Myc oncoproteins are phosphorylated by casein kinase ... - Europe PMC

The EMBO Journal vol.8 no.4 pp. 1 1 1 1 - 1 1 19, 1989

Myc oncoproteins kinase 11

are

phosphorylated by casein

Bernhard Luscher, Elizabeth A.Kuenzel t, Edwin G.Krebs' and Robert N.Eisenman Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104 and IHoward Hughes Medical Institute and Department of Pharmacology, University of Washington, Seattle, WA 98195, USA Deceased

on

January 20, 1988

Communicated by H.Diggelmann

Casein kinase II (CK-II) is a ubiquitous protein kinase, localized to both nucleus and cytoplasm, with strong specificity for serine residues positioned within clusters of acidic amino acids. We have found that a number of nuclear oncoproteins share a CK-II phosphorylation sequence motif, including Myc, Myb, Fos, Ela and SV40 T antigen. In this paper we show that cellular myc-encoded proteins, derived from avian and human cells, can serve as substrates for phosphorylation by purified CK-II in vitro and that this phosphorylation is reversible. One- and two-dimensional mapping experiments demonstrate that the major phosphopeptides from in vivo phosphorylated Myc correspond to the phosphopeptides produced from Myc phosphorylated in vitro by CK-II. In addition, synthetic peptides with sequences corresponding to putative CK-II phosphorylation sites in Myc are subject to multiple, highly efficient phosphorylations by CK-11, and can act as competitive inhibitors of CK-II phosphorylation of Myc in vitro. We have used such peptides to map the phosphorylated regions in Myc and have located major CK-II phosphorylations within the central highly acidic domain and within a region proximal to the C terminus. Our results, along with previous studies on myc deletion mutants, show that Myc is phosphorylated by CK-II, or a kinase with similar specificity, in regions of functional importance. Since CK-II can be rapidly activated after mitogen treatment we postulate that CK-II mediated phosphorylation of Myc plays a role in signal transduction to the nucleus. Key words: casein kinase II/Myc/nuclear oncoproteins/ phosphorylation/signal transduction

Introduction A great deal of evidence has accumulated indicating a role for myc in the control of cell proliferation and differentiation. This notion is supported by the fact that alterations at the c-myc locus, such as amplification, chromosomal translocation, mutation, retroviral transduction and insertional mutagenesis, have been found in diverse neoplasms in a wide range of species (for reviews see Cole, 1985; Cory, 1986; Eisenman and Thompson, 1986). None the less the molecular basis of myc function is as yet unknown.

The myc encoded proteins (henceforth referred to as Myc) have been demonstrated to be short-lived phosphoproteins that are localized predominantly in the nucleus. Phosphate is present in both viral and cellular Myc and phosphoamino acid analyses of Myc isolated by immunoprecipitation indicate that the phosphate is covalently bound to seryl and threonyl residues (Bister et al., 1980; Ramsay et al., 1982; Hann et al., 1983; Hann and Eisenman, 1984). Some progress has been made in localizing the sites of phosphorylation on Myc by utilizing a series of mutants of the v-myc-containing retrovirus MC29 (Bister et al., 1987). These MC29 mutants, arising through spontaneous internal deletions within the v-myc coding region, display an altered transformation phenotype (Ramsay et al., 1980; Ramsay and Hayman, 1982). Phosphopeptide mapping of the v-myc-encoded proteins from wild-type and mutant virusinfected cells indicated that all of the mutants were deleted at a major phosphorylation site(s) (Ramsay et al., 1982; Bister et al., 1987). Nucleotide sequence analysis of the mutants allowed assignment of the phosphorylation to a region lying between residues 669 and 739 in MC29 v-myc (Bister et al., 1987). This corresponds in c-myc to a conserved segment spanning the second and third exon boundary. The present study was prompted by the realization that the clusters of acidic amino acids interspersed with Ser and Thr residues, which are present in the major phosphorylated region, correspond to excellent consensus phosphorylation sites for casein kinase II (CK-II) (for recent references see Walton et al., 1985; Chan et al., 1986; Marin et al., 1986; Kuenzel et al., 1987). This ubiquitous kinase, which has been purified from a number of tissues, has been shown to localize in both the nucleus and the cytoplasm (Hathaway and Traugh, 1982) and to be activated following growth factor stimulation (Sommercorn et al., 1987; Klarlund and Czech, 1988). In addition, on the basis of homology with yeast cell division control proteins, it has been suggested that CK-II may be involved in some aspect of the control of cell proliferation (Takio et al., 1987; Chen-Wu et al., 1988). Since phosphorylation appears to be a major mechanism used by the cell to reversibly modify the activity of its proteins (for reviews see Hunter and Cooper, 1985; Edelman et al., 1987; Sibley et al., 1987) identification of the kinase(s) which phosphorylates Myc may contribute to our understanding of how this nuclear oncoprotein is regulated. We therefore investigated whether Myc serves as a substrate for CK-II phosphorylation both in vivo and in vitro.

Results Phosphorylation of Myc by CK-II The specificity of CK-II has been well defined by studies using both protein (Pinna et al., 1979; Tuazon et al., 1979; Walton et al., 1985; Chan et al., 1986) and synthetic peptide 1111

B.Luscher et al.

