Proc. Nati. Acad. Sci. USA Vol. 83, pp. 8191-8195, November 1986 Cell Biology Protein tyrosine phosphorylation in the cell cycle of BALB/c 3T3 fibroblasts (antiphosphotyrosine antibody/growth factor/cydoheximide) ALEX 0. MORLA AND JEAN Y. J. WANG* Department of Biology, C-016, University of California at San Diego, La Jolla, CA 92093 Communicated by S. J. Singer, July 14, 1986

ABSTRACT Cell cycle-dependent regulation of protein affinity antibody revealed that tyrosine-phosphorylated pro- tyrosine phosphorylation in normal BALB/c 3T3 fibroblasts teins are concentrated at cell-substratum contact points and was examined by immunoblotting with a high-affinity antibody intercellular junctions (19). In this report, we describe the specific for phosphotyrosine. At least 15 different tyrosine- detection of tyrosine-phosphorylated proteins in synchro- phosphorylated proteins are found in normal 3T3 cells. The nized mouse 3T3 cells. The levels of P-Tyr-containing pro- level oftyrosine phosphorylation is higher in growing cells than teins were found to be regulated by serum as well as by the in quiescent cells. However, a prominent tyrosine-phosphoryl- cell cycle in mouse fibroblasts. ated protein ofMr 150,000 is present in quiescent cells, and its level is inversely proportional to the growth rate of these fibroblasts. Stimulation of quiescent cells with serum causes a METHODS major, yet transient, increase in tyrosine phosphorylation. The Cell Culture. BALB/c 3T3 cells, clone A31, were cultured immediate tyrosine phosphorylation reactions in response to in Dulbecco's modified Eagle's medium (DMEM) supple- serum stimulation are independent of protein synthesis, but mented with 10% heat-inactivated calf serum (CS, obtained tyrosine phosphorylation reactions occurring later in the G, from GIBCO). Growth arrest was achieved by incubating phase of the cell cycle are inhibited by cyclohexinmide. Thus, cells (106 per 10-cm dish) in DMEM plus 0.5% CS for 48 hr. tyrosine phosphorylation ofproteins in normal 3T3 cells occurs At time zero, the arrested cells were stimulated with 20%o CS, predominantly at the Go to G1 transition of the cell cycle. platelet-derived growth factor (PDGF, kindly provided by Maintenance of steady-state tyrosine phosphorylation is de- L. T. Williams, Howard Hughes Medical Institute, Univer- pendent on the presence of serum, but at least one tyrosine sity of California, San Francisco), or 20% CS plus 100 ,ug of phosphorylation reaction occurs in the absence of cell growth. cycloheximide per ml as indicated. At given time points after serum stimulation, cell monolayers were washed twice with Reversible phosphorylation ofproteins is an important mech- Dulbecco's phosphate-buffered saline without Ca2+ or Mg2+ anism for regulation of protein function. In normal cells, a (PBS) and incubated at 40C in PBS plus 5 mM EDTA for 1 very small percentage of protein phosphorylation occurs on min. Cells were scraped offthe plate with a rubber policeman tyrosine residues (1, 2). However, several retrovirus-trans- and collected by centrifugation for 30 sec in an Eppendorf formed cell lines contain high levels of tyrosine-phosphoryl- centrifuge. This procedure retrieved all of the cellular com- ated proteins (1-3). The oncogenes of those retroviruses ponents. Detergent extraction of scraped plates did not yield encode tyrosine kinases that are essential to their transform- any detectable P-Tyr proteins. The collected cells were lysed ing activity (2, 4, 5). Tyrosine phosphorylation ofproteins has in NaDodSO4 sample buffer (67 mM Tris'HCl, pH 6.8/10 mM also been implicated in the regulation ofcellular responses to EDTA/2% NaDodSO4/10% glycerol/0.3% 2-mercaptoetha- mitogens, as