Interaction Between the Protein Kinase B-Raf and the A-Subunit of the IIS Proteasome Regulator1

(CANCER RESEARCH 58. 2986-2990, July 15, I998|

Advances in Brief

Interaction between the Protein Kinase B-Raf and the a-Subunit of the IIS Proteasome Regulator1

Andreas Kalmes,2 Garsten Hagemann,2 Christoph K. Weber, Ludmilla Wixler, Tillman Schuster, and Ulf R. Kapp*

InstituÃfÃ¼rMedizinische Strahlenkunde und Zettforschung (MSZ). University of WÃ¼rzburg,97078 WÃ¼rzburg,Germany Â¡C.H.. C. K. W.. L. W.. T. S.. U. K. R.], and Department of Surgery, University of Washington, Seattle. Washington 9SI95 Â¡A.KJ

Abstract using the yeast two-hybrid system. In addition to the already known B-Raf-binding proteins (MEK and several 14-3-3 isoforms), we iso Protein kinases of the Raf family act as signal-transducing elements lated PA28a, the subunit of the 1IS proteasome regulator (11, 12), as downstream of activated cell surface receptors and are involved in the a novel B-Raf-binding partner. regulation of proliferation, differentiation, and cell survival. Whereas the role of c-Raf-1 as a mitogen-activated protein/extracellular signal-regu The 11S regulator is one of two known activators of the 20S lated kinase activator within the mitogenic cascade is well established, less proteasome (13). Both of these activators seem to possess different is known about the mammalian Raf isoforins A-Raf and B-Raf. Here we functions in vivo. The PA700 activator complex (19S regulator) binds report that B-Raf binds to PA28a, one of two subunits of the 1IS regulator to the 20S proteasome core complex to form the active 26S protea of proteasomes. PA28<* was isolated as a B-Raf-binding protein in a yeast some, which mediates the ATP- and ubiquitination-dependent degra two-hybrid screen of a PC 12 cDNA library. Both proteins can be coim- dation of cytosolic and nuclear proteins (13). In contrast, the US munoprecipitated after transient expression in 293 cells. No association regulator functions independently of both ATP and ubiquitination and could be found between PA2Sa and A-Raf or c-Raf-1. B-Raf binds to a has been demonstrated to have a function in MHC class I-mediated region in PA28a that is important for its proteasome-activating function. antigen presentation (14). Attachment of the 1IS regulator to the 20S proteasome core complex influences the specificity of the proteasomal Introduction degradation machinery, thereby causing an enhancement of the rep The serine/threonine kinases of the Raf family act as components of ertoire of antigenic peptides presented on the cell surface without the Ras/Raf/MEK4/extracellular signal-regulated kinase cascade in affecting total protein turnover (14, 15). signal transduction from membrane-associated receptors to transcrip Materials and Methods tion factors in the nucleus ( 1). In mammalian cells, the Raf family consists of three members: (a) c-Raf-1; (b) A-Raf; and (c) B-Raf. All Plasmid Constructions and Generation of the PC12 cDNA Library. three Raf family members share a conserved structure with three The two-hybrid vectors pPC86 and pPC97 were a generous gift from D. evolutionary conserved regions, CR1 to CR3. The CR1 and CR2 Nathans. The full-length B-Raf cDNA was subcloned in pPC97 as a fusion domains are part of the regulatory amino-terminal part of the Raf with the Gal4 DNA-binding domain and in pPC86 as a fusion with the Gal4 proteins, whereas CR3 comprises the carboxyl-terminal kinase do transactivation domain. B-Raf deletion mutants; B-Raf (1-445) was generated main (1). The CR1 domain contains the Ras-binding domain and a by subcloning a B-raf SaWXhol fragment from pPC97-B-Raf in the SalÃ¬and Bglll sites of pPC97. with