Fig. 1. Phosphorylation of Myc by CK-II. (A) Myc was immunoprecipitated from unlabeled BK3A cells using anti-v-Myc 12C peptide serum (Hann et al., 1983) and the resulting immunocomplexes were incubated as described in Materials and methods with or without CK-II. The reactions were stopped by washing the samples once in RIPA buffer (except for lane 1). Lane 1: CK-II alone; lane 2: Myc immunoprecipitate alone; lane 3: anti-Myc antibodies blocked with cognate Myc peptide; lane 4: Myc immunoprecipitates were incubated with CK-II without additional treatment; lane 5: CK-11 incubation after pre-treatment of the Myc immunocomplexes with alkaline phosphatase; lane 6: Myc immunoprecipitates were incubated with CK-II and the immunoprecipitate treated with alkaline phosphatase following the kinase reaction; lane 7: CK-II incubation in the presence of 0.6 uM heparin; lanes 8 and 9: CK-II incubation in the presence of 1 mM myc222-238 peptide or 0.1 mM myc222-238 peptide, respectively. Samples were analyzed on a 10% SDS-polyacrylamide gel. The arrowhead indicates the position of p46C-mYC. (B) Lamin A was immunoprecipitated with an anti-lamin A monoclonal antibody from BK3A cells. Lane 1: the immunocomplex was incubated with CK-II. Lane 2: immunoprecipitate from 32P-labeled cells.

substrates (Pinna et al., 1984; Meggio et al., 1984; Kuenzel and Krebs, 1985; Marin et al., 1986; Kuenzel et al., 1987). Sequences phosphorylated by CK-II contain serines or threonines that are followed by acidic residues. While the most critical determination is an acidic residue three positions to the carboxy terminus (Marin et al., 1986; Kuenzel et al., 1987), other nearby acidic residues can improve the kinetic constants. Thus sequences containing a Ser/Thr located within clusters of acidic residues would represent ideal CK-LI phosphorylation sites. We have identified proteins containing such sequences by computer-assisted search of a protein sequence database. While several of the known CK-II substrates were found, the most striking result of the search was the identification of several nuclear oncoproteins including Myc, Myb, Fos, SV40 large T antigen and the Ela transforming protein. We noted that for Myc one of the putative CK-II sites was contained within a region which was known to be phosphorylated (Bister et al., 1987) and to be functionally important (Ramsay et al., 1982). The c-myc gene encodes multiple proteins, major ones arising from alternative translational initiations within the same reading frame (avian Myc p59/p62; human Myc p64/p67) (Hann et al., 1988), as well as a minor protein (p46) which apparently arises from internal initiation within the Myc open reading frame (Liischer and Eisenman, 1988; B.Luscher and M.W.King, unpublished data). To determine

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whether Myc could serve as a substrate for CK-II, immunocomplexes containing the c-myc encoded proteins were incubated with the purified enzyme in the presence of [_y-32P]ATP. As shown in Figure IA, phosphate was incorporated into major bands of molecular mobilities of 59 and 62 kd, as well as a minor band of 46 kd. All these correspond to the Mr values for the chicken c-myc translation products. Phosphate was also incorporated into the ,B subunit of CK-II, due to autophosphorylation (Figure lA, lane 1). Phosphorylated Myc did not appear in immunocomplexes formed with anti-Myc antibodies that had been pre-incubated with the Myc peptide immunogen originally used to generate the antibodies (i.e. 'blocked') (Figure IA, lane 3). The phosphorylation reaction was dependent on the addition of CK-II (Figure lA, lane 2) and therefore was not catalyzed by a protein kinase co-precipitating with Myc. Heparin inhibited the reaction -7-fold (Figure IA, lane 7) as expected for one catalyzed by CK-LI (Hathaway and Traugh, 1982). No phosphorylation of the immunoglobulins present in the immunocomplexes was observed indicating that CK-II is not simply phosphorylating every protein in the complex. Furthermore CK-II did not detectably phosphorylate the abundant nuclear protein lamin A in anti-lamin A immunocomplexes (Figure 1B, lane 1) even though the protein was efficiently precipitated (Figure 1B, lane 2) and the CK-II

