Regulation of a ribosomal protein S6 kinase activity by the Rous

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 82, pp , November 1985 Cell Biology Regulation of a ribosomal protein S6 kinase activity by the Rous sarcoma virus transforming protein, serum, or phorbol ester (pp6ov-rc expression/growth factors/phorbol 12-myristate 13-acetate/protein kinase C) JOHN BLENIS AND R. L. ERIKSON Department of Cellular and Developmental Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA Contributed by R. L. Erikson, July 18, 1985 ABSTRACT Protein kinase capable of phosphorylating 40S ribosomal protein S6 on serine residues has been detected in chicken embryo fibroblasts. This activity appears to be regulated in direct response to expression of pp6ovsrc in chicken embryo fibroblasts infected with a temperature-sensitive transformation mutant of Rous sarcoma virus. Partially purified S6 kinase was highly specific for S6 in 40S ribosomal subunits. The S6 kinase was not inhibited by calcium or by the heat-stable inhibitor of camp-dependent protein kinase, nor was it activated by phosphatidylserine, diacylglycerol, and calcium. Thus, it is distinct from protein kinase C and camp-dependent protein kinase, which are capable of phosphorylating S6 in vitro. The tumor-promoter phorbol 12-myristate 13-acetate also stimulated ribosomal protein S6 kinase activity in serumstarved chicken embryo fibroblasts, whereas phorbol, the inactive analog of phorbol 12-myristate 13-acetate, had no effect. S6 kinase activity stimulated by expression of pp60vzrc, by phorbol 12-myristate 13-acetate, or by serum growth factors exhibited similar chromatographic properties upon ion-exchange chromatography. These results suggest that a common protein kinase may be activated by three diverse stimuli all involved in regulating cell proliferation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. Modification of proteins by reversible phosphorylation has been shown to alter many cellular biochemical processes. The regulation of protein function through the phosphorylation of serine and/or threonine residues is well documented (1). During the past few years a number of oncogene products have been shown to be protein kinases specific for tyrosine residues (for reviews, see refs. 2 and 3), and a similar activity is associated with the membrane receptors for several growth factors (4). Changes in protein function as a result of phosphorylation on tyrosine residues have not been demonstrated. Nevertheless, a strong correlation exists between the expression of tyrosine-specific protein kinase activity and altered metabolism in cells transformed by retroviruses or stimulated by growth factors. Several transforming gene products and growth factors also stimulate the modification of proteins on serine residues presumably by altering the activity and/or the level of serine-specific protein kinase(s) and/or phosphoprotein phosphatase(s). One such protein of potential physiological importance is ribosomal protein S6. The phosphorylation of S6 is rapidly increased by treatment of quiescent cells with serum, with a variety of mitogenic agents, or with the tumor promoter phorbol 12-myristate 13-acetate (PMA) (5-14). Furthermore, even in the absence of such stimulating agents, S6 remains highly phosphorylated in cells transformed by several oncogenic viruses (15-17). Several lines of evidence link elevated S6 phosphorylation to initiation of protein synthesis, suggesting that this may be one of several events involved in the control of cell proliferation (11, 18-22). As a result, much effort has been directed recently to identification of the activity or activities responsible for altering S6 phosphorylation, and several protein kinases and cell-free lysates have been shown to phosphorylate S6 in vitro (23-31). Phosphorylation of S6 in quiescent cells stimulated by PMA, by serum growth factors, or following the expression of pp60vsrc [the transforming protein of Rous sarcoma virus (RSV)] occurs on similar phosphopeptides suggesting that a common mechanism of phosphorylation may be involved (13). A role for protein kinase C in S6 phosphorylation has been suggested because PMA activates this phospholipidand Ca2l-dependent kinase (32). It is unclear, however, whether this kinase is directly responsible for physiological S6 phosphorylation (23, 27). In this report, we describe properties of the S6 protein kinase activity stimulated by oncogenic transformation of chicken embryo fibroblasts (CEF). To regulate the degree of transformation of the cells, CEF were infected with a temperature-sensitive (ts) transformation mutant of RSV. Such cells (ts-cef) are transformed at the permissive temperature of 35 C but display normal morphology and response to serum-deprivation at-the nonpertnissive