the Xho\ and Bgll\ sites blunted. A cDNA fragment cysteine-rich region involved in the binding of Raf to both Ras and representing B-Raf (145-447) was amplified by PCR using primers 5'-AG- 14-3-3 proteins (2, 3). Signaling via c-Raf-1 involves binding Raf to ATGTGGCGTCGACCAACCCCAAG-3' (upstream, introducing a Sail site) activated GTP-bound Ras at the plasma membrane and subsequent and S'-CAGGAATCTCCCAGCGGCCGCTCGAGTCCCGTCTAG-S' (down steps that currently are not fully understood (1). stream, introducing a Noti site) and subcloned Sali/Noti in pPC97. We and others have recently shown that A-Raf can bind to CK2jÃŸ, PC 12 mRNA was isolated from mitotic PC 12 cells by guanidine isothio- the regulatory subunit of protein kinase CK2 (4, 5). Binding CK2ÃŸto cyanate preparation of total RNA and subsequent purification of mRNA using A-Raf after coexpression in Sf9 cells leads to the activation of A-Raf. the PolyATtract mRNA isolation system (Promega). To generate PC 12 cDNA, we used the Promega Riboclone kit according to the manufacturer's instruc c-Raf-1 and B-Raf, however, are not activated by CK2ÃŸin the same assays (4). Although the potential physiological role of this interaction tions, using 2.5 fig of random primers and 20 /xg of PC 12 mRNA. After ligation of EcoRI adapters, PC 12 cDNA was ligated to EroRI-digested remains to be clarified, these results provide a first example of a Raf pPC86HA3 vector DNA and transformed into XL2-blue supercompetent Esch- isozyme-specific protein-protein interaction. ericlna coli (Stratagene). Colonies (8.5 X IO6) were harvested, and plasmid B-Raf has been shown to be a major MEK activator in brain DNA was prepared according to standard procedures. The two-hybrid library extracts (6, 7), NIH3T3 cells (8), and NGF-stimulated rat pheochro- vector pPC86HA3 is a derivative of pPC86 constructed by inserting a triple mocytoma PC 12 cells (9, 10). In an attempt to identify proteins hemagglutinine tag (HA tag) into the Sail site of the multiple cloning region. interacting specifically with B-Raf, we screened a PC 12 cDNA library The 5' end of the coding region of the PA28a gene was isolated by reverse transcription-PCR using primers 5'-CATGAATTCCTGCAGATGGCTA- CACTGAGGGTCCAT-3' (upstream) and 5'-CTTGATCTCGGGCTTTAG- Received 5/14/98; accepted 5/29/98. GCGTTG-3' (downstream). The resulting PCR product was subcloned in the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with T cloning vector pMOS Blue, resulting in plasmid p28T.l. A full-length 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by Grants RA642/I-1, WE2023/2-1, and SFB 172 from the Deutsche PA28a clone was generated by subcloning a Pstl/Aval fragment from p28T. 1 containing the PA28a amino-terminal sequence with an Aval/EcoRl fragment Forschungsgemeinschaft. 2 A. K. and C. H. contributed equally to this work. from library clone B12.18 in pUCIS, resulting in plasmid pUC-PA28FL. For 1To whom requests for reprints should be addressed, at Institut fÃ¼rMedizinische expression in E. coli, full-length PA28a cDNA was isolated from pUC- Strahlenkunde und Zellforschung (MSZ), University of WÃ¼rzburg,Versbacher Strasse 5, PA28FL as a Pstl/EcoRl fragment and ligated to pGEX-KG digested with 97078 WUrzburg, Germany. Phone: 49-931-201-5140; Fax: 49-931-201-3835. 4 The abbreviations used are: MEK. mitogen-aclivated protein/extracellular signal- Smal and EcoRI after degradation of the Psn overhangs. For expression in Sf9 regulated kinase kinase; NGF. nerve growth factor; HA, hemagglutinin; ÃŸ-Gal.ÃŸ-galac- insect cells, the PA28a Pstl/EcoRl fragment from pUC-PA28FL was sub- tosidase. cloned in pVL1392 to generate pVL-PA28FL. For expression in mammalian 2986