Myc phosphorylation by casein kinase 11

was active in autophosphorylation (Figure LB, lane 1) again suggesting some specificity for CK-II phosphorylation in this assay. To test whether the sites that were modified by CK-ll could be dephosphorylated by known phosphatases, Myc was first incubated with radioactive ATP and CK-II, the kinase reaction was then stopped by washing the immunocomplexes with RIPA buffer followed by treatment with alkaline phosphatase. The alkaline phosphatase treatment removed 80% of the phosphate previously transferred to Myc by CK-II (Figure IA, lanes 4 and 6). Furthermore, when the immunocomplexes were treated with alkaline phosphatase prior to CK-II addition, a 2-fold increase in phosphorylation and a slight increase in electrophoretic mobility was detected (Figure IA, lane 5). These results suggest that some of the in vivo sites dephosphorylated by alkaline phosphatase can serve as sites for CK-II phosphorylation. To ascertain whether Myc from different sources could serve as substrate for CK-H, immunoprecipitates from avian and human cells containing various c-myc locus rearrangements were generated and then treated with CK-II in the immunocomplex assay. As shown in Figure 2, CK-I1 phosphorylated Myc derived from all the different cell lines. In the same experiment, portions of the cells were pulselabeled with [35S]methionine. Comparison of the 35_-labeled bands with the 32P-labeled bands, showed that all of the different c-myc translation products were phosphorylated (Figure 2). The CK-II phosphorylation of Myc reached a plateau and no shift in its apparent mol. wt on SDS polyacrylamide gels was observed (data not shown). The relatively lower level of phosphorylation of Myc in Ramos cells can be accounted for in part by the fact that the steady state levels of Myc are lower in these cells than in Manca as determined by immunoblot assay (data not shown). In addition, differences in the steady state levels of endogenous phosphorylation of Myc in these two cell lines may contribute to the quantitative differences in their ability to accept phosphate in the in vitro reaction. We have also determined that the retrovirally transduced Myc proteins (v-Myc) produced in cells infected with the acute leukemia virus MH2 are also efficiently phosphorylated by CK-II in the immunocomplex assay (data not shown). Analyses of in vitro and in vivo phosphorylation of Myc To determine whether the regions of Myc that are phosphorylated in vivo correspond to those phosphorylated in vitro by CK-II, one-dimensional phosphopeptide maps of avian Myc labeled with 32p; in vivo and in vitro were compared. Figure 3 shows the peptides generated after V8 protease digestion of Myc labeled by CK-II in vitro. All of the fragments co-migrate with a subset of the peptides produced by the V8 protease digestion of Myc isolated from cells that were metabolically labeled with 32P_ (Figure 3, cf. lanes 2 and 4; 6 and 8). This is true of both the alternative translation products in this cell line. Thus maps of p62c-mYc and p59c-mYc labeled in vivo had all of the phosphopeptides found in the maps of p62C-mYC and p59c-mYc labeled by CK-fl in vitro. Several additional peptides, seen in the lower portion of the gel, are labeled in vivo that are not found to be labeled in the in vitro reactions (Figure 3) suggesting that, in the intact cell, there is at least one additional phosphorylation site on Myc.

Fig. 2. Human and avian Myc substrates for CK-II. Ramos, Manca, S13 and BK3A cells were labeled for 20 min with [35S]methionine and immunoprecipitated with anti-Myc peptide serum (lanes 1-4). In parallel immunoprecipitations from unlabeled cells Myc was phosphorylated in vitro by bovine testis CK-II (110 ng) for 30 min at 37°C (lanes 6-9). Lane 5: no immunoprecipitated Myc was added. Samples were analyzed on 10% SDS-polyacrylamide gels.

To carry out high resolution mapping of the phosphorylation sites we prepared two-dimensional fingerprints of the chicken p59cmYc (data not shown) and human p64c-myc (Figure 4). The p64c-myc is the human equivalent of avian p59C-mYc and was immunoprecipitated from FE-A cells, a human keratinocyte cell line (Kaur and McDougall, 1988). Immunoprecipitates of the in vivo phosphorylated and CK-II in vitro phosphorylated human c-myc encoded proteins are shown in Figure 4 (lanes 1 and 2). The eluted [32P]p64c proteins were subjected to trypsin digestion followed by electrophoresis and chromatography. The resulting phosphopeptides resolved into a complex cluster of spots, the pattern of which is shared between the in vivo and in vitro labeled p64C-mYC species (Figure 4, panels A and B). As found for the V8 maps (Figure 3) the in vivo labeled protein contained additional peptides not detected in the in vitro labeled protein. Figure 4 (panel C) shows the results of a mixing experiment of in vivo and in vitro labeled p64c-myc proteins, demonstrating that no new peptides, other than those observed in the siAgle digests, were present. Similar results were obtained with the chicken p59c-mYc and with the v-myc proteins encoded by MH2 (data not shown). We presume that the heterogeneity detected in the CK-II-specific peptides (Figure 4C, arrows and arrowheads) is due to populations of Myc containing combinations of phosphorylations at the different possible acceptor sites. This is consistent with the fact that synthetic peptides corresponding to the central acidic region undergo multiple phosphorylations (see below). When we prepared phosphopeptide maps of p64c-myc that had been pre-treated with alkaline phosphatase prior to CK-ll phosphorylation in vitro we obtained the same pattern, as for p64c-myc labeled with CK-II in the absence of alkaline

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A

..J.

a/

-

N .w

Fig. 4. Two-dimensional phosphopeptide maps of in vivo and in vitro CK-II phosphorylated Myc. Myc was immunoprecipitated from 32P labeled human FE-A cells (lane 1) or from unlabeled cells and phosphorylated in vitro by CK-II (lane 2). p64C-myc was eluted from gel slices and subjected to tryptic peptide mapping as described in Materials and methods. In panel (C) arrowheads and arrows mark the peptides which are in common to both in vivo and in vitro labeled proteins. Arrows in panels (A)-(C) also mark the peptides further analyzed in Figure 6. Equal amounts of radioactive samples were mapped. The maps shown resulted from equivalent exposure times to X-ray film.

.~~~~~ I

.94

Table I. Kinetic constants for CK-II with Myc peptides as substrates

V1ax b (nmol/min/mg)

KM

(AM)

myc222-238

myc246 -263

myc323-334 Fig. 3. One-dimensional phosphopeptide maps of Myc phosphorylated in vivo and by CK-II in vitro. Myc was immunoprecipitated from BK3A cells that were either labeled with 32p; or unlabeled. The immunoprecipitates from unlabeled cells were phosphorylated in vitro with CK-II. Myc was digested with 5 ng (lanes 1, 3, 5 and 7) or 100 ng (lanes 2, 4, 6 and 8) V8 protease and the fragments analyzed on a 20% SDS-polyacrylamide gel. Arrows indicate the positions of four labeled fragments, common to both p59mYc and p62myc labeled either in vivo or by CK-II in vitro.

phosphatase pre-treatment but with significantly increased incorporation, (data not shown). This result indicates that the phosphopeptides present in in vivo phosphorylated Myc that do not correspond to in vitro CK-ll peptides are not likely to be CK-ll sites that were previously occupied by phosphate. Our mapping data up to this point indicate that the kinase which phosphorylates Myc in the intact cell transfers phosphate to the same regions of the myc protein that CK-II does in vitro. The in vivo maps also show additional peptides not due to CK-II phosphorylation, indicating that other kinases must phosphorylate Myc (see Discussion).