several growth factor receptors have been found nol/0.03% bromophenol blue) at 2 x 107 cells per ml at 100°C to contain tyrosine kinase activity (5-9). Although a single for 10 min. oncogenic tyrosine kinase can induce the transformation of Immunoblots. Cell lysates (25 ul) were electrophoresed in fibroblasts, progression of BALB/c 3T3 fibroblasts through Laemmli (20) NaDodSO4/8% polyacrylamide gels. Proteins the cell cycle requires both competence and progression were transferred onto 0.45-,um nitrocellulose (Schleicher & growth factors, activating more than one receptor tyrosine Schuell) filters in 25 mM Tris/19 mM glycine/0.1% kinase (10-12). Recent studies (13-15) on the distribution of NaDodSO4 plus 20% methanol at 1.5 A for 30 min at room tyrosine kinases in normal tissues indicate that protein temperature with a Hoefer heat-exchange manifold. The be restricted the filters were stained with 0.1% amido black in 45% metha- tyrosine phosphorylation may not to regu- nol/10% acetic acid and destained in 10% methanol/10% lation of cell proliferation, because high levels of the cellular acetic acid. Filters were equilibrated with Tris/NaCl buffer src gene product, pp60csrc, are found in nonproliferating cells (50 mM Tris'HCl, pH 7.5/150 mM NaCl) and then incubated such as neurons, neural retina, and platelets. in Tris/NaCl buffer with 5% bovine serum albumin (BSA, Elucidation ofthe functions oftyrosine kinases depends on Pentex brand, Miles) overnight at room temperature. Then the identification of physiological substrates of these en- the filters were incubated for 3 hr with affinity-purified zymes. It has been shown by Ross and coworkers (16, 17) that anti-P-Tyr antibodies (18) at 0.3 ,ug/ml in Tris/NaCl buffer antibodies specific for phosphotyrosine (P-Tyr) can be used containing 5% BSA and 0.2% Nonidet P-40 (NP-40). After to identify tyrosine-phosphorylated proteins. Recently, a extensive washing with 0.5% BSA and 0.2% NP-40 in high-affinity antibody for P-Tyr was isolated in our labora- Tris/NaCl buffer, the filters were incubated for 40 min with tory (18). This antibody reacted well with tyrosine- "II-labeled protein A (ICN, >30 ,ACi/pg; 1 Ci = 37 GBq) at phosphorylated proteins in normal cells and tissues. Im- 1 ,Ci/ml in Tris/NaCl buffer plus 5% BSA and 0.2% NP-40. munofluorescence staining of normal cells with this high- The filters were again washed extensively with 0.5% BSA

The publication costs of this article were defrayed in part by page charge Abbreviations: P-Tyr, phosphotyrosine; PDGF, platelet-derived payment. This article must therefore be hereby marked "advertisement" growth factor. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

8191 Downloaded by guest on September 26, 2021 8192 Cell Biology: Morla and Wang Proc. Natl. Acad. Sci. USA 83 (1986) and 0.2% NP-40 in Tris/NaCl buffer, air-dried, and autora- A diographed with an x-ray film plus intensifying screen at 0 1 2 3 4 5 6 7 8 9 10 11 -800C. 210 - 180- 150 - RESULTS Cl) 100 - Sensitivity of the Affmiity-Purified Anti-P-Tyr Antibody in 0 Immunoblotting. The level ofP-Tyr in mouse 3T3 fibroblasts x is only 0.03% ofthe total phospho amino acids (21), indicating 75 - * that the levels oftyrosine-phosphorylated proteins are low in these cells. An antibody for P-Tyr would not be useful for the study of tyrosine phosphorylation in untransformed cells unless it can detect low levels ofthese proteins. To determine the sensitivity of our detection method, affinity-purified anti-P-Tyr antibody was hybridized to a v-abl protein purified from bacteria (22). v-abl protein produced in bacteria con- tains 1 mol of P-Tyr per mol of protein (23). Under our B immunoblotting conditions, the anti-P-Tyr