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1998 American Association for Cancer Research. B-RAF BINDS TO PA28o cells, the full-length PA28a sequence was isolated from pVL-PA28FL and The rat PA28a gene encodes a protein of 249 amino acids (12). The subcloned in pCMV5 as a Pstl/BamH\ fragment to generate pCMV5-PA28FL. four PA28a clones isolated in our screen represent four different A triple hemagglutinine tag (HA tag) was introduced into pCMV5-PA28FL by partial polypeptides starting at different amino acid positions behind inserting the tag at the 5' end of the PA28a sequence, resulting in plasmid the amino terminus, all of which contain the carboxyl terminus of PCMV-HAPA28. PA28a [PA28a(51-249). PA28a(75-249), PA28a(88-249), and The PA28a deletion mutant PA28a(51-174) in pPC86HA3 was generated PA28a( 106-249)]. by digesting the PA28a library clone B12.18, starting at PA28a amino acid No PA28a cDNA clones were isolated in screens of the same position 51. with Bglll and by subsequent religation. By this procedure, a library with A-Raf or c-Raf-1 as bait,6 suggesting that a Raf isozyme- nucleotide sequence encoding carboxyl-terminal amino acids 175-249 was deleted. To generate the PA28a deletion mutant PA28a(l-120), a correspond specific interaction had been identified. To further address this ques ing PA28a fragment was isolated from the PA28a full-length clone in tion, we tested the binding of PA28a with A-Raf and c-Raf-1 in direct pGEX-KG after digestion with BamHl and Ava\ and subcloned into the Bgl\\ two-hybrid binding assays. As shown in Fig. 1/4, no interaction could site of pPC86HA3 by blunt-end ligation after end-filling the 5' overhangs. be detected. Both Raf isoforms scored positive in combination with pcDNA3-14-3-30 containing an ammo-terminal myc tag was a generous Ras or MEK, the upstream activator and downstream effectors. In the gift from W. J. Fanti. same assays, PA28a did not bind to Ras or MEK (Fig. \A). Yeast Two-Hybrid Library Screening. Yeast cultures were grown at 30Â°Cunder standard conditions in liquid or on solid media based on either Mapping of the Binding Domains. In a first attempt to map the PA28a-binding domain in B-Raf, we found that PA28a binds to the YPD or minimal SD media. amino-terminal regulatory part of the B-Raf polypeptide but not to the The yeast strain HF7c was sequentially transformed with first the bait carboxyl-terminal kinase domain in two-hybrid interaction assays plasmid and then with the cDNA library. Transformants were grown on SD medium lacking the amino acids leucine. tryptophane. and histidine. After 3 (Fig. 10). However, a B-Raf deletion mutant lacking the region days, growing clones were tested for activation of the lacZ reporter gene in a amino-terminal of the CR1 domain lost the ability to bind to PA28a ÃŸ-Galfilter-assay. Clones that were positive at this step were further verified in this assay (Fig. IÃŸ).Because this part of the protein represents one by retransforming the isolated library plasmid into HF7c together with differ of the regions that is not conserved between the different mammalian ent bait plasmids. Clones showing a positive result for the ÃŸ-Galassay only in Raf isoforms (1), this finding is in agreement with and might explain the presence of Raf as bait were scored as positive. the observed isozyme specificity. Mammalian Cell Culture and Transient Expression in Mammalian Cells. The 293 cells were grown under standard conditions (37Â°C,5%CO2) in The shortest PA28Â« clone isolated in our screen encodes a PA28a polypeptide lacking the amino-terminal 105 amino acids DMEM (Life Technologies, Inc.) supplemented with 10% heat-inactivated [PA28a(106-249); Fig. IÃŸ].To further map the B-Raf-binding fetal bovine serum (Hyclone) and 100 units/ml streptomycin and penicillin (Life Technologies, Inc.). The 293 cells were transfected using the calcium region in PA28a, we tested different PA28 deletion mutants in phosphate method as described previously (16). At 48 h after transfection, cells two-hybrid interaction assays. B-Raf bound to PA28a(51-174) were washed twice with PBS and lysed in lysis buffer [50 mM Tris-Cl (pH 7.5). lacking the carboxyl-terminal 75 amino acids, but not to PA28a(l- 137 mM NaCl. 1% Triton X-100, 10 mM sodium pyrophosphate, 25 mM sodium 120) (Fig. IÃŸ).We conclude from these data that the B-Raf- glycerophosphate, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 0.1% 2-mercap- binding region in PA28a is located in the middle portion of PA28a toethanol, 1 mM sodium vanadate, 0.01 % leupeptin. 