temperature of 41 C. Thus, after the appropriate temperature shift these cells should yield useful information on the activity of various enzymes that may be involved in growth control and cellular proliferation. Upon transfer of cultured ts-cef from the nonpermissive to permissive temperature or from the permissive to nonpermissive temperature, S6 phosphorylation and S6 kinase activity increased or decreased with kinetics similar to those of the activation and inactivation of pp6ov-src. This S6 kinase activity was chromatographically identical to S6 kinases from normal cells stimulated with PMA or with serum. The inter-relationships between pp60v-src expression, phorbol ester, and growth factors with respect to S6 phosphorylation and growth control are discussed. MATERIALS AND METHODS Cell Culture. CEF were prepared from virus-free 11- to 12-day-old chicken embryos (SPAFAS). Culture conditions for CEF or cells infected with a temperature-sensitive transformation mutant of RSV (72-4, obtained from H. Handfusa, Rockefeller University, New York) have been described (13). Biosynthetic labeling of cells and isolation of ribosomes were as previously described (13) except that cells were prelabeled with 1-2 mci (1 mci = 37 MBq) of H332PO4 (carrier free, ICN) per 100-mm tissue culture dish for 6-12 hr prior to temperature shift for all time-course experiments. Preparation of Cell-Free Lysates and Chromatographic Fractionation of S6 Protein Kinase. Data presented here were Abbreviations: RSV, Rous sarcoma virus; CEF, chicken embryo fibroblasts; ts-cef, chicken embryo fibroblasts infected with a temperature-sensitive transformation mutant of RSV; PMA, phorbol 12-myristate 13-acetate. 7621

2 7622 Cell Biology: Blenis and Erikson obtained from cells cultured at 41'C that were allowed just to reach confluence, were serum-starved for hr, and then were transferred to the permissive temperature (350C) in the case of ts-cef, or were incubated with dialyzed calf serum [10% (vol/vol) final concentration], phorbol (100 ng/ml), or PMA (100 ng/ml) in the case of normal cells. We have found that when cells, cultured to a~very high density (obtained by feeding confluent monolayers with fresh medium), were serum-starved for 24 hr, the kinetics of activation of S6 kinase and S6 biosynthetic labeling with H332PO4 were slightly slower, and those of inactivation of kinase activity were slightly faster than. those that are presented here. Cell-free lysates were prepareq by washing mnonolayers twice with cold lysis buffer (10 mm potassium phosphate/i mm EDTA/10 mm EGTA/10 mm MgCl2/100 mm glycerol 2- phosphate/1 mm sodium vanadate/5 mm dithiothreitol/40 pug of phenylmethylsulfonyl fluoride per ml, ph 6.5), gently scraping the cells off the dish in 1 ml of lysis buffer, and homogenizing cells in a Dounce homogenizer with 25 strokes of a tight-fitting pestle. After centrifugation at 10,000 x g for 15 min, the resulting supernatant was adjusted to 10% (vol/vol) glycerol and centrifuged at 100,000 X g for 2 hr. The supernatant (cell-free lysate) was stored at -50C. Cells or cell-free lysates were never frozen before analysis of S6 kinase. S6 kinase activity in cell lysates was fractionated by cation-exchange chromatography (Mono S, Pharmacia FPLC system), anion-exchange chromatography (Mono Q, Pharmacia), and gel filtration (Spherogel-TSK, Altex, Berkeley, CA). Partially purified enzyme was concentrated by dialysis against 10 MM potassium phosphate/i mm EDTA/10 mm EGTA/15 mm MgCl2/5 mm dithiothreitol/50% (vol/vol) glycerol, ph 6.5, and stored at -20'C. Assay of S6 Protein Kinase. 40S ribosomal subunits were prepared from Xenopus laevis ovaries as described by Erikson and Maller (25). S6 in these ribosomes is almost completely dephosphorylated and, thus, provides a convenient substrate (33). Phosphotransferase activity in the various lysates was determined with equal amounts of lysate protein, 0.16 A260 unit of 40S ribosomal subunits, 15 mm MgCl2, 50 AxM ATP (20 UCi of [y32p]atp), and 20 mm Tris HCl, ph 7.5. Radiolabeled ATP was prepared by published methods (34). Reaction mixtures (25,ul) were incubated for 15 min at 30 C and were stopped by the addition of 6.5,ul of five times concentrated gel electrophoresis sample buffer (35) followed by boiling for 5 min. Gel Electrophoresis. Proteins were resolved by NaDod- S04/12% polyacrylamide gel electrophoresis as described by Laemmli (35). Coomassie blue-stained gels were dried and exposed to DuPont Cronex-4 film with DuPont Lightning Plus intensifying screens. Radiolabeled S6 was identified, excised, and associated radioactivity was quantitated by liquid scintillation spectrometry. RESULTS Transformation-Dependent S6 Kinase Activity. In ts-cef, phosphotylation of ribosomal protein S6 increases toward maximal levels (4-5 mol of phosphate/mol of S6) in response to pp60-src activity (13, 15). To determine whether this growth factor-independent, transformation-dependent process reflected a change in the specific activity of a S6 kinase, cell-free lysates were analyzed for their ability to directly and specifically phosphorylate S6 in 40S ribosomal subunits. Fig. LA (lanes 1-4) shows the results obtained when S6 kinase activity in a cell-free lysate of ts-cef grown at 41 C was compared to the S6 kinase activity in a lysate from transformed cells (35TC). Differential phosphorylation of S6 (Mr -32,000) was consistently detected and was generally 3- to 6-fold greater in lysates from cells cultured at the permissive Proc. Natl. Acad. Sci. USA 82 (1985) 5 6 B _ S6 FIG. 1. Identification of transformation-dependent S6 kinase activity. (A) Cell-free lysates (10 Aul, equal amounts of protein) from ts-cef, cultured at 41'C (lanes 1 and 3) or 35 C (lanes 2 and 4) for 2 hr, were incubated without or with 40S ribosomal subunits (0.16 A260 unit) in the presence of 50 AiM ATP (20 itci of [y32p]atp per assay) and 15 mm MgCl2 for 15 min at 30TC. Reactions (25 Al) were stopped by the addition of 6.5 dl of five-times-concentrated electrophoresis sample buffer, and radiolabeled proteins were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and. autoradiography. Cell-free lysates from ts-cef, cultured at 41'C or 350C, were further purified, in parallel, by anion- and cation-exchange chromatography and gel filtration. Assuming equal recovery on all columns, equal amounts of partially purified S6 kinase activity from 41TC ts-cef (lane 5) or 35TC ts-cef (lane 6) were incubated with 408 ribosomal subunits and analyzed as above. (B) An autoradiogram was obtained from two-dimensional thin layel electrophoretic analysis of phosphoamino acids that were prepared from S6 labeled in vitro by the partially purified S6 kinase preparation as in A, lane 6, S6 denotes the location of a Mr 32,000 phosphoprotein which comigrates with biosynthetically labeled ribosomal protein S6 on NaDodSO4/PAGE and with Coomassie blue-stained ribosomal protein S6 on two-dimensional gels. ps, phosphoserine; pt, phosphothreonine; py, phosphotyrosine. temperature for transformation than at temperature (assayed 2 hr after temperature shift, 410 to PT py the nonpermissive 350C). Several proteins were phosphorylated in the cell lysates in the presence or absence of 40S subunits (Fig. LA, lanes 1-4); however, differential phosphorylation of these substrates was not observed. The observed difference iii specifil activity was maintained even after S6 kinase activity from normal and transformed cell lysateg was partially purified by anion- and cation-exchange chromatography and gel filtration chromatography (Figs. LA, laneg 5 and 6). The reaction of 40S subunits with the partially purified preparations of 41 C and 35 C enzymes (Fig. LA, lanes 5 and 6) also demonstrates the high degree of S6 specificity of these preparations. The phosphorylation of S6 in Vitro occurred solely at serine residues (Fig. 1B). Several protein kinases have been reported to phosphorylate S6 in 40S subunits in vitro (23-31). Of these, two are of particular interest as they are regulated by the putative second messengers camp (camp9dependent protein kinase) or diacylglycerol (protein kinase C). In Fig. 2A, lanes 1-4, the partially-purified S6 kinase.is compared to camp-dependent protein kinase in the presence and Absence of the heat-stable inhibitor of camp-dependent protein kinase (36). Both kinases have equal phosphotransferase activity toward S6 (lanes 1 and 2), but, at equal concentrations ofthe heat-stable inhibitor of camp-dependent protein kinase, the ability of camp-dependent protein kinase to phosphorylate S6 was completely inhibited whereas the transformation-dependent S6 kinase activity was unaffected (lanes 3 and 4, respectively). Fig. 2B indicates that the transformation-dependent S6 kinase activity was unaffected by concentrations of up to

3 Cell Biology: Blenis and Erikson A B C @ 1 F- 0 > ' U) (D U)- x Proc. Natl. Acad. Sci. USA 82 (1985) 7623 (O U) z J LLJ to Cal x 2 FIG. 2. Effect of heat-stable protein kinase inhibitor, calcium, and phospholipid on S6 kinase activity. Assays were performed as described in Fig. 1. (A) Lanes 1 and 2, assay of equivalent phosphotransferase activity toward 40S ribosomal protein S6 by purified catalytic subunit of camp-dependent protein kinase and partially purified S6 kinase (from ts-cef, 350C), respectively; lanes 3 and 4, identical reactions as above with the inclusion of an equal concentration of the heat-stable inhibitor of camp-dependent protein kinase. (B) In vitro S6 kinase assay with partially purified S6 kinase without calcium (lane 1) or with 500,uM CaCl2 (lane 2, final concentration of Ca2" that is in excess of EGTA). (C) Partially purified S6 kinase isolated from ts-cef cultured at 41TC (lanes 1 and 2) or transferred to 35TC (lanes 3 and 4) and assayed in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 120 /xg of phosphatidylserine per ml, 4,ug of diacylglycerol per ml, and 50 AuM CaCl2 in excess of the EGTA. Lipids (lox) were added from an emulsion prepared by sonication in 20 mm Tris HCl, ph 7.6 (three times for 3 min) in a Heat System/Ultrasonics (Plainview, NY) (W-375) sonicator set at 20% of full scale. 