antibody could 0 1 2 3 4 5 A 7 8 9 10 11 210 - detect as little as 1 ng of this protein (Fig. 1). Thus, our 180 - ^ .2 immunoblotting method can detect a stoichiometrically 150 - 100 - I= tyrosine-phosphorylated protein of Mr 100,000 in a crude sl=srw lysate of 106 cells if the protein is present at 6000 copies per cell. 75 - Cell 60 - Cycle Regulation of Tyrosine Phosphorylation. The 55 - :. ::: #; :. .. :.; method of. immunoblotting was used to study protein tyrosine 43 - phosphorylation in BALB/c 3T3 fibroblasts. These cells 40 - - were synchronized by serum starvation and induced to enter 38 P the cell cycle by the addition of20% serum (see Methods). As CO) expected, serum stimulation caused the appearance of sev- 0 eral tyrosine-phosphorylated proteins. The most prominent xI. * we>twof. .0ECl.> band corresponded to an apparent Mr of 180,000 (Fig. 2A, 12 13 14 15 16 17 18 19 20 21 22 24 lane 1). Three other bands at Mr 210,000, 100,000 and 75,000 210 - 180 - were also stimulated by serum (Fig. 2A, lane 1). The 150 appearance of these four P-Tyr proteins could be detected as 100 - early as 5 min after the addition of either serum or purified to PDGF the growth-arrested cells (data not shown). Thus, 75-- Ve q U

these serum-stimulated P-Tyr proteins are to be the 60 - .- , likely z, -- qw substrates for the PDGF receptor tyrosine kinase (24-26). 55- I. Longer exposure ofthe same blot showed that several fainter 43 - 40 - 4;*....l bands, at Mr 60,000, 55,000, and 38,000, were also induced by serum (Fig. 2B, lane 1; see also Fig. 4). As cells progressed through the G1 phase of the cell cycle, the intensity of all of the serum-stimulated bands decreased to a basal level (Fig. 2A, lanes 1-9). On the other hand, intensity of the Mr FIG. 2. Tyrosine-phosphorylated proteins in synchronized 60,000-65,000 and 40,000-43,000 P-Tyr protein bands in- BALB/c 3T3 cells. Cells were harvested at the given times after serum stimulation and analyzed by immunoblotting. Number above creased with time in G1 to a steady-state level (Fig. 2B, lanes each lane corresponds to hours after addition of 20%o serum to 2-9 for the Mr 60,000-65,000 bands, lanes 5-9 for the Mr quiescent cells. Autoradiographic exposure time was 5 hr for A and 40,000-43,000 bands). 20 hr for B.

Nine hours after the addition of serum, a stable pattern of -15 P-Tyr protein bands was obtained (Fig. 2B, lanes 9-24). The pattern found in the 9- to 24-hr samples is virtually indistinguishable from that found in lysates of unsynchro- nized cells. The same pattern ofP-Tyr proteins is also found in cells arrested at the S phase of the cell cycle by a double thymidine block. Release of these S phase-arrested cells did not induce any discernible change in the P-Tyr proteins (data not shown). Fifteen is a minimal number of tyrosine- phosphorylated proteins in these fibroblasts. The antibody detected several faint P-Tyr proteins of Mr 36,000, 32,000, 31,000, 27,000, 25,000, and 19,000 when the cell lysates were 120 6030 15 8 4 2 1 0.5 0.3 0.1 analyzed in higher-percentage polyacrylamide gels. The in- tensity of a pair of P-Tyr protein bands at Mr 30,000-32,000 FIG. 1. Sensitivity of immunoblotting with anti-P-Tyr antibody. were found in several experiments to increase at 18 hr after Serial dilutions of the protein produced in bacteria from plasmid the addition of serum; however, this increase was not ptabll3O (22), a 1-mol/mol P-Tyr protein, were analyzed by immu- consistently observed. noblotting as described in Methods. Amount (ng) of protein loaded Quiescent 3T3 cells contained low levels of the P-Tyr is indicated below each lane. Exposure time is 20 hr. proteins found in growing cells. Most of the bands found in Downloaded by