0.01 % aprotinin, and 2 mM between amino acids 120 and 174. pepstatin] for 10 min on ice. The lysates were precleared by centrifugation at B-Raf Binds to PA28a in Mammalian Cells. For further analysis 10,000 X g at 4Â°Cfor 10 min. of the observed interaction between B-Raf and PA28a, we isolated the Immunoprecipitations and Western Blots. Precleared detergent lysates missing portion of the PA28a coding sequence from PC 12 mRNA by of Sf9 cells or 293 cells were subjected to immunoprecipitations after the addition of 25 fi\ of protein A-agarose (Boehringer Mannheim) and the reverse transcription PCR. This partial sequence was used to generate indicated antisera at 4Â°Cfor 2 h. Immune complexes were washed twice in a full-length HA-tagged PA28a clone (HA-PA28a) in pCMVS. To lysis buffer and once in HEPES buffer [25 mM HEPES (pH 7.5), 25 mM test whether B-Raf can bind to full-length PA28a, and whether the sodium glycerophosphate. 1.5 mM EGTA, 5% glycerol, and 1 mM DTT] for association of both proteins also occurs in mammalian cells, B-Raf subsequent immune complex kinase assays or washed three times in lysis and HA-PA28a were coexpressed in human embryonic kidney 293 buffer for subsequent Western blot analysis. cells and assayed for complex formation in coimmunoprecipitation For Western blot analysis, proteins were separated by SDS-PAGE and experiments. As shown in Fig. 2A (middle panel), HA-PA28Â« could transferred onto a nitrocellulose membrane (Schleicher & Schuell). Immuno- be coimmunoprecipitated with B-Raf antiserum on coexpression of detection was performed using the enhanced chemiluminescence system (Am- ersham) according to the manufacturer's protocol, using the antisera as indi both proteins. In a reciprocal setting, B-Raf could be immunoprecipitated with the HA tag-specific monoclonal antibody 12CA5, depend cated in the text. ing on the presence of HA-PA28a (Fig. 2A, lower panel). 14-3-3 proteins have been discussed as possible regulators of Raf-proteins Results (17). B-Raf has been isolated as a protein complex with 14-3-3 from Isolation of PA28a in a B-Raf Two-Hybrid Screen. Using the brain extracts (7) and has been reported to interact with several 14-3-3 yeast two-hybrid system, 7.5 X IO6 clones of a PC 12 cDNA library isoforms in the yeast two-hybrid system (18). We have isolated were screened with full-length B-Raf as bait. Of 91 His- and lacZ- different 14-3-3 isoforms as interaction partners of B-Raf in our positive clones, 38 clones were finally scored as positive, based on the two-hybrid screen. The 14-3-3 proteins bind to phosphorylated serine fact that the His- and lacZ-positive phenotype of the yeast transfor residues in the CR2 domain (serine 259) and at the carboxyl-terminal mants was dependent on the coexpression of B-Raf as bait. end of the kinase domain (serine 621) of c-Raf-1 (19). These binding These positive clones encoded MEK-1 (4 clones), 14-3-3ÃŸ(11 sites are conserved between the different Raf-isoforms. It has been clones), 14-3-3Â£(12 clones), 14-3-3e (2 clones), 14-3-30 (2 clones), proposed that simultaneous binding of 14-3-3 proteins to inactive 14-3-3T) (3 clones), and PA28a (4 clones). Two clones coding for c-Raf-1 at both sites might lead to a closed conformation of Raf-1 (17) PA28a were isolated in a second two-hybrid screen of a different that might interfere with the binding of c-Raf-1 to other proteins. To cDNA library.5 test whether binding 14-3-3 to B-Raf has any effect on the interaction