500 AxM CaCl2. Results in Fig. 2C demonstrate that both the 41'C (lanes 1 and 2) and 350C (lanes 3 and 4) partially purified S6 kinase were unaffected by the inclusion of phosphatidylserine, diacylglycerol, and Ca2+ in the reactions. Under identical conditions, histone H1 phosphorylation by protein kinase C was activated about 10- to 20-fold (data not shown), in agreement with published results (37). Regulation of S6 Protein Kinase Activity in ts-cef. To determine whether a change in S6 kinase specific activity correlated with changes in pp60v-s' activity and biosynthetic phosphorylation of S6 in cultured cells, the kinetics of activation of the S6 kinase were measured following transfer of cultured cells from the nonpermissive (410C) to the permissive (350C) temperature for transformation (Fig. 3). This was done by preparing cell-free lysates from serumstarved ts-cef at various times following temperature shift and assaying for S6 kinase activity by using equal amounts of lysate protein with unphosphorylated 40S subunits in the presence of MgCl2/[y-32P]ATP as described above. Rapid activation of the kinase activity was observed with maximal activation usually observed within 1 hr. Furthermore, the increase of kinase activity following temperature shift (410C to 350C) was followed closely by a steady increase in phosphorylation of S6 in cultured serum-starved ts-cef (Fig. 3) as well as a shift from the unphosphorylated species to the more highly phosphorylated species of S6, as determined by two-dimensional gel electrophoresis of ribosomes (data not shown). The shift to the highly phosphorylated species of S6 was nearly complete as determined by Coomassie blue staining of gels. Maximal activation and expression of pp60v-src usually requires approximately 2 hr (38) following transfer of cultured ts-cef to the permissive temperature. However, the more rapid activation of the S6 kinase is not surprising, since experiments with inducible promoters linked to v-src demonstrate that only a fraction of the FIG. 3. Transformation-dependent activation of S6 kinase activity and biosynthetic phosphorylation of S6 in cultured ts-cef. Cell-free lysates from ts-cef (4.4 ug of protein per assay) were incubated with 40S ribosomal subunits, [y32p]atp, and MgCl2 and were analyzed. Following NaDodSO4/PAGE and autoradiography of the dried gel, the S6 band was excised and radioactivity was measured by scintillation spectroscopy. Data are plotted as percent maximal S6 kinase activity obtained during the 2-hr time course versus time after transfer of culture dishes from the nonpermissive (410C) to the permissive (350C) temperature (o). Incorporation of 32p into S6 in cultured ts-cef was determined by excising radiolabeled S6 from dried two-dimensional polyacrylamide gels (11). Data are plotted as percent maximal phosphorylation of ribosomal protein S6 obtained during the 2-hr time course versus time after temperature shift (e). The kinetics of activation of S6 kinase activity and S6 phosphorylation were slightly affected by cell density and, therefore, in this comparison equivalent cell densities were used. pp60vl-c levels usually found in infected CEF is required for oncogenic transformation (39). Transfer of cells cultured at the permissive temperature (350C) to the nonpermissive temperature (410C) for 30 min results in a 50% reduction of pp60v-src tyrosine-specific protein kinase activity (38). Following a shift of ts-cef from 35TC to 41TC the S6 kinase also showed a 50% inactivation within 30 min (Fig. 4). The rate of the inactivation of the S6 kinase strongly suggests that a close link exists between pp60v-src and S6 kinase. pp60vw, Serum Growth Factors, and PMA Stimulate Similar S6 Kinases. S6 protein kinase activity was assayed in cell-free lysates prepared from serum-deprived normal CEF stimulated with serum or with PMA. As shown in Fig. 5, incubation of serum-deprived quiescent cells with 10% serum 100 < 80 LiJ z TIME, min TIME, hr FIG. 4. Transformation-dependent inactivation of S6 kinase. ts-cef were transferred to 350C for 5 hr. At zero time plates were transferred to the nonpermissive temperature (410C) and at various times cell lysates were prepared and assayed for S6 kinase as described in Fig. 3.