guest on September 26, 2021 Cell Biology: Morla and Wang Proc. Natl. Acad. Sci. USA 83 (1986) 8193 A B C D 24 72 0 1 mmI 1 F r - 1 5 9 2 6 10 3 7 11 4 8 12 210- .";z m 180 - 150- 210 -r b :c4 ....f, 180- .jJ x 100- V, i.. 1 50 - 1 100 - * Ot' CIO 75- 0 -4 Tx f x 75 - 4% i 4 60 - N hi 40 - 4.j1 ,a FIG. 5. Level of M, 150,000 P-Tyr protein in unsynchronized FIG. 3. Specificity of immunoblotting with anti-P-Tyr antibody. BALB/c 3T3 cells. Cells were plated and then incubated under the BALB/c 3T3 cells were harvested at the given times after serum following conditions: lane 24, 24 hr in 10o serum; lane 72, 72 hr in stimulation and analyzed by immunoblotting. Numbers above lanes 10%o serum; lane 0, 48 hr in 0.5% serum; lane 1, 48 hr in 0.5% serum correspond to time points as in Fig. 2. Blots were probed with followed by 1 hr in 20% serum. Cells were harvested and analyzed anti-P-Tyr antibody alone (A) or in the presence of 40 mM as described in Methods. Autoradiographic exposure time was 10 hr. phosphoserine (B) or P-Tyr (C) or without antibody (III-labeled protein A only; D). Autoradiographic exposure time was 15 hr. test whether the serum-stimulated appearance of these tyrosine-phosphorylated proteins was dependent on protein the stable pattern could not be detected in serum-starved synthesis, cycloheximide was added to the arrested cells cells unless a very long exposure (>48 hr) was made of the simultaneously with the 20% serum. The presence of cyclo- blots. However, P-Tyr proteins of Mr 200,000, 170,000, heximide did not prevent the stimulation of early-appearing 150,000, and 95,000 were detected in lysates of growth- bands at Mr 210,000, 180,000, 100,000, 75,000, 60,000, 55,000, arrested fibroblasts with a 20-hr autoradiographic exposure and 38,000 (Fig. 4). However, the increase in the levels ofthe (Fig. 2B, lane 0). The Mr 150,000 band was the most Mr 60,000-65,000 and 40,000-43,000 proteins, which oc- prominent P-Tyr protein in quiescent cells. curred later in G1, was abolished in cycloheximide-treated Specificity of the Anti-P-Tyr Antibody. The bands detected cells (Fig. 4). Thus, the stimulation of these late-appearing by the anti-P-Tyr antibody were completely eliminated by the P-Tyr proteins seems to require protein synthesis. The addition of40 mM P-Tyr to the incubation mixture (Fig. 3C). addition of cycloheximide also had an effect on the decay of Phosphoserine or phosphothreonine at the same concentra- the early-appearing P-Tyr proteins. The intensity of these tion had no effect (Fig. 3B shows competition with P-Ser). bands remained high even after 20 hr (Fig. 4B). Quantitation Incubation with 125I-labeled protein A alone did not label any by densitometry showed the half-life of the early-appearing of bands was about 2- to 3-fold longer in the presence of these protein bands (Fig. 3D). The possibility that these cycloheximide. Curiously, the stabilizing effect of cyclohex- bands might contain phosphohistidine was ruled out by their imide on early-appearing P-Tyr proteins was not observed in resistance to treatment with 0.5 M HCl at 490C for 10 min, a cells stimulated with purified PDGF in the absence of serum. condition under which >99% of both w- and r-phosphohis- Addition of chloroquine (0.5 mM) with serum did not inhibit tidine are dephosphorylated (27). the decay of the early-appearing P-Tyr proteins, indicating Effect of Cycloheximide on Tyrosine Phosphorylation. To that the decay observed between hour 1 and hour 10 was not due to protein degradation by the lysosomal pathway. A B Unlike the serum-stimulated bands, the decay of the Mr I IrI 150,000 band was not inhibited in cycloheximide-treated cells 0 1 5 10 15 20 1 5 10 15 20 (Fig. 4). A slight