5C. Hagemann, A. Kalmes. L. Wixler. T. Schuster, and U. R. Rapp, unpublished 6U. Frey, A. Kalmes, C. Hagemann, L. Wixler, T. Schuster, and U. R. Rapp, observations. unpublished observations. 2987

B His'/LacZ* baitB-Rafc-Raf-1A-RafRasMEKpreyPA28ct CRI CR2 CR3 B-RafFL 1 (51-249)+----Ras+++n.d.n.d.MEK+++n.d.n.d.B-Raf(l-445) B-Raf (378-765)

B-Raf( 145-447)

PA28a(51-249) SU/////////7I249 PA28a( 106-249) /\

PA28a(51-174) \ /////A

PA28a (1-120) /////

Fig. I. Specific interactions of B-Raf and PA28Â« in two-hybrid binding assays. Yeast strain HF7c was cotransformed with the indicated combinations of bait and prey, and transformants were tested for His and /3-Gal prototrophy. A, PA28tÂ»interacts with B-Raf, but not with c-Raf-1, A-Raf. Ras, or MEK. B. full-length B-Raf (B-RafFL) and different B-Raf deletion mutants were tested as bait with library clone B12.18 [PA28a( 106-249)] as prey (lop panel), and different partial PA28or clones were tested as bait in combination with B-Raf-FL as prey (bottompanel). B-Raf(l-445) and B-Raf( 145-447) were positive in combination with Ras but not with MEK, and B-Raf(378-765) was positive with MEK, but not with Ras as prey, confirming the expression and proper folding of these mutants.7 n.d., not determined. of PA28a with B-Raf, we coexpressed these proteins in 293 cells and -0, and -T}.Papin et al. (18) have recently reported the isolation of assayed for complex formation in coimmunoprecipitation experi 14-3-3-f, -T), and -6 in a two-hybrid screen using the B-Raf kinase ments. As shown in Fig. 2B, both PA28a and 14-3-3 can be detected domain as bait. In several two-hybrid screens with c-Raf-1 as bait, in B-Raf immune complexes on coexpression of all three proteins. only 14-3-3-ÃŸand 14-3-3-f were isolated (24-26). Whether the The relative amounts of PA28a bound to B-Raf in these assays are not isolation of additional 14-3-3 isoforms with B-Raf as bait reflects a different after coexpression with B-Raf alone compared to those after potential Raf and 14-3-3 isoform-specific physiological diversity re coexpression with B-Raf and 14-3-3/3. mains to be clarified. In this screen, we did not isolate any clones for B-Raf Does Not Phosphorylate PA28a. Phosphorylation of Ras, the immediate upstream activator of Raf. However, in a second PA28a at serine residues has been reported to be important for its screen, seven H-Ras clones were isolated with B-Raf as bait.5 function as a proteasome activator (20). MEK-1 is the only physio In addition, we report the interaction of B-Raf with a subunit of the logical B-Raf substrate described thus far. To test whether PA28a 1IS proteasome regulator, PA28a, in the yeast two-hybrid system and might be an alternative substrate for B-Raf, we used different prepa in mammalian cells. PA28a binds to a region in the B-Raf amino- rations of PA28a expressed either in E. coli, Sf9 cells, or in 293 cells as potential protein substrates in B-Raf immune complex kinase terminal CR1 domain. This part of the protein is present in neither A-Raf nor c-Raf-1, but it is highly conserved between B-Raf proteins assays. Whereas the Raf substrate MEK was efficiently phosphorylated in these assays, no phosphorylation by B-Raf of any of the of different vertebrate species (1, 27) and thus might explain the different PA28a preparations was observed.7 However, because no B-Raf-specific binding. B-Raf and PA28a were also isolated as a protein complex after kinase phosphorylating PA28a in vivo or in vitro is known to date, we cannot exclude the possibility that this result is inherent to our PA28a transient expression in 293 cells. This complex was stable under stringent conditions (0.1 % SDS)7 that were able to abolish binding of preparations. c-Raf-1 to 14-3-3 proteins (28), indicating a relatively strong binding. Discussion In PC 12 cells, B-Raf has been shown to be a major MEK activator responsible for the sustained extracellular signal-regulated kinase In this study, we report the identification of B-Raf-binding proteins by screening a PC 12 cDNA library using the yeast two-hybrid system. activation in response to NGF stimulation, which leads to the differ Positive clones represented three classes, two of which encoded entiation of PC12 cells into a neuronal phenotype (9, 10). Inhibition of proteins already known to interact with B-Raf, MEK-1 and 14-3-3. No proteasomes by lactacystin is capable of inducing cell cycle arrest in clones for MEK-2 have been identified in this screen. In NIH3T3 cells MG-63 human osteosarcoma cells and neurite outgrowth in a NGF- and in epidermal growth factor-stimulated HeLa cells, MEK-1, but nonresponsive mouse neuroblastoma cell line; however, it does not not MEK-2, has been reported to be the major Raf-activated mitogen- have the same effects in PC 12 cells (29). activated protein kinase kinase (21, 22), although MEK-2 is activated Like c-Raf-1, which has been shown to act in concert with Bcl-2 as by oncogenic v-Raf (21, 23), and MEK-2 has previously been shown a suppressor of apoptosis in interleukin 3-deprived 32D.3 cells (30), to be able to interact with both c-Raf-1 and B-Raf in the yeast B-Raf involvement in the protection of cells against programmed cell two-hybrid system (18). death has been discussed. B-Raf knockout mice die from massive We detected several 14-3-3 isoforms in this screen: 14-3-3-ÃŸ,-f, -e, apoptosis in endothelial cells during embryonic vasculogenesis (31), suggesting a role for B-Raf as a survival factor. Whether or not B-Raf 7 A. Kalmes. C. Hagemann, and C. K. Weber, unpublished observations. fulfills this function in a Bcl-2-dependent manner is unknown. Recent 2988

