Oncogene (2000) 19, 2846 ± 2854 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Hyperphosphorylation and increased proteolytic breakdown of c-Myb induced by the inhibition of Ser/Thr

Juraj Bies1,2, Sona Feikova 1,2, Donald P Bottaro3 and Linda Wol€*,2

1Laboratory of Molecular Virology, Cancer Research Institute, Slovak Academy of Sciences, 833 92 Bratislava, Slovakia; 2Laboratory of Cellular Oncology, National Cancer Institute, NIH, Bethesda, Maryland, MD 20892-4255, USA; 3Laboratory of Cellular and Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland, MD 20892-4255, USA

The c-myb proto-oncogene encodes a nuclear phospho- Kalkbrenner et al., 1990). The DNA-binding domain protein that plays a crucial role in normal hematopoiesis. is the most conserved region among Myb family It is a short-lived transcription factor rapidly degraded members and is comprised of three imperfect repeats by the 26S proteasome. Although it has been shown that (R1 ± 3) of 50 ± 52 amino acids each (Sakura et al., instability determinants reside in its carboxyl terminus, 1989; Howe et al., 1990). The second and third repeats the molecular mechanism of c-Myb degradation is recognize and speci®cally bind to the consensus unknown. Here, we report the ®rst evidence that sequence PyAAC(G/T)G (Biedenkapp et al., 1988; plays a role in targeting the protein to Howe and Watson, 1991; Ogata et al., 1996). A highly the proteasome. Inhibition of cellular / hydrophilic transactivation domain is required for protein activity by okadaic acid resulted in increased transcription of reporter genes (Sakura et hyperphosphorylation of c-Myb and extremely rapid al., 1989; Kalkbrenner et al., 1990). This region of c- turnover. The hyperphosphorylation resulted in a protein Myb interacts with the coactivator CBP, a histone with altered properties that was indicative of conforma- acetyltransferase, which serves to bridge c-Myb to the tional changes. Its mobility on gel electrophoresis was basal transcription machinery (Dai et al., 1996; altered as well as its recognition by speci®c monoclonal Oelgeschlager et al., 1996). The ability to transactivate antibody. The altered hyperphosphorylated protein still target genes is also regulated through a large negative bound to DNA with an anity similar to that of the regulatory domain. Within this region the putative hypophosphorylated form. Phosphorylation of three leucine zipper motif has been identi®ed as a region previously identi®ed sites, 11, 12, and 528, does capable of binding cellular cofactors that modulate the not appear to be involved in the proposed changes in activity of the protein (Kanei-Ishii et al., 1992; Tavner conformation or stability. However, phosphoamino acid et al., 1998). In addition, the EVES motif located in the analyses of the hyperphosphorylated form of c-Myb COOH terminus has been suggested to modulate the revealed increased c-Myb phosphorylation mainly on activity of c-Myb by inhibiting the binding of p100 to threonine residues that correlated with other okadaic the DNA-binding domain (Dash et al., 1996). acid-induced alterations of c-Myb. These ®ndings Post-translational modi®cation of transcription fac- indicate that Ser/Thr phosphatases prevent conforma- tor, such as c-Myb, can modulate regulatory activity tional changes that may play an important role in through changes in subcellular localization, DNA- controlled degradation of c-Myb. Oncogene (2000) 19, binding anity, transactivation potential and proteo- 2846 ± 2854. lytic stability (Calkhoven and Ab, 1996). c-Myb is phosphorylated at multiple sites in vivo (Bading et al., Keywords: c-Myb; ; phosphorylation; 26S 1989; Luscher et al., 1990; Aziz et al., 1995) and at proteasome; Ser/Thr phosphatases least some of these sites have been demonstrated to be physiologically important. Serines 11 and 12, located in the NH2 terminus, have been identi®ed as phosphor- Introduction ylation sites in vivo and are phosphorylated by caseine II (CKII) in vitro (Luscher et al., 1990). The c-myb proto-oncogene encodes a highly conserved Phosphorylation of these residues results in a decrease 75 ± 80 kDa transcription factor which is expressed in sequence speci®c binding to DNA and also inhibits predominantly in immature cells of hematopoietic cooperativity with the transcription factor NF-M origin (Wol€, 1996; Weston, 1998). Analyses of (Luscher et al., 1990; Oelgeschlager et al., 1995). Ser deletion mutants performed in several laboratories 528 (murine numbering, Bender and Kuehl, 1986), have established a functional map of c-Myb. It consists which is in the negative regulatory domain, is also of an NH2-terminal DNA-binding domain (Klemp- phosphorylated in vivo by the 42 kDa MAP kinase or nauer and Sippel, 1987; Biedenkapp et al., 1988; another related directed kinase (Aziz et al., Sakura et al., 1989), a central acidic transactivation 1995). Ser to Ala substitution at this site results in an domain (Nishina et al., 1989; Weston and Bishop, increased ability of c-Myb to activate transcription 1989; Ibanez and Lipsick, 1990) and a COOH-terminal from some, but not all Myb-responsive promoters negative regulatory domain (Sakura et al., 1989; (Miglarese et al., 1996). It was also reported that phosphorylation of c-Myb increases during and c-Myb isolated from mitotic cells lost the ability to *Correspondence: L Wol€ bind DNA, suggesting that changes in phosphorylation Received 17 December 1999; revised 30 March 2000; accepted status may alter c-Myb activity during the cell cycle 4 April 2000 (Luscher and Eisenmann, 1992). Phosphorylation-dependent instability of c-Myb J Bies et al 2847 The steady state level of any protein is the result of the balance between the rates of its production and degradation. We previously showed that c-Myb is degraded by the 26S proteasome in hematopoietic cells, and that the region responsible for its very short half- life is located in the COOH-terminus (Bies and Wol€, 1997; Bies et al., 1999). In the present study, we looked at the e€ect of phosphorylation on the proteolytic processing of the protein by examining its turnover in cells treated with okadaic acid (OA), an inhibitor of cellular Ser/Thr protein phosphatases. Treatment of cells with OA resulted in rapid c-Myb hyperpho- sphorylation, altered properties suggesting conforma- tional changes, and accelerated turnover of the protein by the 26S proteasome. Phosphoamino acid analyses of the hyperphosphorylated form of c-Myb revealed increased phosphorylation mainly on threonine. We concluded that OA-sensitive protein phosphatases regulate c-Myb activity through modulation of the protein half-life.