Phosphorylation of synthetic peptides with sequences derived from the primary structure of Myc Quantitative studies on the kinetic parameters of the phosphorylation of Myc by CK-II could not be carried out in the reactions involving Myc immunoprecipitates from cells because there was no method for accurately determining the concentration of Myc in such immunoprecipitates. Furthermore, in these reactions the substrate is not free in solution but is bound in a complex of antibody-protein A-Sepharose. In order to study both the kinetics of phosphorylation and the nature of the phosphorylation sites 1114

4.29 (7)' 20 (1) 3920 + 120 (2)

10.43

4

26-104

116 146;310

CK-II from bovine lung or testes was assayed with varying concentrations of the peptides using the P81 paper assay as described previously (Kuenzel and Krebs, 1985). The reaction temperature was 37°C and the pH was 7.6. Kinetic constants were determined by Lineweaver - Burk analysis. aThe number of experiments is given in parentheses. bDifferent preparations of CK-II were used in these experiments. Therefore the range of Vmax values is given.

in Myc, we analyzed CK-II phosphorylation of synthetic peptides corresponding to defined regions of the Myc sequence. Residues 229-238 in avian Myc had been identified in the initial search for potential CK-ll substrates. Accordingly, a peptide of the sequence RRRPPTTSSDSEEEQEEDEE was synthesized and purified. This contains amino acids 222-238 in avian Myc, with an additional three arginine residues on the N terminus to simplify assays of peptide phosphorylation (Casnellie et al., 1982). Arginines located at least three positions to the N terminus from a serine or threonine have been shown to have no effect on the kinetic constants for phosphorylation by CK-II (Kuenzel et al., 1987). This peptide (myc222-238) served as a substrate for CK-II with a KM of 10 ItM (Table I). Avian Myc contains two regions in addition to residues 222 -238 that could serve as potential CK-II phosphorylation sites. These are residues 246-263 (EANESESSTESSTEASEE; this peptide also contained three Rs at its N terminus) and 323-334 (RTSDSEENDKRR). Peptides modeled after each of these sequences were also found to be phosphorylated although with higher KM values than for myc222-238 (Table I). CK-II when assayed for its activity towards a peptide previously reported to be a substrate, RRREEESEEE (Kuenzel et al., 1987) displayed a KM of 500 AM.


Table II. Survey of protein kinases for activities towards peptides modeled after the myc protein sequence Protein kinases cAMP-dependent protein kinase protein kinase C skeletal muscle MLCK casein kinase I

casein kinase II

pmol phosphate incorporation (pmol/min) control substrate myc222-238 308 310 379 3.7 87 20 22 133 88

0 0 1.4 0 0 0 0 266 732

myc 246-263

myc322-332

0 0 0 0 0 0 0 165 398

0 ND 0 0 0 ND 0 53 ND

Protein kinases were assayed as described (Kuenzel and Krebs, 1985). The control substrates used for the kinases are as follows: kemptide synthetic peptide (50 1M) for the cAMP-dependent protein kinase, histone III-S (0.5 mg/ml) for protein kinase C, the myosin light chain synthetic peptide (50 uM) for skeletal muscle myosin light chain kinase and casein (1 mg/ml) for casein kinases I and II. All three peptides were assayed with all kinases at a final concentration of 1 mM. Background counts were determined by assaying each kinase in the absence of added substrate and subtracted. ND: not determined. Each row represents a separate experiment.

Because there are mutiple seryl and threonyl residues in these peptides, it was of interest to know the number and identity of amino acids that were modified. This question was addressed for peptide myc222-238, which has five potential phosphate-acceptor sites, two threonines and three serines. We carried out a reaction time course that was extended until the amount of phosphate incorporated into the peptide reached a plateau. We calculated that -4 mol of phosphate were introduced per mol of peptide (data not shown). Thus, several of the potential sites in this peptide may be phosphorylated. This is also consistent with phosphoamino acid analyses of the human myc240-262 synthetic peptide and both human and chicken Myc proteins labeled in vivo and in vitro with CK-II which indicated that predominantly serine with lower amounts of threonine were phosphorylated (data not shown). When peptide myc222-238 was added to reactions containing immunoprecipitates of Myc, it competed with Myc as a substrate for CK-II phosphorylation (Figure 1, lanes 8 and 9). Competitive inhibition required relatively high concentrations of peptide however, with 1 and 0.1 mM inhibiting 17- and 4-fold, respectively. The other two Myc peptides also competed with Myc as substrates, but again, relatively high concentrations were required (not shown). There is good evidence that the region of Myc that contains the majority of the in vivo phosphate encompasses the sequences found in myc222-258 and myc246-253 (Bister et al., 1987). The results shown in Table I demonstrate that these sequences are phosphorylated by CK-II, but it was also of importance to determine whether these regions were permissive for phosphorylation by many other kinases. Therefore we tested four other kinases for reactivity with the Myc peptides. The data, presented in Table II, indicate that the cyclic AMP (cAMP)-dependent protein kinase, protein kinase C, skeletal muscle myosin light chain kinase and casein kinase I show no significant reaction with any of the putative Myc CK-II peptide substrates while all were capable of phosphorylating their control substrates. Of all the kinases tested only CK-II displayed reactivity with either Myc peptides or protein (see Discussion). Localization of regions phosphorylated by CK-ll The fact that synthetic peptides corresponding to CK-II consensus sequences in Myc can serve as good specific