acceleration of the decay of the Mr 150,000 band was observed in some experiments, but it was not 210 - I consistently found with cycloheximide treatment. The Mr 180 - VW 150,000 species was the most prominent tyrosine-phospho- 150 - --.A I ", rylated protein in growth-arrested cells, and the addition of 100 - pol wv C.,) serum caused a decrease in its intensity (Fig. 4). Nine hours after serum stimulation, the Mr 150,000 band was at one-tenth 0Tx 75- ^ the level found in serum-starved cells (Fig. 2A). The level of x this band could be correlated with the growth rate of 3T3 60 - fibroblasts. Cells in the exponential phase of growth (24 hr 55 - after seeding and sparse) contained a low level of this P-Tyr 43 - protein (Fig. 5, lane 24). A confluent culture (72 hr after 40 f seeding, with no feeding) contained a higher level of the Mr 38 150,000 P-Tyr protein (lane 72), and a growth-arrested culture contained the highest level (lane 0). FIG. 4. Effect of cycloheximide on tyrosine phosphorylated proteins in synchronized BALB/c 3T3 cells. Cells were synchro- DISCUSSION nized by serum starvation and then stimulated with serum alone (A) or with serum plus 100 ,ug ofcycloheximide per ml (B). Lane numbers Immunoblotting with a high-affinity anti-P-Tyr antibody correspond to time points as in Fig. 2. Tyrosine-phosphorylated allowed the detection of tyrosine-phosphorylated proteins in proteins were detected by immunoblotting. Autoradiographic expo- lysates of normal mouse 3T3 fibroblasts. The antibody was sure time was 20 hr. shown to be specific for tyrosine-phosphorylated proteins Downloaded by guest on September 26, 2021 8194 Cell Biology: Morla and Wang Proc. Natl. Acad. Sci. USA 83 (1986) (18, 19). Examination of P-Tyr proteins in synchronized 3T3 phorylation is dependent on the presence of serum factors. cells by immunoblotting with this antibody established sev- However, one prominent P-Tyr protein of Mr 150,000 is eral observations. (i) The most dramatic alterations in found in quiescent cells. By immunofluorescence staining tyrosine phosphorylation are found when growth-arrested with the same anti-P-Tyr antibody, Maher et al. (19) have cells are stimulated with serum-i.e., at the Go to G1 shown that P-Tyr proteins are concentrated at focal contact transition of the fibroblast cell cycle. Several tyrosine- points ofnormal fibroblasts. The staining ofadhesion plaques phosphorylated proteins appear upon serum stimulation in is not affected by serum deprivation; i.e., the focal contact the absence of protein synthesis. By immunoprecipitating points ofquiescent 3T3 cells contain P-Tyr proteins. Because 32P-labeled proteins with their anti-P-Tyr antibody, Ek and the most prominent P-Tyr protein band in the lysates of Heldin (26) found an increase in tyrosine-phosphorylated quiescent 3T3 cells is the Mr 150,000 protein, it is possible proteins in human fibroblasts in response to PDGF stimula- that this protein is associated with the adhesion plaques. It is tion. The major PDGF-stimulated P-Tyr protein is the Mr interesting that the decay of the Mr 150,000 protein in 180,000 receptor for PDGF (24, 26, 28, 29). They also serum-stimulated cells is not inhibited by cycloheximide or detected P-Tyr proteins of Mr 200,000-300,000, 115,000, chloroquine. This observation indicates that the decay ofthis 72,000, 54,000, 45,000, and 35,000, which were believed to be P-Tyr protein is different from that of the P-Tyr proteins substrates of the PDGF receptor kinase (26). In our study, associated with a mitogenic response. The Mr 150,000 P-Tyr stimulation with serum or purified PDGF induced a major protein might be degraded through a cycloheximide-insensi- P-Tyr protein band at Mr 180,000 and