Results

Treatment of M1 myeloid cells with inhibitors of Ser/Thr protein phosphatases induces hyperphosphorylation and increased proteolytic turnover of c-Myb Protein phosphorylation is maintained through the balanced activities of protein and phosphatases. To determine if alteration of the phosphorylation state of the cell a€ects the stability of c-Myb, we treated myeloblastic M1 cells with okadaic acid (OA), a potent cell permeable inhibitor of serine/threonine phospha- tases. In the ®rst experiment, 1 mM OA was added during pulse labeling and was present throughout the entire chase. At the indicated time points, c-Myb protein was immunoprecipitated, separated by poly- acrylamide gel electrophoresis, and analysed by ¯uor- ography. As shown in Figure 1, OA treatment of M1 cells caused a dramatic decrease in c-Myb protein Figure 1 Okadaic acid induces the hyperphosphorylation and expression to the point where it was undetectable. increased proteolytic degradation of c-Myb. (a) M1 cells were labeled for 45 min in medium containing 35S-/ However, simultaneous treatment of cells with OA and and chased for the indicated time. Okadaic acid (OA, 1 mM), an ALLN, an inhibitor of 26S proteasome, restored inhibitor of Ser/Thr phosphatases and ALLN (100 mM), an immunoprecipitable c-Myb. The protein obtained in inhibitor of the 26S proteasome, were added to the cells at the the presence of both inhibitors showed a reduced beginning of the pulse and were present during the entire chase. The control cells were treated with the vehicle (DMSO, 0.5%). c- electrophoretic mobility on SDS ± PAGE. Treatment of Myb were immunoprecipitated, separated on an 8% immunoprecipitated c-Myb in vitro with calf intestinal SDS ± PAGE and visualized by ¯uorography as described in phosphatase restored its normal mobility, suggesting Materials and methods. Some immunoprecipitates, as indicated, that slower migration of c-Myb was due to phosphor- were also treated in vitro with calf intestinal phosphatase (CIP) to ylation-induced conformational changes (Figure 1a). remove phosphates from modi®ed amino acids. The mobilities of prestained molecular weight standards in kDa are indicated on The proteolytic processing was so ecient in this the left. (b) M1 cells were metabolically labeled and okadaic acid experiment that we were unable to calculate the half- (OA, 1 mM) was added only during the chase. At the indicated life of the hyperphosphorylated form of c-Myb. For times c-Myb was immunoprecipitated and analysed as described this reason, we set up a pulse chase experiment, where in Figure 1. Quantitative analysis was performed on a PhosphorImager 425 and IMAGEQUANT software. Each point OA was added only at the end of the pulse, instead of on the graph represents the relative amount of c-Myb during the throughout the pulse. Cells were immediately chased pulse-chase experiment; the level of protein at the end of pulse and the level of radio-labeled c-Myb was analysed by was assigned to 100% immunoprecipitation and ¯uorography. As shown in Figure 1b, the stability of c-Myb was signi®cantly reduced immediately after OA addition. In OA-treated To explore the kinetics of hyperphosphorylation and cells, after a 60 min chase, we detected less than 10% degradation of c-Myb in cells after inhibition of Ser/ of the protein, whereas in untreated cells we were able Thr phosphatases, we treated COS7 cells, expressing to detect more than 50%. A similar e€ect was observed full-length c-Myb (FL-c-Myb), with OA for various when M1 cells were treated with calyculin A, another lengths of time. Cells were lysed and protein lysates inhibitor of Ser/Thr phosphatases (data not shown). were subjected to Western blot analyses using anti-c-

