphorlyation of GAP by virtue of this mechanismof protein sorting. PlasmamembraneGAP could be internalized with the EGF-R upon growth stimulation. This, however, would have to occur following the initial phosphorylation of phospholipaseCr and phosphatidylinositol 3-kinase.,since activated EGF-R from rat acinar cell surface fractions have been shownto co-immunoprecipitatethesetwo proteinsearly in acinarcell growth stimulation 1177
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2051165 604435kDa Figure 4. Distribution of GAP following isolation of the nuclear membrane. Parotid gland cell nuclei were isolated as described previously (6). Ten pg of the nuclear envelope were separated by 8% SDS polyacrylamide gel and stained for protein by Coomassie Brilliant blue R-250 (Dane1 A) or transferred to nitrocellulose and reacted with antibody to GAP and 125;:labelled protein A (panel B). Molecular weight standards are as in Figure 1. Lane 1, control rat parotid gland nuclear membrane; lane 2, ISO-treated acinar cell nuclear membrane; lane 3, EGF-treated parotid gland acinar cell nuclear membrane.
(9; unpublishedresults). Activation of p21ras could therefore proceed by stimulation of the RasGuanine nucleotide exchange factor (19, 22, 31). As acinar cell hyperplasia is down-regulated, reappearanceof GAP at the cell surfacecould act upon p21‘as-GTP to stimulate GTP hydrolysis and thus inactivate p21rassignal transduction. Alternatively, GAP alone may be taken up into endosomes,independentof EGF-R and recycled to the plasmamembrane. Indeed we have found GAP in low density vesiclesof unstimulatedcontrols (Fig. 3B). In this mechanism,GAP might be storedin endosomesor the Golgi complex asseenin certain receptor densitizationmechanisms (32). Tyrosine phosphorylation could occur in a membranecompartmentindependentof receptor kinaseactivity. GAP itself may have other effector functions besidesinteraction with p21rasduring the early transition to growth. Growth factor stimulation or transformation by tyrosine kinase genes, also leads to the phosphorylation of p62 and ~190 (10-13). Recently, p62 and ~190, which interact with GAP, have been purified and cDNA clones isolated. Analysis of p62 revealed extensive sequencesimilarity to a putative hnRNA protein, GRP33. p62 has the ability to bind single strandedDNA and RNA and appearsto be localized, in part, to the cell nucleus(33). ~190 is localized within the cytoplasm, and nucleus, and its sequenceincludes predicted protein homology to the transcriptionalrepressorof glucorticoid receptor (GRF-l),BDR, n-chimaerin and the GTPase superfamily (34). These two reports speculatethat GAP can transducesignalsfrom p21rasthrough GAP-protein complexesat sitesdistal to the plasmamembrane.Thus the early loss of GAP from the plasmamembrane,and from the cytoplasm, in stimulated acinar cells may be directly related to theseother cellular functions requiredfor the transitionfrom quiescenceto active proliferation asobservedhereby the localization of increasedGAP to the nuclearmembrane.
Acknowledgments This work was supported by NIH grants DE 08778 and DE 00291 (MHB); DE 02110 (CAS); and DK 33326 (WAD). The authors thank Dr. JacksonGibbs for his generousprovision 1178
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of antibody to bovine GAP 677 and 638. Acknowledgment is given to Ms. Marilyn Lietz for preparationof the manuscript.
References :: 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
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