and proteasome subunits LMP-2 and LMP-7 (37). The binding of the Ras effector B-Raf to a region in PA28a overlapping a domain critical for proteasome activation might point to an additional pathway for the 1r down-regulation of antigen presentation in Ras-transformed cells via the attenuation of proteasome activity. IP: B-Raf B-Raf IB: B-Raf Acknowledgments We thank D. Nathans (The John Hopkins University School of Medicine. Baltimore. MD) for plasmids pPC86 and pPC97, W. J. Fanti (Chiron Corpo ration, Emeryville. CA) for pcDNA3-14-3-30. and B. Bauer and R. Krug for IP: B-Raf technical assistance. PA28a IB: PA28a References

1. Daum. G.. Eisenmann Tappe, I., Fries, H. W., Troppmair. J., and Rapp, U. R. The ins and outs of Raf kinases. Trends Biochem. Sci., 19: 474-480, 1994. B-Raf IP: PA28a 2. Warne, P. H., Viciana, P. R.. and Downward. J. Direct interaction of Ras and the IB: B-Raf amino-terminal region of Raf-1 in vitro. Nature (Lond.). 364: 352-355. 1993. 3. Zhang, X. F.. Settleman. J., Kyriakis, J. M. Takeuchi Suzuki, E., Elledge. S. J.. Marshall, M. S.. Binder. J. T.. Rapp, U. R.. and Avruch. J. Normal and oncogenic p21""' proteins bind to the amino-terminal regulatory domain of c-Raf-l. Nature (Lond.). 364: 308-313, 1993. 4. Hagemann. C., Raimes. A.. Wixler, V., Wixler, L., Schuster, T.. and Rapp. U. R. The regulatory subunit of protein kinase CK2 is a specific A-Raf activator. FEBS Lett.. B 403: 200-202, 1997. 5. Boldyreff, B., and Issinger, O. G. A-Raf kinase is a new interacting partner of protein kinase CK2 ÃŸsubunit. FEBS Lett., 403: 197-199, 1997. 6. Catling, A. D., Reuter. C. W., Cox, M. E., Parsons. S. J.. and Weber. M. J. Partial V vv purification of a mitogen-activated protein kinase kinase activator from bovine brain. Identification as B-Raf or a B-Raf-associated activity. J. Biol. Chem.. 269: 30014- 30021. 1994. 7. Yamamori, B., Kuroda. S., Shimizu. K.. Fukui, K.. Ohtsuka. T.. and Takai, Y. Purification of a Ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain cytosol and its identification as a complex of B-Raf and 14-3-3 proteins. IP: B-Raf PA28a J. Biol. Chem., 270: 11723-11726. 1995. IB: PA28a 8. Reuler. C. W., Catling, A. D.. Jelinek. T.. and Weber. M. J. Biochemical analysis of MEK activation in NIH3T3 fibroblasts. Identification of B-Raf and other activators. J. Biol. Chem., 270: 7644-7655. 1995. 9. Traverse, S.. and Cohen, P. Identification of a latent MAP kinase kinase kinase in PC12 cells as B-Raf. FEBS Lett.. 350: 13-18, 1994. 14-3-3ÃŸ IP: B-Raf 10. Wixler, V., Smola, U., Schuler, M., and Rapp, U. Differential regulation of Raf IB: 14-3-3P isozymes by growth versus differentiation inducing factors in PC 12 pheochromocy- toma cells. FEBS Lett., 385: 131-137. 1996. 11. Chu Ping. M.. Vu. J. H.. Proske. R. J.. Slaughter. C. A., and DeManino, G. N. Fig. 2. Interaction of B-Raf and PA28a in 293 cells. The 293 cells were transfected Identification, purification, and characterization of a high molecular weight. ATP- with the indicated plasmids. After 48 h. proteins were immunoprecipitated from detergent dependent activator (PA700) of the 20S proteasome. J. Biol. Chem.. 269: 3539-3547. lysates with the indicated antisera and assayed by Western blot analysis. A, B-Raf was 1994. coimmunoprecipitated with PA28or and vice versa only when both proteins were ex 12. Ahn, J. Y., Tanahashi, N.. Akiyama. K.. Hisamatsu. H.. Noda. C.. Tanaka. K.. Chung. pressed. B, binding of B-Raf to PA28a is not affected by the presence of 14-3-3 proteins. C. H.. Shibmara, N.. Willy. P. J.. Mott, J. D.. Slaughter. C. A., and DeManino. G. N. Primary structures of two homologous subunits of PA28. a â€¢¿y-interferon-inducible IP, immunoprecipitation; IB, immunoblot; Vector, empty pCMVS vector. protein activator of the 20S proteasome. FEBS Lett., 366: 37-42, 1995. 