Oncogene Phosphorylation-dependent instability of c-Myb J Bies et al 2848 Myb antibody. Representative results are shown in We wanted to test whether hyperphosphorylated Figure 2. The ®rst appearance of a slower migrating Myb could bind to DNA and, if it was bound, whether form of c-Myb occurred between 10 and 15 min after it adopted a di€erent conformation on DNA than the start of OA treatment. Ninety minutes of treatment hypophosphorylated c-Myb. For this purpose we with the inhibitor caused a signi®cant decrease in the performed an electromobility shift assay (EMSA) using level of protein. Since the di€erence in size appears to cell lysates prepared from transfected COS7 cells. Mild be greater than expected to result from the addition of conditions for preparation of cell lysates were used to phosphate groups we suggest that hyperphosphoryla- avoid protein denaturation (Oelgeschlager et al., 1995). tion results in conformational changes. This would As shown in Figure 4a, a single DNA-protein complex imply further that phosphorylation-induced conforma- was formed as detected in EMSA following incubation tional changes in c-Myb precede its accelerated of the MRE-A DNA with extracts prepared from proteolytic processing. In addition, co-treatment of COS7 cells expressing c-Myb in the absence of OA OA with either an inhibitor of protein synthesis, treatment. The presence of c-Myb in the complex was cycloheximide (CHX), or an inhibitor of mRNA demonstrated by a supershift after incubation of the synthesis, actinomycin D (ActD), did not block EMSA reaction in the presence of monoclonal anti-c- phosphorylation-induced alterations in mobility and Myb antibody (against the COOH terminus of murine degradation of c-Myb (Figure 2). Furthermore, treat- c-Myb, Upstate Biotechnology). In addition, rabbit ment with CHX or ActD alone did not cause any polyclonal anti-c-Myb antiserum (directed against the change in the mobility of the protein. Therefore DNA-binding and transactivation domains of murine mRNA or protein synthesis are not required for the c-Myb; Bies et al., 1996) blocked the binding of MRE- observed e€ect of OA on the conformation and A to c-Myb. There was no change in the mobility or stability of c-Myb. intensity of the protein-DNA complex when rabbit preimmune serum was used in the reaction (Figure 4a). The speci®city of this binding reaction was demon- Further evidence for OA-induced conformational changes strated by a cold competition assay, where preincuba- in c-Myb tion of lysates with 20- and 100-fold excess of As mentioned above, we observed that hyperpho- unlabeled MRE-A oligonucleotide diminished the sphorylated c-Myb had a decreased mobility in PAGE. detection of c-Myb-32P-labeled MRE-A complex (Fig- Further support for the phosphorylation-induced ure 4a). To speci®cally examine the e€ect of hyperpho- conformational changes came from an analysis of sphorylation on c-Myb's ability to bind MRE-A, we accessibility of domains of the protein to monoclonal prepared cell lysates from COS7 cells expressing FL- antibodies. Western blotting was performed using Myb treated with OA and ALLN (to prevent lysates from OA treated or untreated COS7 cells. degradation of the hyperphosphorylated protein) or Although monoclonal antibodies directed against the ALLN alone. c-Myb from OA-treated or untreated NH2 terminus (aNH2-Myb) and the COOH-terminus cells was able to bind DNA with almost identical (aCT-Myb) strongly recognized the protein from eciency. The similar DNA binding of hypo- and untreated cells, there was a pronounced di€erence in hyperphosphorylated forms indicates that the increased the reactivity of the domain speci®c monoclonals to the phosphorylation does not cause its release from DNA protein from OA-treated cells (Figure 3). The mono- in cells as a prerequisite for increased degradation clonal antibody raised against the NH2 terminus of c- (Figure 4b). In addition, the use of an anti-c-Myb Myb reacted strongly with the most slowly migrating, monoclonal antibody that recognizes an epitope in the hyperphosphorylated form of Myb from OA-treated COOH terminus suggested further that a unique cells, whereas the antibody raised against the COOH- conformation of the protein may be formed in cells terminus did not (Figure 3). These results indicate that treated with OA. As shown in Figure 4b, MRE-c-Myb c-Myb can respond to its phosphorylation status by complex involving the hypophosphorylated form of c- conformational alterations that inhibit access to the Myb was completely shifted by aCT-Myb antibody, carboxyl terminus. whereas only 35% of the hyperphosphorylated com-

Figure 2 Okadaic acid-induced hyperphosphorylation and de- gradation of c-Myb is independent of the synthesis of new RNA and protein. COS7/FL-Myb cells were treated for the indicated Figure 3 Phosphorylation-induced alteration in c-Myb reduces times with OA (1 mM) alone or together with either actinomycin recognition of hyperphosphorylated c-Myb protein by mono- D (ActD; 5 mg/ml) or cycloheximide (CHX; 15 mg/ml). In control clonal antibody directed against COOH terminus. Level of c-Myb experiments, COS7/FL-c-Myb were also treated with ActD (5 mg/ and its phosphorylation status in cellular lysates used in EMSA ml) or CHX (15 mg/ml) alone. At the indicated times, the cells was evaluated by immunoblotting with anti-c-Myb monoclonal were lysed, and the quantity of c-Myb and its phosphorylation Ab directed against the amino-terminal region (aNH2-Myb) or status were determined by immunoblotting with anti-c-Myb carboxyl terminus (aCT-Myb) and developed by ECL kit monoclonal Ab and developed using an ECL kit (Amersham) (Amersham)