other bands corre- tive pathway, or its dephosphorylation may be mediated by sponding to molecular weights similar to those described by phosphatases other than those involved in the response to Ek and Heldin (26). Thus the initial effect of serum stimula- growth factors. In unsynchronized populations of 3T3 cells, tion on tyrosine phosphorylation is likely due to the activa- we found the intensity ofthe Mr 150,000 band to be inversely tion of the PDGF receptor kinase. proportional to the growth rate ofthe cells. Thus, stimulation (ii) The intensity of the serum-stimulated P-Tyr protein of cell proliferation is correlated with both an increase and a bands decreases as cells progress through the cell cycle. This decrease in protein tyrosine phosphorylation in 3T3 cells. decay is not affected by chloroquine but can be partially In summary, an antibody for P-Tyr has revealed the major inhibited by cycloheximide. Binding ofgrowth factors to their tyrosine-phosphorylated proteins in untransformed mouse receptors is known to induce the endocytosis and degradation 3T3 fibroblasts. Most ofthe tyrosine-phosphorylation events of the receptors (i.e., the down-regulation of receptors; refs. in these cells are dependent on the presence of serum; 30 and 31). Cycloheximide has been shown to reduce the however, a few tyrosine-phosphorylated proteins are found down-regulation of the insulin receptor by slowing the in serum-starved cells. These tyrosine-phosphorylated pro- degradation of the receptor protein (32). It is possible that the teins can be purified by immunoaffinity column chromatog- decay of the serum-stimulated proteins is due to degradation raphy employing the anti-P-Tyr antibody, as has been done of the receptor kinase through a pathway that is sensitive to in the purification of PDGF receptor (33). A detailed char- cycloheximide but not to chloroquine. It is also possible that acterization of these tyrosine-phosphorylated proteins may the decay is caused by the action of tyrosine phosphatases lead to the understanding ofthe role oftyrosine kinases in the and that cycloheximide can inhibit either their synthesis or regulation of cellular functions. their activation. The inhibitory effect of cycloheximide was This work was supported by grants from the National Science not observed when the cells were stimulated with a saturating Foundation (PCM-8314300), the American Cancer Society, and the dose of purified PDGF. Perhaps the activation of the decay Camille and Henry Dreyfus Foundation. J.Y.J.W. is a Searle process requires new protein synthesis when cells are stim- Scholar. ulated with lower levels of PDGF, as are present in serum. A maximal stimulation of the PDGF receptor might activate 1. Hunter, T. & Sefton, B. M. (1980) Proc. Natl. Acad. Sci. USA 77, 1311-1315. degradation of the receptor kinase to an extent that it 2. Cooper, J. A. & Hunter, T. (1983) Curr. Top. Microbiol. overcomes the need for new protein synthesis and therefore Immunol. 107, 125-162. the inhibitory effect of cycloheximide. 3. Cooper, J. A. & Hunter, T. (1981) Mol. Cell. Biol. 1, 394-407. (iii) Although the major increase in tyrosine phosphoryl- 4. Bishop, J. M. (1983) Annu. Rev. Biochem. 52, 301-354. ation is observed immediately upon the addition of serum to 5. Hunter, T. & Cooper, J. A. (1985) Annu. Rev. Biochem. 54, quiescent cells, several P-Tyr proteins appear with a lag time. 897-930. For example, the Mr 65,000 and 43,000 proteins did not attain 6. Cohen, S., Carpenter, G. & King, L. R. (1980) J. Biol. Chem. their highest levels until 3-5 hr after serum addition. The 255, 4834-4842. intensity of a few other faint bands, at Mr 112,000 and 7. Ek, B., Westermark, B., Wasteson, A. & Heldin, C.