13. Peters. J. M. Proteasomes: protein degradation machines of the cell. Trends Biochem. Sci., 19: 377-382. 1994. 14. Groettrup, M.. Soza, A., Eggers, M.. Kuehn. L., Dick, T. P., Schild. H.. Rammensee. studies suggest a connection between proteasome activity and apop- H. G., Koszinowski. U. H., and Kloetzel. P. M. A role of the proteasome regulator tosis. Depending on the cellular background, inhibition of proteaso- PA28a in antigen presentation. Nature (Lond.). 381: 166-168, 1996. 15. Dick. T. P.. Ruppert, T.. Groettrup. M.. Kloetzel. P. M. Kuehn. L., Koszinowski, mal activity by lactacystin or other specific proteasome inhibitors U. H.. Stevanovic. S.. Schild. H., and Rammensee. H. G. Coordinated dual cleavages either induced (32, 33) or inhibited (34, 35) programmed cell death. induced by the proteasome regulator PA28 lead to dominant MHC ligands. Cell. 86: Whether the interaction of B-Raf with PA28a is involved in the 253-262. 1996. 16. Troppmair. J.. Bruder. J. T.. MuÃ±oz.H.. Lloyd. P. A.. Kyriakis. J.. Banerjee. P.. function of B-Raf as a potential survival factor and/or a mediator of Avruch. J., and Rapp, U. R. Mitogen-activated protein kinase/extracellular signal- differentiation of NGF-stimulated PC 12 cells remains to be investi regulated protein kinase activation by oncogenes. serum, and 12-O-tetradecanoyl- phorbol-13-acetate requires Raf and is necessary for transformation. J. Biol. Chem., gated. 269: 7030-7035. 1994. The B-Raf-binding region in PA28a is located in the middle 17. Aitken, A. 14-3-3 and its possible role in coordinating multiple signaling pathways. Trends Cell. Biol.. 6: 341-347. 1996. portion of the protein between amino acids 120 and 174. Recently, 18. Papin. C.. Denouel. A.. Calothy, G.. and Eychene. A. Identification of signalling Zhang et al. (36) have identified a region in PA28a between amino proteins interacting with B-Raf in the yeast two-hybrid system. Oncogene. 12: acids 141 and 149 that is critical for its proteasome-activating func 2213-2221. 1996. 19. Muslin. A. J.. Tanner. J. W., Allen. P. M.. and Shaw. A. S. Interaction of 14-3-3 with tion, but not for the binding of PA28a to the proteasome (36). The signaling proteins is mediated by the recognition of phosphoserine. Cell. X4: 889- 1IS regulator has been shown to alter the specificity of the proteaso- 897, 1996. 20. Li, N.. Lerea, K. M., and Etlinger, J. D. Phosphorylation of the proteasome activator mal degradation machinery, enhancing the variety of antigenic pep- PA28 is required for proteasome activation. Biochem. Biophys. Res. Commun.. 225: tides presented on the cell surface in combination with MHC class I 855-860. 1996. 21. Jelinek, T., Catling, A. D., Reuter, C. W., Moodie. S. A., Wolfman. A., and Weber. molecules (14, 15). In Ras-transformed cells and in a variety of human M. J. RAS and RAF-1 form a signaling complex with MEK-1 but not MEK-2. Mol. tumor cells, a suppression of the antigen presentation machinery has Cell. Biol.. 14: 8212-8218. 1994. been observed, which is at least in part due to the down-regulated 22. Wu. X.. Noh. S. J.. Zhou, G.. Dixon. J. E.. and Guan. K. L. Selective activation of MEK1 but not MEK2 by A-Raf from epidermal growth factor-stimulated HeLa cells. expression of MHC class I, peptide transporters TAP-1 and TAP-2, J. Biol. Chem.. 271: 3265-3271. 1996. 2989