Oncogene Phosphorylation-dependent instability of c-Myb J Bies et al 2849

Figure 4 DNA binding activity of hypo- and hyperphosphorylated c-Myb. (a) Cell lysates from COS7 (Control) or COS7/FL-Myb cells were incubated with 32P-labeled MRE-A oligonucleotide and analysed by EMSA as described in Materials and methods. The c- Myb-DNA complex (b), detected only in the COS7/FL-Myb lysate, was e€ectively competed with 20 and 1006molar excess of unlabeled MRE oligonucleotide and supershifted with c-Myb-speci®c monoclonal antibody (aNH2-Myb) or polyclonal antiserum (aCOOH-Myb), but not with normal rabbit serum (NRS). (b) Band shift reaction of 32P-labeled MRE oligonucleotide with c-Myb isolated from COS7/FL-Myb cells treated (+) or untreated (7) with OA was incubated in the absence or presence of monoclonal antibody recognizing an epitope in COOH terminus (aCT-Myb). F indicates free probe and B indicates bound probe plex was shifted in the same reaction. This suggests was not in¯uenced by OA treatment. Quanti®cation of that the aCT-Myb antibody cannot recognize some these data revealed that, after a 90 min chase, more than phosphorylation-induced forms of c-Myb. 65% of CT6-MybD(266aa) protein remained in the presence or absence of OA. In contrast, the phosphor- ylation mutants behaved like wt with about 30% of the Comparison of wild-type and mutant forms of c-Myb protein remaining after 90 min, in the absence of OA c-Myb is known to be phosphorylated on Ser11, 12 or treatment, and less than 10% in the presence of OA. 528 in vivo (Bading et al., 1989; Luscher et al., 1990; Aziz Therefore, the more stable form of c-Myb with a deleted et al., 1995), however, it has not been determined COOH-terminus (Bies et al., 1999) is also resistant to the whether this posttranslational modi®cation a€ects its e€ects of OA treatment. In addition, phosphorylation of proteolytic stability. To address this question, we serines 11, 12, or 528 is not required for the observed compared the half-lives of wild-type (wt) protein with increase in c-Myb turnover. We also found that mobility that of mutants where Ser 11, 12 or 528 had been changes following hyperphosphorylation were identical replaced with alanine to prevent phosphorylation (FL- for wt FL-Myb and FL-Myb(S11,12A) and FL- Myb(S11,12A); FL-Myb(S528A)). We also included in Myb(S528A) mutants (Figure 6). this experiment a COOH-terminally truncated form of c- Myb CT6-MybD(266aa) (Bies et al., 1999), which is Inhibition of Ser/Thr phosphatases in cells increases the missing the leucine zipper and negative regulatory phosphorylation of c-Myb on Thr residues domain and resembles the stable oncogenic form of c- Myb expressed in leukemia (Bies and Wol€, 1997). For To compare the state of c-Myb phosphorylation in these experiments we used COS7 cells, because the cells untreated or treated with a Ser/Thr protein protein can be transiently expressed at high levels and c- phosphatase inhibitor, transiently transfected COS7 Myb is degraded by the 26S proteasome in these cells at a cells were metabolically labeled with 32P-orthophos- rate similar to that observed in hematopoietic cells (Bies phate in phosphate-free medium for 6 h. Immunopre- et al., 1999). Exponentially growing COS7 cells, cipitated protein from 32P-labeled cells treated with OA transiently transfected with plasmid DNAs containing and ALLN (to prevent degradation of hyperphos- wt c-Myb and mutants, were metabolically labeled 36 h phorylated forms of c-Myb) or ALLN alone were after transfection and chased in complete medium either fractionated by SDS ± PAGE, transferred to the in the presence or the absence of OA. The stabilities of wt membrane and analysed by direct exposure to X-ray and mutated forms of the protein were analysed by ®lm. As expected, inhibition of phosphatases in immunoprecipitation (Figure 5). The CT6-MybD(266aa) conjunction with proteasome inhibition resulted in had a marked increase in resistance to the proteasome the appearance of a slower migrating form of 32P- compared to wt or the other mutants and, in addition, it labeled c-Myb. There was, in addition, an increase in

Oncogene Phosphorylation-dependent instability of c-Myb J Bies et al 2850

Figure 5 The COOH-terminal region of c-Myb is required for okadaic acid-induced degradation. COS7 cells transiently expressing FL-Myb, FL-Myb(S11,12A), FL-Myb(S528A) or CT6-MybD(266aa) were metabolically labeled and analysed in a pulse-chase experiment in the presence or absence of okadaic acid (OA, 1 mM). DMSO was used at the concentration 0.5% in control reactions. The gels were quantitatively analysed as described in Figure 2. Each bar on the graph represents the relative amount of c-Myb after 90 min of chase. The level of protein at the beginning of the pulse was assigned to 100%