4 7624 Cell Biology: Blenis and Erikson Proc. Natl. Acad Sci. USA 82 (1985) a a pạ '*4*1 a*s6 - I _~~ - z FIG. 5. Identification of activated S6 kinase activity in CEF incubated with PMA or serum growth factors. Cell-free lysates were prepared and analyzed as described in Fig. 1. Lane 1, ts-cef at 410C; lane 2, ts-cef at 350C (2 hr); lane 3, CEF; lane 4, CEF incubated with 10% (vol/vol) serum for 1 hr; lane 5, CEF incubated with 100 ng of phorbol per ml for 1 hr; and lane 6, CEF incubated with 100 ng of PMA per ml for 1 hr. An equal amount of protein (4-10 /ig) was used in each pair of assays. for 1 hr resulted in increased S6 kinase activity (lane 4) in comparison to untreated cells (lane 3). Similarly, treatment of serum-deprived CEF for 1 hr with PMA (100 ng/ml) also resulted in elevation of S6 kinase activity (lane 6) whereas, the inactive analog ofpma, phorbol (100 ng/ml) had no effect (lane 5). In an initial effort to compare the S6 kinase activities influenced by serum growth factors, a tumor promoter, or expression of an active oncogene product, cell lysates were subjected to ion-exchange chromatography, and the fractions were assayed for S6 kinase activity. Under the experimental conditions described here, the S6 kinase from the three types of cells eluted from a Pharmacia Mono Q anion-exchange column at salt concentration of =225 mm (Fig. 6 A-C), and from a Pharmacia Mono S cation-exchange column at 240 mm NaCl (Fig. 6 D-F). Analysis of the low level of S6 kinase activity detectable in unstimulated CEF or ts-cef cultured at 41 C also revealed the same chromatographic behavior on both ion-exchange columns. In addition to the similar behavior of these S6 kinases by ion-exchange chromatography, we have observed identical elution profiles from gel filtration columns (data not shown). DISCUSSION In this report we describe a ribosomal protein S6 kinase regulated by the expression of the RSV transforming gene product, pp60v-src. Transfer of ts-cef from the nonpermissive to the permissive temperature resulted in a rapid increase of S6 kinase activity. Following transfer from the permissive to the nonpermissive temperature, S6 kinase activity rapidly decreased with kinetics parallel to the inactivation of pp6ov-src protein kinase. In contrast to pp60v src, which is a tyrosine-specific protein kinase unable to significantly phosphorylate 40S subunits, a partially purified preparation of the S6 protein kinase phosphorylated S6 in vitro on serine residues. In ts-cef, metabolic labeling of S6 with H332P04 after shift to the permissive temperature was correlated with the increase in S6 protein kinase activity suggesting that the enzymatic activity may have a significant physiological role in S6 phosphorylation. The S6 protein kinase is distinct from two other wellstudied serine-specific protein kinases in that it is not affected by the heat-stable inhibitor of the cyclic AMP-dependent protein kinase and that it is phospholipid- and Ca2+-independent, two requirements for protein kinase C. In this regard it FRACTION NUMBER FIG. 6. Comparison of S6 kinase activity stimulated by pp6ov-src expression, serum growth factors, or PMA. Cell-free lysates from serum-starved ts-cef cultured at 35 C for 28 hr and lysates from serum-starved CEF cultured for 1 hr with 10% (vol/vol) serum or 100 ng of PMA per ml were fractionated by anion- and cation-exchange chromatography using the Pharmacia FPLC system with Mono Q or Mono S columns. Mono Q column buffer was 20 mm Tris HCl (ph 7.5, 4 C)/5.5 mm EGTA/2 mm EDTA/15 mm MgCl2/10 mm 