Fig. 5. Two-dimensional phosphopeptide maps of in vitro phosphorylated human Myc and synthetic peptides digested with trypsin and V8 protease. Myc was immunoprecipitated from unlabeled FE-A cells, phosphatase treated, phosphorylated in vitro with CK-II, and the labeled p64c-myc band eluted and subjected to digestion with trypsin and V8 protease. The Myc synthetic peptides were HPLC-purified, phosphorylated with CK-II as described in Materials and methods, and digested with trypsin and V8 protease. Equal amounts of radioactive samples were used for mapping. The maps shown resulted from equivalent exposure times to X-ray film. Panel (F) diagrams the map position of all the phosphopeptides. Filled spots represent co-migrating phosphopeptides from Myc and Myc synthetic peptides. Open spots represent minor phosphorylations that are found only in either Myc or Myc peptides (see text), the spot marked P corresponds to free phosphate.

substrates for CK-II (Tables I and II) has allowed us to use such peptides to more accurately determine which regions within Myc are phosphorylated. We synthesized two peptides homologous to the major CK-II consensus region in human Myc. The first corresponded to residues 240-262 (myc240-262: LHEETPPTTSSDSEEEQEDEEEI) and the second to residues 342-357 (myc342-357: CTSPRSSDTEENVKRR) (see Figure 7 for location). These represent human Myc segments homologous to the avian Myc peptides (myc222-238 and myc323 -334 respectively) described above. Their length was chosen so as to provide convenient sites for trypsin and/or V8 protease digestion. The HPLC-purified

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B.Luscher et al.

from peptide

myc342-357 suggested that this region might

account for the major phosphorylation observed in both the

Fig. 6. Two-dimensional phosphopeptide maps of in vitro phosphorylated human Myc and myc342 -357 synthetic peptide digested with trypsin. Myc was immunoprecipitated from unlabeled FE-A cells, phosphatase treated, phosphorylated in vitro with CK-II and then eluted p64cmYc digested with trypsin. Equal amounts of radioactive sample were used for mapping. The maps shown resulted from equivalent exposure times to X-ray film.

Fig. 7. Localization of CK-II phosphorylation regions in human Myc. Sequences corresponding to the synthetic peptides used for mapping are shown as well as other landmarks. Underlined residues represent possible acceptor sites for CK-1I phosphorylation. The MyoD (and achaete-scute) homology region has been postulated to comprise a DNA binding helix-loop-helix structure (Murre et al., 1989). Likewise the C-terminal region marked 'leu-motif' has been hypothesized to fold into an a-helix with leucine side chains projecting from one face. Two such structures might interlock to form dimers (Landschulz et al., 1988). Nuc. Ie. refers to a sequence required for nuclear localization of c-Myc (Dang and Lee, 1988). The arrow indicates the position of the exon 2-3 border.

peptides were radioactively phosphorylated by CK-II, digested first with trypsin and then with V8 protease, and two-dimensional phosphopeptide maps prepared (Figure 5, panels B and C). The peptides each generated several cleavage products (myc 42-357 spots 1 -2; myc240-262: major spots 3-4, minor spots 5-6 and free phosphate, P; Figure 5, panels B, C and F) which are most likely due to partial proteolytic cleavages or to differences in the stoichiometry of phosphorylation. We next determined whether the labeled cleavage products from the CK-II phosphorylated synthetic peptides co-mapped with phosphorylated Myc. Human p64myc was radioactively phosphorylated with CK-II in vitro, digested with trypsin and V8, and mapped alone (Figure 5, panel A) or after mixing with peptide myc240-262 (panel D) or peptide myc34 -357 (panel E). The results indicate that peptide spots 1 -4 co-migrate with four major peptides produced from in vitro phosphorylated Myc. Minor spots 5-6, which are associated only with the myc240-26 peptide and not the protein (Figure 5, A, B, D, and F) are presumably due to partial cleavages as mentioned above. Minor spots 7- 10 in CK-II Myc presumably also arise from partial cleavages within the protein or possibly from sub-molar phosphorylations at other CK-II sites. Both the intensity and map positions of the spots produced 1 1 16

in vivo and in vitro phosphorylated Myc digested with tryspin (Figure 4, arrows; and Figure 6A arrows). We therefore prepared a tryptic digest of myc342-357 radioactively phosphorylated with CK-II and mapped it in parallel (Figure 6A and B), and mixed with (Figure 6C), p64c-myc. The maps clearly demonstrate co-migration of the major and minor tryptic cleavage products of p64c-myc with myc342 5 These data confirm and extend the phosphopeptide mapping experiments in Figures 4 and 5 and strongly argue that the major in vivo phosphorylated regions in Myc are related to CK-ll phosphorylation sites located within residues 240-262 and 342-357 of human Myc (Figure 7).