methods. As presented in Figure 7b, there was an obvious increase in the phosphorylation of Thr residues as a result of OA treatment. Quantitative analyses of phosphoamino acid contents on TLC plates revealed that the increase in phosphorylation of threonine residues in the hyperphosphorylated form of Myb was more than 350%. We then determined whether the increased stability of the COOH-terminally truncated mutant CT6-MybD(266aa) (Figure 5) corre- lated with a loss of phosphorylation on Thr. As shown in Figure 7c the ratio of phosphorylated serines and in FL-Myb and a less truncated mutant CT4-MybD(151aa) was similar. However, there was a 35% decrease in Thr phosphorylation of the CT6- MybD(266aa) protein (for quantitation, the level of P- Thr was normalized to the level of P-Ser; this may Figure 6 Mutation of serine 11, 12, or 528 to alanine does not actually give an understimate of the per cent decrease prevent the hyperphosphorylation-induced conformational P-Thr if P-Ser levels decreased somewhat as well). The changes in c-Myb caused by OA treatment. COS7 cells, region of murine c-Myb spanning amino acids 371 ± transiently transfected with FL-Myb, FL-Myb(S11,12A), or FL- 485 is highly conserved among c-Myb proteins from Myb(S528A) were untreated (7) or treated (+) with OA (1 mM) together with ALLN (100 mM) for 90 min and lysed. Protein di€erent species and could contain a threonine residue samples were separated by SDS ± PAGE, transferred to a which is phosphorylated in cells after inhibition of Ser/ nitrocellulose membrane, probed with anti-c-Myb monoclonal Thr phosphatases and could be responsible for Ab (aNH2-Myb) and developed using an ECL kit (Amersham) initiating of c-Myb targeted degradation. To test this hypothesis, we constructed individual mutants in which the overall amount of radioactive phosphate incorpo- one of 11 Thr were mutated to Ala and tested for rated into c-Myb when cells were treated with OA alterations in mobility and proteolytic stability. All of (Figure 7a). Next, we looked at which amino acids of the 11 mutated c-Myb proteins had similar proteolytic the protein were phosphorylated in cells treated with half-lives as wt c-Myb in untreated or OA treated cells OA and ALLN or ALLN alone. Phosphorylated (data not shown), suggesting that modi®cation of any proteins were hydrolyzed and phosphoamino acid single Thr residue in this region has no e€ect on analysis was performed as described in Materials and stability of c-Myb. However, we cannot rule out the

Oncogene Phosphorylation-dependent instability of c-Myb J Bies et al 2851

Figure 8 Tryptic peptide mapping of hypo- and hyperpho- sphorylated c-Myb. 32P-labeled hypo (P-c-Myb) and hyperpho- sphorylated c-Myb (PP-c-Myb) protein was digested with trypsin and the resulting tryptic peptides were separated by reverse phase HPLC on a C18 column. Radioactivity contents in separate fractions was assessed by Cerenkov counting

fractions 26, 46, and 52. We also found decreased radioactivity in fractions 22 and 38 ± 42, which could be a consequence of surface alterations due to the hyperphosphorylation of c-Myb. Phosphoamino acid analysis of peptides in fractions revealed that peptides in fractions 26 and 52 were phosphorylated on Thr. The peptide in fraction 26 contained only phospho-Thr and the one in 52 was phosphorylated on both Ser and Figure 7 Okadaic acid increases the phosphorylation of threo- Thr (data not shown). Phosphoamino acid analyses nine. (a) COS7 cells expressing FL-Myb were labeled in phosphate-free medium with inorganic 32Pi-orthophosphate for together with tryptic peptide mapping of hyperpho- 6 h. ALLN alone, or together with OA, was added for the last sphorylated c-Myb suggest that there is more than one 2 h and cells were lysed as described in Materials and methods. phosphorylated Thr residue in cells under strict control 32P-labeled c-Myb protein was immunoprecipitated, separated by of Ser/Thr phosphatases. SDS ± PAGE, transferred to a PVDF membrane and exposed to X-ray ®lm. (b) The radioactive bands depicted in panel A were cut out, treated with constant boiling HCl for 2 h at 1108C, and separated electrophoretically on thin-layer cellulose plates as Discussion described in Materials and methods. The positions of the phosphoserine (P-Ser), phosphothreonine (P-Thr), and phospho- Over the last few years, phosphorylation has been (P-Tyr) markers were visualized by ninhydrin staining and are indicated on the right. (c) Comparative phosphoamino implicated in the control of DNA-binding and acid analysis of FL-Myb, CT4-MybD(151aa) and CT6-Myb- transactivation activity of c-Myb (Bading et al., 1989; D(266aa) was performed as described in (b) Luscher et al., 1990; Aziz et al., 1995; Oelgeschlager et al., 1995; Miglarese et al., 1996). In data presented here, we have demonstrated for the ®rst time that possibility that multiple phosphorylation sites are hyperphosphorylation of c-Myb results in an altered required for the observed mobility changes and form with a slower mobility in gel electrophoresis and destabilization of protein. this alteration is correlated temporally with its rapid To determine if treatment of cells within OA resulted degradation by the 26S proteasome. It was established in phosphorylation of new sites in c-Myb, or increased in our laboratory previously, that the 26S proteasome the stochiometry of residues that were already is responsible for the degradation of c-Myb (Bies and phosphorylated, hypo- and hyperphosphorylated forms Wol€, 1997). This study provides new insight into the of c-Myb were digested with trypsin and the resulting mechanism involved in the targeting of the protein to peptides were separated by reverse-phase HPLC as this multimeric proteolytic complex. described in Materials and methods. Separated pep- On the basis of these studies we speculate that tides were monitored for radioactivity by Cerenkov hyperphosphorylation-induced conformational changes counting. As shown in Figure 8, similar pro®les of in c-Myb may expose epitopes on the surface of the radioactive tryptic peptides were obtained from the protein that are required for recognition and proteolytic hypo- and hyperphosphorylated forms of c-Myb. This processing. Alternatively, phosphorylation site(s) under result implies that OA increased the level of phosphor- strict control of a putative Ser/Thr phosphatase could ylation of c-Myb on residues already directly serve as a recognition signal for the proteolytic modi®ed in untreated cells rather than inducing de machinery. There are several examples where phosphor- novo sites of phosphorylation. Therefore, OA induced a ylation of speci®c amino acids in a protein substrate quantitative change in the phosphorylation of c-Myb. triggers an enzymatic cascade ultimately leading to Obvious increases in radioactivity were observed in destruction by proteasome. For example site speci®c