23. Wu, J., Harrison, J. K., Dent, P., Lynch, K. R., Weber, M. J., and Sturgill, T. W. 30. Wang, H.. Myashita, T., Takayama, S., Sato, T., Torigoe, T., Krajewski, S., Tanaka, Identification and characterization of a new mammalian milogen-activated protein S., Hovey, L., Troppmair, J., Rapp. U. R., and Reed, J. C. Apoptosis regulation by kinase kinase, MKK2. Mol. Cell. Biol., 13: 4539-4548, 1993. interaction of Bcl-2 protein and Raf-1 kinase. Oncogene, 9: 2751-2756, 1994. 24. Freed, E., Symons, M., Macdonald, S. G., McCormick, F., and Ruggieri, R. Binding 31. Wojnowski, L., Zimmer, A. M., Beck, T. W., Hahn, H., Bemal. R., Rapp, U. R., and of 14-3-3 proteins to the protein kinase Raf and effects on its activation. Science Zimmer, A. Endothelial apoptosis in Braf-deficient mice. Nat. Genet., 16: 293-297, (Washington DC), 265: 1713-1716, 1994. 1997. 25. Irie, K., Gotoh, Y., Yashar. B. M., Errede, B., Nishida, E., and Matsumoto, K. 32. Drexler, H. C. Activation of the cell death program by inhibition of proteasome Stimulatory effects of yeast and mammalian 14-3-3 proteins on the Raf protein function. Proc. Nati. Acad. Sci. USA, 94: 855-860, 1997. kinase. Science (Washington DC), 265.- 1716-1719, 1994. 33. Lopes, U. G., Erhardt, P., Yao, R., and Cooper, G. M. p53-dependent induction of 26. Fanti, W. J., Muslin, A. J., Kikuchi, A., Martin, J. A., MacNicol, A. M., Gross, R. W., apoptosis by proteasome inhibitors. J. Biol. Chem.. 272: 12893-12896, 1997. and Williams, L. T. Activation of Raf-1 by 14-3-3 proteins. Nature (Lond.), 371: 612-614, 1994. 34. Grimm, L. M., Goldberg, A. L., Poirier, G. G., Schwartz, L. M., and Osborne, B. A. Proteasomes play an essential role in thymocyte apoptosis. EMBO J., /5: 3835-3844, 27. Naumann, U., Hoffmeyer, A., Flory. E.. and Rapp, U. R. Raf protein serine/threonine kinases. In: F. Marks (ed.). Protein Phosphorylation, pp. 203-236. Weinheim, Ger 1996. many: Wiley-VCH, 1996. 35. Sadoul. R., Fernandez, P. A., Quiquerez, A. L., Martinou, I., Maki, M., Schroter, M., 28. Michaud, N. R., Fabian. J. R.. Mathcs, K. D., and Morrison, D. K. 14-3-3 is not Becherer. J. D.. Irmler, M.. Tschopp, J., and Martinou. J. C. Involvement of the essential for Raf-1 function: identification of Raf-1 proteins that are biologically proteasome in the programmed cell death of NGF-deprived sympathetic neurons. activated in a 14-3-3- and Ras-independent manner. Mol. Cell. Biol.. IS: 3390-3397. EMBO J.. 15: 3845-3852, 1996. 1995. 36. Zhang. Z.. Clawson, A., Realini. C., Jensen, C. C., Knowlton, J. R., Hill, C. P., and 29. Fenteany. G., Standaert, R. F., Reichard, G. A., Corey, E. J., and Schreiber, S. L. A Rechsteiner, M. Identification of an activation region in the proteasome activator ÃŸ-lactonerelated to lactacystin induces neunte outgrowth in a neuroblastoma cell line REGa. Proc. Nati. Acad. Sci. USA, 95: 2807-2811, 1998. and inhibits cell cycle progression in an osteosarcoma cell line. Proc. Nati. Acad. Sci. 37. Seliger, B., Maeurer, M. J., and Ferrone. S. TAP offâ€”tumors on. Immunol. Today, USA. 91: 3358-3362, 1994. 18: 292-299, 1997.

2990

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1998 American Association for Cancer Research. Interaction between the Protein Kinase B-Raf and the α-Subunit of the 11S Proteasome Regulator

Andreas Kalmes, Carsten Hagemann, Christoph K. Weber, et al.

Cancer Res 1998;58:2986-2990.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/58/14/2986

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/58/14/2986. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.