-H. (1982) Nature (London) 295, 419-420. 80,000-85,000 also increased later in the cell cycle. Those 8. Sherr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F., increases are inhibited by cycloheximide, indicating that Look, A. T. & Stanley, R. E. (1985) Cell 41, 665-676. protein synthesis is required. These late-appearing P-Tyr 9. Jacobs, S., Frederick, C. K., Jr., Earp, H. S., Svoboda, proteins might be substrates for the receptor kinase of M. E., Van Wyk, J. J. & Cuatrecasas, P. (1983) J. Biol. Chem. insulin-like growth factor I, which has been shown to act at 258, 9581-9584. the restriction point of the G1 phase (12). The finding ofP-Tyr 10. Antoniades, H. N., Scher, C. D. & Stiles, C. D. (1979) Proc. proteins during G1 progression is consistent with the fact that Natl. Acad. Sci. USA 76, 1809-1813. more than one receptor tyrosine kinase is required for 3T3 11. Scher, C. D., Shepard, R. C., Antoniades, H. N. & Stiles, cells to complete the cell cycle (6-9, 13, 14). It is possible that C. D. (1979) Biochim. Biophys. Acta 560, 217-241. the substrates of the progression growth-factor receptor 12. Campisi, J. & Pardee, A. B. (1984) Mol. Cell. Biol. 4, 1807-1814. kinases are synthesized during Go to G1 transition and that 13. Brugge, J. S., Queral, A. E., Cotton, P. C., Barrett, J. N., tyrosine phosphorylation of these proteins is important for Nonner, D. & Keane, R. W. (1985) Nature (London) 316, the progression through the restriction point. The constant 554-557. level ofP-Tyr proteins found from hour 9 through hour 24 and 14. Golden, A., Nemeth, S. P. & Brugge, J. S. (1986) Proc. Natl. in S phase-arrested cells indicates that a steady state ofthese Acad. Sci. USA 83, 852-856. P-Tyr proteins is maintained throughout the cell cycle. 15. Sorge, L. K., Levy, B. T. & Maness, P. F. (1984) Cell 36, (iv) Most of the P-Tyr proteins found iwexponentially 249-257. growing cells are present at a very low level in serum-starved 16. Ross, A. H., Baltimore, D. & Eisen, H. N. (1981) Nature cells. Thus, the maintenance of steady-state tyrosine phos- (London) 294, 654-656. Downloaded by guest on September 26, 2021 Cell Biology: Morla and Wang Proc. Nati. Acad. Sci. USA 83 (1986) 8195

17. Frackelton, A. R., Jr., Ross, A. H. & Eisen, H. N. (1983) 26. Ek, B. & Heldin, C.-H. (1984) J. Biol. Chem. 259, Mol. Cell. Biol. 3, 1343-1352. 11145-11152. 18. Wang, J. Y. J. (1985) Mci. Cell. Biol. 5, 3640-3643. 27. Hultquist, D. E., Moyer, R. W. & Boyer, P. D. (1966) Bio- 19. Maher, P., Pasquale, E. B., Wang, J. Y. J. & Singer, S. J. chemistry 5, 322-331. (1985) Proc. Nad. Acad. Sci. USA 82, 6576-6580. 28. Glenn, K., Bowen-Pope, D. R. & Ross, R. (1982) J. Biol. 20. Laemmli, U. K. (1970) Nature (London) 227, 680-685. Chem. 257, 5172-5176. 21. Sefton, B. M., Hunter, T., Beeman, K. & Eckhart, W. (1980) 29. Heldin, C.-H., Ek, B. & Ronnstrand, L. (1983) J. Biol. Chem. Cell 20, 807-816. 258, 10054-10061. 22. Wang, J. Y. J. & Baltimore, D. (1985) J. Biol. Chem. 260, 30. Carpenter, G. & Cohen, S. (1976) J. Cell Biol. 71, 159-171. 64-71. 31. Gordon, P., Carpentier, J., Cohen, S. & Orci, L. (1978) Proc. 23. Foulkes, J. G., Chow, M., Gorka, C., Frackelton, A. R., Jr., Natl. Acad. Sci. USA 75, 5025-5029. & Baltimore, D. (1985) J. Biol. Chem. 260, 8070-8077. 32. Knutson, V. P., Ronnett, G. V. & Lane, M. D. (1985) J. Biol. 24. Frackelton, A. R., Jr., Tremble, P. M. & Williams, L. T. Chem. 260, 14180-14188. (1984) J. Biol. Chem. 259, 7909-7915. 33. Daniel, T. 0., Tremble, P. M., Frackelton, A. R., Jr., & 25. Cooper, J. A., Bowen-Pope, D. F., Raines, E., Ross, R. & Williams, L. T. (1985) Proc.82,5Natl. Acad. Sci. USA Hunter, T. (1982) Cell 31, 263-273. 2684-2687. Downloaded by guest on September 26, 2021