Oncogene Phosphorylation-dependent instability of c-Myb J Bies et al 2852 phosphorylation of E (Won and Reed, 1996), most sensitive region to the enzymatic activity of cyclin D1 (Diehl et al., 1997) and inhibitor p27Kip1 (Vlach endoproteinase Glu-C, trypsin or chymotrypsin (Krieg et al., 1997) marks these regulators of the cell cycle for et al., 1995; Bies and Wol€, unpublished result). modi®cation by and rapid degradation by the Phosphorylation-dependent conformational changes 26S proteasome. The speci®city of this process is could expose hydrophobic surfaces otherwise hidden in achieved by the recently identi®ed group of proteins the protein. This region, once exposed on the surface, found in ubiquitin ligase complexes. These proteins could directly serve as a recognition signal for proteolytic contain conserved structural motifs such as WD40 or machinery, or cause a switch between more than one WW, which can directly bind phosphorylated Ser or Thr interacting protein partner in cells. It has been proposed residues and recruit other proteins necessary for that some proteins contain a surface of prefolded destruction of the substrate by the 26S proteasome structure that is an important determinant for degrada- (Laney and Hochstrasser, 1999; Koepp et al., 1999). tion. This is exempli®ed by the yeast mating type Our data suggest that conformational changes in c- transcription factor a2 (reviewed in Laney and Hoch- Myb after OA treatment are required for accelerated strasser, 1999) and is in contrast to the short peptide proteolytic processing of c-Myb. The slower mobility sequences de®ned for recognition of proteins such as of c-Myb on SDS ± PAGE was detected much earlier IkBa (Chen et al., 1995; Brown et al., 1995). Thr than increased proteolytic processing. In addition, full- phosphorylation of c-Myb in vivo during mitosis was length c-Myb underwent a more dramatic structural demonstrated previously (Luscher and Eisenman, 1992). alteration than the more stable CT6-MybD(266aa). However, the relationship of this result to ours is unclear. Results from phosphoamino acid analyses and tryptic They reported that c-Myb isolated from chicken pre-B peptide mapping suggest phosphorylation of more than cell lymphoma arrested with nocodazole in G2/M phase one Thr residue in c-Myb after OA treatment. of cell cycle was phosphorylated on unidenti®ed Thr Phosphoamino acid analyses of tryptic peptides of residue(s) and also did not bind DNA. In contrast, we hyperphosphorylated protein identi®ed phosphorylated demonstrated that the hyperphosphorylated c-Myb from Thr residues in two separate fractions. We also OA treated cells was able to bind MRE-A with an detected a decrease in Thr phosphorylation by apparent anity similar to that of the hypophosphory- removing sequences between 371 aa (CT6-Myb- lated form. However, one cannot exclude the possibility D(266aa) and 485 aa (CT4-MybD(151aa). The region that the protocol used for puri®cation of mitotic c-Myb between aa 371 ± 485 is very conserved and contains in the other study could cause the irreversible denatura- several Thr residues which are potential phosphoryla- tion of the hyperphosphorylated form of c-Myb, as tion sites for di€erent protein kinases. In regard to this, DNA binding activity could not be rescued by it was reported by Aziz et al. (1993) that unidenti®ed phosphatase treatment in vitro (Luscher and Eisenman, Thr residue(s) in this region can be phosphorylated in 1992). In regard to phosphorylation during mitosis, we vitro by p42mapk. Also, we observed that truncation in have been unable to detect a signi®cant di€erence in the this region results in its increased half-life (Bies et al., half-life of c-Myb protein isolated either from interphase 1999). Therefore, we focused on this region and or nocodazole treated M1 cells (J Bies and L Wol€, prepared mutants of all 11 Thr. However, mutation unpublished observations). of any single Thr from the region spanning aa 371 ± Our ®ndings on the increased stability of the COOH- 485 failed to stabilize or prevent phosphorylation- truncated protein is intriguing in light of results from induced mobility changes of c-Myb protein after several laboratories that COOH-terminally truncated c- treatment of cells with OA (data not shown). There- Myb exhibits increased DNA binding anity, transac- fore, we can hypothesize that phosphorylation of more tivation and transforming capacity (Sakura et al., 1989; than one residue may be required for the proposed Hu et al., 1991; Grasser et al., 1991; Dash et al., 1996). conformational changes. Alternatively, phosphoryla- The mechanism through which NRD exerts its negative tion in the amino terminus may result in a conforma- e€ect on these activities is not clear. Also, several tional change which a€ects the carboxyl-terminal part cellular proteins capable of binding to the NRD of c- of the protein. Finally, we cannot completely rule out Myc were detected (Kanei-Ishii et al., 1992; Tavner et the possibility that a c-Myb interacting protein is al., 1998). What role, if any, these interactions play in phosphorylated and responsible for its degradation. determining the half-life of c-Myb is not clear at It was suggested previously that c-Myb is capable of present and will require further experimentation. adopting an alternative conformation (Ness, 1996). The In summary, our data demonstrate that disruption of EVES motif in COOH-terminus can interact with the a phosphorylation pathway has a signi®cant e€ect on DNA binding domain of c-Myb and this intramolecular the phosphorylation of c-Myb and its half-life. Studies interaction and consequent conformational change can are in progress to precisely map phosphorylated Thr a€ect the function of c-Myb as a transcriptional residue(s) in c-Myb and to determine their role in the activator. This intramolecular interaction can be proposed conformational changes and subsequent competitively inhibited by binding of p100 protein which degradation. contains an identical EVES motif (Dash et al., 1996). Experiments with linker insertion mutants in the carboxyl terminus also implied that conformational changes are important in regulation of c-Myb activity Materials and methods (Dubendor€ and Lipsick, 1999). That the COOH- terminus can adopt an open conformation and be Cell lines and culture conditions sensitive to proteolysis when bound to DNA is in The murine myeloid leukemia cell line, M1 (Liebermann and agreement with in vitro proteolytic clipping experiments. Ho€man-Liebermann, 1989), was maintained in RPMI 1640 The COOH-terminus of c-Myb has been identi®ed as the medium supplemented with 10% heat-inactivated horse