2-mercaptoethanol/10% (vol/vol) glycerol. The Mono S column buffer was 10 mm potassium phosphate/s mm EGTA/1 mm EDTA/10 mm MgCl2/10 mm 2-mercaptoethanol/10%o (vol/vol) glycerol, ph 6.5. Approximately 500,ug of lysate protein was applied to each column. Both columns were eluted with a mm linear gradient of NaCl, and 1-ml fractions were collected. Aliquots (10 IL) of alternate fractions were assayed. A-C, Anion-exchange chromatography; D-F, cation-exchange chromatography. A and D, Serumstarved ts-cef (35 C for 28 hr); B and E, serum-starved CEF incubated with 10o (vol/vol) calf serum for 1 hr; C and F, serum-starved CEF incubated with 100 ng of PMA per ml for 1 hr. is of interest that S6 kinase activity stimulated by incubation of CEF with PMA was chromatographically similar to that stimulated by pp6ov-src or serum addition. PMA can activate protein kinase C; however, this enzyme is not the major in vitro kinase for S6 phosphorylation in PMA-stimulated cells. The most highly purified and specific S6 kinase reported to date, purified from Xenopus eggs (25), is also clearly distinct from those two well-studied serine-specific protein kinases; however, its relationship to the S6 kinase from CEF remains to be determined. A report on a serum-stimulated S6 protein kinase in lysates of 3T3 cells suggested S6 phosphorylation was inhibited by Ca2+ (28), whereas the activity described here from CEF is unaffected by Ca2+ in cell-free lysates (data not shown) or after partial purification (Fig. 2). Additional studies on the two enzymes should resolve this apparent discrepancy. It is important to determine the mechanism of S6 kinase activation. The data presented here raise a number of possibilities concerning the pathways that lead to the stimulation of S6 kinase activity. Whereas pp6ov-src or growth factors in serum may utilize tyrosine phosphorylation of various polypeptides to exert their control, the only known receptor for phorbol ester is protein kinase C (for review, see ref. 40). This enzyme phosphorylates serine or threonine residues in protein substrates. Activation of protein kinase C is known to rapidly stimulate the phosphorylation of a Mr 42,000 protein on tyrosine (41-43), and, therefore, this

5 Cell Biology: Blenis and Erikson enzyme must stimulate a tyrosine-specific protein kinase or inhibit a phosphotyrosine-specific protein phosphatase. Thus, there exists a potential for the regulation of the S6 kinase activity by reversible tyrosine phosphorylation. Alternatively, the activation of growth factor receptors or oncogene products may stimulate the formation of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (44), which would lead to a more active protein kinase C, and the S6 kinase may be activated by the protein kinase C phosphorylation of serine or threonine. Understanding the nature of the S6 kinase, which is cellular in origin and which is activated by proteins and agents that play an obvious role in malignant transformation and growth control, may yield information on the biochemical events regulating cell division. We thank Eleanor Erikson and Thomas A. Langan for comments on the manuscript, Gordon Foulkes for a gift of the heat-stable inhibitor of camp-dependent protein kinase, and James Maller and Yossi Graziani for a gift of camp-dependent protein kinase. This research was supported by Public Health Service Grant CA from the National Institutes of Health and by a grant from the American Business Cancer Research Foundation. J.B. was supported by a Damon Runyon-Walter Winchell Postdoctoral Fellowship and now holds a fellowship from the National Institutes of Health. R.L.E. is an American Cancer Society Professor of Cellular and Developmental Biology. 