Discussion CK-ll phosphorylation sites in Myc It has been known for some time that c-myc- and v-mycencoded proteins are phosphorylated. Previous studies with v-myc deletion mutants had raised the possibility that at least one set of phosphorylations may correlate with transformation (Ramsay et al., 1982; Bister et al., 1987). However neither the sites of phosphorylation on Myc nor the enzymes mediating Myc phosphorylation have been identified. In this report we present evidence that CK-II phosphorylates Myc. We show that Myc can serve as a substrate for purified CK-II in vitro and that this phosphorylation is reversible (Figures 1 and 2). One- and two-dimensional phosphopeptide maps derived from the in vitro phosphorylated Myc are very similar to maps generated from phosphorylated Myc isolated from cells (Figures 3 and 4). In addition we have used synthetic peptides derived from Myc CK-II recognition sequences in mapping studies to show that these peptides comprise sites in whole Myc protein which are phosphorylated in vivo and by CK-II in vitro. These data demonstrate that the sites of in vitro and in vivo phosphorylation lie within the same phosphopeptides, all of which contain CK-II substrate consensus sequences. Given the known restricted specificity of the enzyme (Kuenzel et al., 1987; Marin et al., 1986; and see below), our results suggest that CK-II, or a kinase with very similar specificity, phosphorylates Myc in vivo. Our evidence strongly suggests that the in vivo and in vitro sites of CK-II phosphorylation lie in the highly acidic region at the exon 2-3 border and in a segment proximal to the C terminus (see Figure 7). The acidic region, consisting largely of glutamic acids interspersed with serine and threonine residues, was initially identified in a search for CK-II substrate consensus sequences. In addition we found that synthetic peptides corresponding to segments of the Myc acidic region competitively inhibited phosphorylation of Myc in immunocomplexes in vitro and served as excellent substrates for CK-ll phosphorylation (Table I). The peptides, comprising residues 222-238 and 246-253 of the chicken c-myc protein, displayed KM values in the low tzM range (Table I). These are 10- to 100-fold lower than previously obtained with the standard peptide used for CK-II assays (Kuenzel and Krebs, 1985), and are among the lowest reported to date for any CK-II substrate. The Myc peptides are not simply good non-specific kinase substrates, since of five purified kinases tested only CK-II phosphorylated the Myc peptides to a significant extent (Table II). This result is consistent with the previous findings of a highly restricted


specificity for CK-II (Keunzel and Krebs, 1985; Walton et al., 1985; Chan et al., 1986). In Myc the anionic sequence at the exon 2-3 border (residues 222-238 in avian, and 240-262 in human Myc, see Figure 7) is highly conserved from Xenopus to human, and its essential character is maintained between the human c-myc and the N- and L-myc encoded proteins. Such a high degree of conservation may be indicative of functional significance. Some support for the idea that the acidic region may be functionally important comes from studies on the phenotype of MC29 v-myc mutants containing relatively large internal deletions which include the acidic region (Ramsay et al., 1980, 1982; Ramsay and Hayman, 1982; Bister et al., 1987). In contrast to wild-type MC29, the mutants fail to transform chicken macrophages although both mutant and wild-type viruses can transform quail macrophages and form foci on chicken and quail fibroblasts. However fibroblast transformation is also altered in the mutants since colony formation in soft-agar is suppressed (C.Tachibana and R.Eisenman, unpublished data). A similar loss in the ability to transform chicken, but not quail, macrophages was found for smaller deletions introduced into the acidic region of MC29 (Heaney et al., 1986; Biegalke et al., 1987). One of these deletions (CH202) extends from residue 220 to 239 and thus nearly corresponds to one of the synthetic peptide substrates used here (myc222 -238). Another mutant possessing the altered transformation phenotype on chicken macrophages is deleted from residue 239-249, a segment which lies between and overlaps both the acidic synthetic peptide substrates used (avian myc222-238 and myc246-26., note that the latter peptide sequence is not conserved in human Myc). Thus mutations of the acidic region lead to complex, but nevertheless definite, alterations in the transformation potential of v-myc. By contrast, deletion of the acidic region from human c-myc had little effect on its ability to induce cooperative transformation of rat embryo cells in the presence of activated ras (Stone et al., 1987). Taken at face value it would seem that the acidic region might be involved in some but not all of the phenotypes ascribed to Myc. A test for the role of the acidic region clearly must await a biochemical assay for Myc function. Our mapping data also indicate that a region close to the C-terminus, comprising residues 342-357, can serve as a target for CK-II phosphorylation (see Figure 7). This segment is interesting both in terms of its structural homologies and its location. It has been noted that this region constitutes a structural motif common to adenoviral EIA, papovaviral large-T and human papillomavirus E7 proteins as well as the product of the yeast mitotic regulatory gene CDC 25 (Figge et al., 1988; Figge and Smith, 1988). In EIA and SV40 large-T antigen this region is required for association with the protein product of the retinoblastoma susceptibility gene, Rb (Whyte et al., 1988a, 1989; DeCaprio et al., 1988). It is also required for EIA, T antigen and Myc transforming function (Kalderon and Smith, 1984; Stone et al., 1987; Whyte et al., 1988b). Furthermore this phosphorylated region is on the N-terminal border of another structural motif which has been found to be present in several regulatory proteins including MyoD, achaete-scute, and immunoglobulin enhancer binding proteins (Murre et al., 1989). It has been postulated that this domain constitutes an amphipathic helix-loop-helix involved in DNA binding and dimerization (Murre et al., 1989). Thus phosphorylation at