Oncogene Phosphorylation-dependent instability of c-Myb J Bies et al 2853 serum. COS7 cells (simian virus 40 large T antigen pleteTM) as well as the phosphatase inhibitors sodium ¯uoride transformed CV-1 cells) were cultured in Dulbecco's modi®ed (50 mM), sodium pyrophosphate (30 mM) and sodium Eagle medium (DMEM) with 10% fetal calf serum. The orthovanadate (100 mM). Cell lysates were sonicated, cen- inhibitors of Ser/Thr phosphatases, the 26S proteasome, trifuged at 13 000 r.p.m. for 15 min at 48C and protein RNA synthesis, and protein synthesis were used in the cell extracts were fractionated by SDS ± PAGE on an 8% gel and culture experiments as described in the ®gure legends and electrophoretically transferred to a nitrocellulose membrane Results section. (Schleicher & Schuell). Immunoblots were incubated with anti-c-Myb monoclonal antibodies (a kind gift from Tim Bender, University of Virginia, Charlottesville, Virginia and Reagents purchased from Upstate Biotechnology) and detected using The peptide aldehyde N-acetyl-L-leucinyl-L-leucinyl-norleuc- an ECL kit (Amersham) in accordance with the manufac- inal (ALLN, Sigma) and okadaic acid (OA, Sigma) were turer's instructions. dissolved in DMSO and used at concentrations of 1 mM and 100 m , respectively. The inhibitor of protein synthesis, M Phosphoamino acid analysis and tryptic peptide mapping cycloheximide (CHX, Sigma), and RNA synthesis, actinomy- cin D (ActD, Sigma), were dissolved in PBS and used at the Phosphoamino acid analysis was performed as described indicated concentrations. previously (Biesova et al., 1997). COS7 cells, transiently expressing full-length c-Myb, were rinsed twice with Tris- bu€ered saline (20 m Tris-HCl [pH 7.4], 137 mM NaCl, Plasmid constructs M 2.7 mM KCl) and labeled in phosphate-free Dulbecco's The expression vector encoding murine c-Myb under the modi®ed Eagle medium with 0.5 mCi/ml carrier-free 32Pi control of CMV promoter was prepared by subcloning a 2 kb orthophosphate (ICN) for 6 h. During the last 90 min of BamHI ± XbaI fragment of murine c-myb cDNA (Bender and metabolic labeling, the cells were treated with OA (1 mM) and Kuehl, 1986) into pcDNA3.1(+) vector (Invitrogen). Pre- ALLN (100 mM) or ALLN alone. Phosphorylated forms of c- mature termination mutants CT4-MybD(151aa) and CT6- Myb were immunoprecipitated, fractionated on SDS ± PAGE MybD(266aa) were constructed as described previously (Bies and transferred to an Immobilon-P membrane (Millipore). et al., 1999). A vector containing the mutant of c-Myb, where The part of the membrane with immobilized 32P-labeled c- serines 11, 12 was replaced with alanines FL-Myb(S11,12A) Myb protein was excised and hydrolyzed in 200 ml of 6N HCl was kindly provided by Bernard Luscher (Medizinische (constant boiling) (Sigma) at 1108C for 2 h. Hydrolyzed Hochschule Hannover, Hannover, Germany) and a construct samples were diluted with water, frozen in dry ice, lyophilized containing mutant of serine 528 to alanine FL-Myb(S528A) in a Speedvac (Savant), and resuspended in 10 ml of water was provided by Tim Bender (University of Virginia, containing 0.1 mg/ml of each phosphoamino acid standard, Charlottesville, Virginia). phosphoserine, phosphothreonine and phosphotyrosine (Sig- ma). The samples were spotted onto thin-layer cellulose plates (Sigma) and subjected to electrophoresis at 48CinpH Transient transfection 3.5 bu€er (pyridine-glacial acetic acid-water, 5 : 50 : 945) for Plasmids encoding c-Myb proteins were transiently trans- 1 h at 950 V. The migration of phosphoamino acid standards fected into COS7 cells using DOSPER liposomal transfection was revealed by staining with ninhydrin (Sigma) and 32P- reagent (Boehringer Mannheim) according to the manufac- labeled phosphoamino acids were visualized by autoradio- turer's instructions. Brie¯y, COS7 cells were plated at 36105 graphy. cells per 100 mm dish 1 day prior to transfection. The For tryptic peptide mapping, immunoprecipitated 32P- following day, the medium was changed and cells were labeled c-Myb protein was separated on SDS ± PAGE and transfected with 10 mg of plasmid DNA and 30 mlof transferred to the nitrocellulose membrane (Schleicher & DOSPER reagent. Protein analyses were performed 36 ± Schuell). The piece of membrane with immobilized c-Myb 48 h post-transfection. was incubated for 30 min at 378C in 0.5% PVP-360 100 mM acetic acid, extensively washed in 50 mM ammonium bicarbonate and digested in the same bu€er with 10 mgof Metabolic labeling of proteins, immunoprecipitation and gel TPCK-trypsin (Sigma) for 2 h at 378C. Peptides were diluted electrophoresis with water, frozen in dry ice and lyophilized in a Speedvac Protein stability was evaluated in a pulse-chase experiment as (Savant). Lyophilized peptides were dissolved in water and described previously (Bies and Wol€, 1997). Brie¯y, the cells analysed by reversed-phase HPLC (Hewlett-Packard 1090L) were starved in methionine/cysteine free medium, labeled for using a narrow bore C-8 column (2.16250 mm, 5 micron 45 min (pulse) with Trans 35S label (ICN) and chased in pre- particle size; Advantage) and a water-acetonitrile gradient warmed complete medium without radioactive amino acids. (5 ± 60% in 1 h) in the presence of 0.1% TFA. Absorbance At the indicated times, the cells were disrupted in cold lysis from 200 ± 600 nm was monitored continuously using a bu€er (20 mM Tris-HCl [pH 7.5], 50 mM NaCl, 0.5% NP40, Waters 996 photodiode array detector. Fractions were 0.5% SDS, 0.5% sodium deoxycholate) supplemented with a collected at 1 min intervals and radioactivity measured by mix of proteases inhibitors (CompleteTM, Boehringer Man- Cerenkov counting. nheim), sonicated and immunoprecipitated with rabbit anti-c- Myb antiserum (Bies et al., 1996). Immunocomplexes were Electrophoretic gel mobility shift assay (EMSA) separated by SDS ± PAGE on an 8% gel and visualized by ¯uorography. Quantitative analyses of radioactive signals EMSA with COS7/FL-Myb cell lysates was performed as were performed on a PhosphorImager 425 using IMAGE- described by Oelgeschlager et al. (1995). Brie¯y, COS7 cells QUANT software (Molecular Dynamics). expressing the c-Myb protein were lysed in EMSA lysis bu€er (10 mM Tris-HCl [pH 7.05], 50 mM NaCl, 30 mM sodium pyrophosphate, 50 m NaF, 5 mM ZnCl , 100 mM Na VO , Immunoblotting M 2 3 4 1% Triton X-100) supplemented with a cocktail of proteases Untreated cells or cells treated with an inhibitor of Ser/Thr inhibitors (CompleteTM). The lysate was vortexed for 30 s, protein phosphatases, OA, and an inhibitor of the 26S centrifuged at 48C for 30 min at 14 000 r.p.m. and stored at proteasome, ALLN, for the indicated times, were washed 7808C. For EMSAs, the Myb responsive element (MRE) twice in phosphate-bu€ered saline, and lysed in lysis bu€er from the mim-1 promoter (site A) (Ness et al., 1989) was supplemented with a cocktail of protease inhibitors (Com- generated by the hybridization of synthesized oligonucleo-