1. Krebs, E. G. & Beavo, J. A. (1979) Annu. Rev. Biochem. 48, Erikson, R. L., Purchio, A. F., Erikson, E., Collett, M. S. & Brugge, J. S. (1980) J. Cell Biol. 87, Hunter, T. & Cooper, J. A. (1984) in Advances in Cyclic Nucleotide and Protein Phosphorylation Research, eds. Greengard, P., Robison, G. A., Paoletti, R. & Nicosia, S. (Raven, New York), Vol. 17, pp Heldin, C.-H. & Westermark, B. (1984) Cell 37, Chambard, J.-C., Franchi, A., Le Cam, A. & Pouyssegur, J. (1983) J. Biol. Chem. 258, Haselbacher, G. K., Humbel, R. E. & Thomas, G. (1979) FEBS Lett. 100, Lastick, S. M. & McConkey, E. H. (1980) Biochem. Biophys. Res. Commun. 95, Nilsen-Hamilton, M., Hamilton, R. T., Allen, W. R. & Potter- Perigo, S. (1982) Cell 31, Nishimura, J. & Deuel, T. F. (1983) FEBS Lett. 156, Smith, C. J., Wejksnora, P. J., Warner, J. R., Rubin, C. S. & Rosen, 0. M. (1979) Proc. Natl. Acad. Sci. USA 76, Thomas, G., Martin-Perez, J., Siegmann, M. & Otto, A. (1982) Cell 30, Thomas, G., Siegmann, M. & Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, Blenis, J., Spivack, J. G. & Erikson, R. L. (1984) Proc. Natl. Acad. Sci. USA 81, Trevillyan, J. M., Kulkarni, R. K. & Byus, C. V. (1984) J. Biol. Chem. 259, Proc. Natl. Acad. Sci. USA 82 (1985) Decker, S. (1981) Proc. Natl. Acad. Sci. USA 78, Blenis, J. & Erikson, R. L. (1984) J. Virol. 50, Maller, J. L., Foulkes, J. G., Erikson, E. & Baltimore, D. (1985) Proc. Natl. Acad. Sci. USA 82, Bommer, V.-A., Noll, F., Lutsch, G. & Bielka, H. (1980) FEBS Lett. 111, Burkhard, S. J. & Traugh, J. A. (1983) J. Biol. Chem. 258, Duncan, R. & McConkey, E. H. (1982) Eur. J. Biochem. 123, Kisilevsky R., Treloar, M. A. & Weiler, L. (1984) J. Biol. Chem. 259, Terao, K. & Ogata, K. (1979) J. Biochem. (Tokyo) 86, Blenis, J., Sugimoto, Y., Biemann, H.-P. & Erikson, R. L. (1985) in Cancer Cells: Growth Factors and Transformation, eds. Feramisco, J., Ozanne, B. & Stiles, C. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. 3, pp Cobb, M. H. & Rosen, 0. M. (1983) J. Biol. Chem. 258, Erikson, E. & Maller, J. L. (1985) Proc. Natl. Acad. Sci. USA 82, Gabrilli, B., Wettenhall, R. E. H., Kemp, B. E., Quinn, M. & Bizonova, L. (1984) FEBS Lett. 175, Le Peuch, C. J., Ballester, R. & Rosen, 0. M. (1983) Proc. Natl. Acad. Sci. USA 80, Novak-Hofer, I. & Thomas, G. (1984) J. Biol. Chem. 259, Perisic, 0. & Traugh, J. A. (1983) J. Biol. Chem. 258, Smith, C. J., Ruppin, C. S. & Rosen, 0. M. (1980) Proc. Natl. Acad. Sci. USA 77, Wettenhall, R. E. H. & Cohen, P. (1982) FEBS Lett. 140, Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. & Nishizuka, Y. (1982) J. Biol. Chem. 257, Nielsen, P. J., Thomas, G. & Maller, J. L. (1982) Proc. Natl. Acad. Sci. USA 79, Johnson, R. A. & Walseth, T. F. (1979) Adv. Cyclic Nucleotide Res. 10, Laemmli, U. K. (1970) Nature (London) 227, Walsh, D. A., Ashby, C. D., Gonzales, C., Calkins, D., Fischer, E. H. & Krebs, E. G. (1971) J. Biol. Chem. 246, Kaibuchi, K., Takai, Y. & Nishizuka, Y. (1981) J. Biol. Chem. 256, Sugimoto, Y. & Erikson, R. L. (1985) Mol. Cell. Biol., in press. 39. Jakobovits, E. B., Majors, J. E. & Varmus, H. E. (1984) Cell 38, Nishizuka, Y. (1984) Nature (London) 308, Bishop, R., Martinez, R., Nakamura, K. D. & Weber, M. J. (1983) Biochem. Biophys. Res. Commun. 115, Cooper, J. A., Sefton, B. M. & Hunter, T. (1984) Mol. Cell. Biol. 4, Gilmore, T. & Martin, G. S. (1983) Nature (London) 306, Berridge, M. J. (1984) Biochem. J. 220,