the CK-II site bordering this domain could potentially influence a critical regulatory region in Myc. Site-directed mutagenesis of the serine residues should allow us to assess this possibility. We also investigated whether other cellular kinases might phosphorylate Myc in an immunocomplex assay. Casein kinase I, glycogen synthase kinase 3, and the raf kinase transferred only low amounts of phosphate to Myc while the cAMP-dependent protein kinase strongly phosphorylated Myc. Phosphopeptide maps of Myc phosphorylated by the cAMP-dependent and raf kinases did not correspond to those derived from in vivo phosphorylated Myc (B. Liischer, unpublished data). These data indicate that at least the majority of Myc in growing cells is unlikely to be significantly phosphorylated by casein kinase I, glycogen synthetase kinase 3, raf or cAMP-dependent kinases. A role for CK-ll phosphorylation of nuclear oncoproteins in signal transduction?

While both the structure and substrate specificity of CK-II have been well studied, information concerning its potential physiological role has only recently begun to emerge (see Hathaway and Traugh, 1982; Edelman et al., 1987 for reviews). Of relevance to this report are the findings that CK-II activity has been detected in a broad range of cell types and, where examined, appears to be localized in both nuclear and cytoplasmic fractions. A number of enzyme substrates have been identified whose activities appear to be either directly or indirectly modulated by CK-II phosphorylation. In addition, a role for CK-II in the cellular response to mitogens has been postulated based on the findings that insulin, insulin-like growth factor and EGF treatment lead to rapid increases in CK-II activity (Sommercorn et al., 1987; Klarlund and Czech, 1988). Growth factor-stimulated activation of CK-II appears to occur in the absence of protein synthesis and thus may be due to factor-induced posttranslational modification of the enzyme. Recent studies in yeast and larger eukaryotes have demonstrated that kinases mediate functions critical to cell cycle progression (Hunter and Cooper, 1985; Edelman et al., 1987; Hunter, 1987; Sibley et al., 1987). It is intriguing that a number of nuclear proteins implicated in cell proliferation, Myc, Myb, Fos, Ela and T antigen, all possess potential CK-II phosphorylation sites. In this report we have demonstrated that Myc is a substrate for CK-II. Other work has shown that both T antigen (Krebs et al., 1988; Grasser et al., 1988) and Myb (Luscher et al., manuscript in preparation) are also phosphorylated by CK-II. It seems possible then that CK-H-mediated phosphorylation of nuclear oncoproteins could represent part of the circuitry involved in mitogenic signal transduction. Knowledge of the detailed relationship between growth factor stimulation and CK-II activation as well as the functional consequences of CK-II induced phosphorylation of nuclear oncoproteins will be required in order to evaluate this model.

Materials and methods Cell lines The avian bursal lymphoma cell lines BK3A and S13 were provided by H.Hihara and M.Linial and have been described elsewhere (Hihara e- al., 1974; Linial et al., 1985). The human Burkitt's lymphoma lines Manca and Ramos were provided by A.Hayday (Yale University) and W.Hayward (Sloan-Kettering Institute), respectively. The human keratinocyte cell line

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B.Luscher et al. FE-A was provided by P.Kaur (Fred Hutchinson Cancer Research Center) (Kaur and McDougall, 1988).

Enzymes CK-ll was purified from bovine lung or bovine testis as described previously (Takio et al., 1987). cAMP-dependent protein kinase was from bovine cardiac muscle and was isolated as described (Bechtel et al., 1977). Other enzymes were provided by the following investigators (all from the University of Washington, Seattle, WA): casein kinase I from bovine lung from Dr James Sommercorn; protein kinase C from Dr Kathryn Meier; skeletal muscle myosin light chain kinase from Dr Peter Kennelly. Alkalkine

phosphatase

was

purchased from Sigma.

Antibodies The production and characterization of the anti-peptide antibodies used to immunoprecipitate Myc from avian and human cell lines have been previously described (Hann et al., 1983; Hann and Eisenman, 1984). They were prepared against peptides corresponding to the 12 C-terminal amino acids of Myc. All sera were affinity purified. Monoclonal antibody A8, against chicken lamin A, was obtained from P.Liu and H.Weintraub.

Peptides Peptides were synthesized on an Applied Biosystems AB430 automatic solid-phase peptide synthesizer. All peptides were purified by reverse phase HPLC and the human Myc peptides verified by amino acid sequencing. Metabolic cell labeling, immunoprecipitation and gel elec-