Oncogene Phosphorylation-dependent instability of c-Myb J Bies et al 2854 tides 5'-TCGACACATTATAACGGTTTTTTAGC-3' and 5'- 0.25 mM EDTA), dried and exposed to X-ray ®lm at room TCGAGCTAAAAAACCGTTATAATGTG-3'. The double- temperature for 4 ± 20 h. stranded oligonucleotide was labeled with a-32P-dCTP (ICN) to a speci®c activity greater than 108 c.p.m./mg by end ®lling using Klenow fragment. The DNA-binding reaction was performed for 30 min at 278C with 1 ml of cell lysate and 0.1 ng of labeled oligonucleotide probe in reaction bu€er Acknowledgments (10 mM Tris-HCl [pH 7.9], 50 mM NaCl, 1 mM EDTA, We thank TP Bender and MR Miglarese for providing 10 mM dithiothreitol, 0.5% nonfat dry milk, 5% glycerol vector containing mutant FL-Myb(S528A) and B Luscher and 1 mg poly(dI-dC)). The DNA-protein complexes were for providing vector containing mutant FL-Myb(S11,12A). separated by PAGE on a 5% gel in 0.256TBE bu€er This work was supported in part by a grant No. 2/5052/98 (12.5 mM Tris-HCl [pH 8.5], 12.5 mM boric acid and from VEGA Slovak Academy of Sciences.

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Oncogene