trophoresis Unlabeled and labeled cells (2 x 107) were lysed in I ml of antibody buffer [20 mM Tris-HCI, pH 7.4, 50 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 (NP-40), 0.5% deoxycholate, 0.5% SDS, 0.5% aprotinin, 10 mM iodoacetamide], and then sonicated for 30 s. The lysates were clarified by centrifugation for 15 min in an Eppendorf table-top centrifuge and 5 Ag of the appropriate affinity-purified antibody, or 5 11 of A8 culture supernatant and 5 ytg of rabbit anti-mouse serum (Pel-Freeze Biologicals), was added to the supernatant. For peptide blocking the antibodies were pre-incubated with excess peptide immunogen for 20 min on ice prior to immunoprecipitation. Immunocomplexes were collected by incubation with protein A - sepharose - CL4B (Sigma). The sepharose beads were washed sequentially with RIPA buffer (10 mM Tris, pH 7.4, 0.15 M NaCl, 1% NP-40, 1 % DOC, 0.1 % SDS, 0.5 % aprotinin), with high salt buffer (2 M NaCI, 10 mM Tris, pH 7.4, 1% NP-40, 0.5% DOC) and twice with RIPA buffer. Labeling of cells with [35S]methionine was performed as described (Luscher and Eisenman, 1988). For labeling with [32P]orthophosphate (ICN), cells were washed twice in 0.1 M NaCl, 10 mM Tris, pH 7.4, resuspended in phosphate-free medium at 2 x 107 cells/ml and labeled for 120 min with 2 mCi 32Pi/ml. FE-A cells were labeled for 2 h in 3 ml phosphate-free medium per 10 cm plate with 5 mCi 32pi. Collected cells were processed as described above. SDS-PAGE was performed as described previously (Hann and Eisenman, 1984). The following prestained molecular size markers (BRL, Inc.) were used: myosin (200 kd), phosphorylase b (97.4 kd), bovine serum albumin (68 kd), ovalbumin (46 kd), a-chymotrypsinogen (25.7 kd), ,3-lactoglobulin (18.4 kd) and lysozyme (14.3 kd). Kinase assays For CK-II reactions immunocomplexes attached to sepharose beads were washed twice in 40 mM Tris, 100 mM NaCI, 2 mM EDTA after the final wash in RIPA buffer. Then 15 41 of kinase buffer (30 mM Tris-HCI, pH 7.4; 100 mM NaCl; 10 mM MgCl2; 4.8 !M [-y_32P]ATP at 300 Ci/mmol) was added followed by 2 ,ul of purified CK-II (0.36 mg/ml from bovine testes; specific activity 2778 nmol/min/mg with RRRDDDSDDD as a substrate). The assays were performed at 37°C for 60 min. The concentrations of reaction components in individual experiments are indicated in the figure legends. Peptide phosphorylation reactions were carried out as described (Kuenzel and Krebs, 1985). The results shown in Figure 2 and Tables I and II were obtained using different preparations of purified CK-II. The methods for assaying the other protein kinases were described or referenced previously (Kuenzel and Krebs, 1985).

Phosphatase

treatment

Immunoprecipitates were washed twice in 100 mM Tris-HCI, pH 8, 50 mM MgCI2, 1% aprotinin and then resuspended in 45 Al of the same buffer. Alkaline phosphatase (5 /1, 0.14 units) was added and incubated for 30 min at 37°C. The reaction was stopped by washing the samples twice in RIPA buffer.

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Peptide mapping For Staphylococcus aureus V8 protease one-dimensional peptide maps, 32P-labeled protein bands were excised from unfixed SDS-polyacrylamide gels and treated according to Cleveland (1977). In brief, gel pieces were swollen for 2 min in 0.125 M Tris, pH 6.8, 1 mM EDTA, 0.1 % SDS, 1 %, 1 mM fl-mecaptoethanol. Swollen gel pieces were placed on top of a 20% SDS -PAGE overlaid with 0.125 M Tris, pH 6.8, 1 mM EDTA, 0.1 % SDS, 1 mM ,B-mercaptoethanol, 20% glycerol and phenol red. S.aureus V8 protease (obtained from ICN) was diluted in buffer used for overlay but containing only 10% glycerol. Samples were run to the bottom of the stacking gel, the electrophoresis stopped for 30 min to allow digestion and then the proteolytic fragments were separated. For two-dimensional peptide mapping 32P-labeled Myc was extracted from unfixed homogenized gel pieces in 50 mM NH4HCO3, pH 7.4, 0.1 % SDS, 1% f3-mercaptoethanol for 5 min at 100°C, then overnight at 37°C. Proteins in the clarified supernatant were precipitated with 15% trichloroacetic acid with 10 /Ag RNase A as a carrier for 1 h at 0°C. Phosphorylated Myc peptides were precipitated in 50% trichloroacetic acid. The precipitated proteins and peptides were treated with performic acid (formic acid:H202, 10: 1) for 90 min, lyophilized and digested twice with 10 ,g TPCK-treated trypsin (Cooper Biomedicals) in 50 mM NH4HCO3. pH 7.4 for 24 h. For double digests the tryptic peptides were digested for an additional 24 h with two additions of 10 ug V8 protease. Peptides were lyophilized twice in H20 and analyzed on cellulose thin-layered plates (Boehringer Mannheim) by electrophoresis at pH 8.9 for 20 min at 1.2 kV and then by ascending chromatography in the second dimension in isobutyric acid: pyridine:acetic acid: butanol: water (65:5:3:2:29) (Scheidtmann et al., 1982).

Acknowledgements We are grateful to David Litchfield for purified CK-H and helpful discussions, Jon Cooper for advice and use of peptide mapping facilities, Pritinder Kaur for the FE-A cell line, and Ernie Tolentino for peptide synthesis and purification. We also thank Linda Breeden, Jon Cooper, Andrius Kazlauskas and Volker Vogt for critical readings of the manuscript; and Jan Smith for secretarial assistance. This work was supported by grants from the National Cancer Institute (NIH) to R.N.E. and the Howard Hughes Institute to E.G.K. B.L. is a postdoctoral fellow of the Swiss National Science Foundation (No. 83.503.0.87).

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Received on August 12, 1988; revised on January 10, 1989

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