Interaction of microtubule‐associated protein 1B light chain (MAP1B‐LC1) and p53 represses transcriptional activity of p53
Interaction of microtubule‐associated protein 1B light chain (MAP1B‐LC1) and p53 represses...
Kim, Jung‐Woong; Lee, So‐Youn; Jeong, Mi‐Hee; Jang, Sang‐Min; Song, Ki‐Hyun; Kim, Chul‐Hong; Kim, You‐Jin; Choi, Kyung‐Hee
2008-01-01 00:00:00
Animal Cells and Systems 12: 69-75, 2008 Interaction of Microtubule-associated Protein 1B Light Chain (MAP1B-LC1) and p53 Represses T ranscriptional Activity of p53 † † Jung-W oong Kim , So-Y oun Lee , Mi-Hee Jeong, Sang-Min Jang, Ki-Hyun Song, Chul-Hong Kim, Y ou-Jin Kim and Kyung-Hee Choi* Laboratory of Molecular Biology , Department of Life Science, College of Natural Sciences, Chung-Ang University , Seoul 156- 756, Korea Abstract: The tumor suppressor and transcription factor Gonzalez-Billault et al., 2002; T akei et al., 2000). MAP1B p 53 i s a key mo dul ator o f cel lul ar str ess re sponse s, and can consists of 2464 amino acids and synthesized as a trigger apoptosis in many cell types including neurons. In polyprotein precursors which is rapidly cleaved to give rise this study, we have shown that Microtubule-associated to the respective heavy and light chains, termed MAP1B- protein 1B (MAP1B) light chain interacts with tumor HC and MAP1B-LC1 (Langkopf et al., 1992). The site of suppressor p53. MAP1B is one of the major cytoskeletal proteolytic cleavage of the MAP1B precursor has been proteins in the developing nervous system and essential in narrowed down to a region in the proximity of amino acid forming axons during elongation. We also demonstrate that both p53 and MAP1B-LC1 interact in the nucleus in HEK 2210 (T ogel et al., 199 9). However , its cleavage proteolytic 293 cells. Indeed, we show that the MAP1B-LC1 negatively enzyme does not yet identified. Both MAP1B heavy and regulates p53-dependent transcriptional activity of a reporter light chain has microtubule binding domain (MTB) and has containing the p21 promoter. Consequently, MAP1B light the capacity to cross-link microtubules directly interacting chain binds with p53 and their interaction leads to the with tubulin and actin (Riederer, 2007). inhibition of doxorubicin-induced apoptosis in HEK 293 Because MAP1B interacts with varieties of other cells. Furthermore, these examinations might be taken into consideration when knock-down of MAP1B-LC1 is used as proteins, there is increasing evidence that MAP1B not only a cancer therapeutic strategy to enhance p53’s apoptotic plays a crucial role in the stability of the cytoskeleton but activity in chemotherapy . also has other cellular functions. To maintain microtubule stability , MAP1B interacts with several other proteins such Key words: MAP1B-LC1, p53, transcriptional activity , apopto- sis, protein interaction as gigaxonin, a protein that links microtubules and intermediate filaments (Allen et al., 2005; Ding et al., 2002), myelin associated glycoprotein (MAG), typical for cytoskeleton formation of glial cells (Dashiell et al., 2002; Microtubule-associated protein 1B (MAP1B) is one of the Franzen et al., 2001) and SCG10, essential in neurite major cytoskeletal proteins in the developing nervous outgrowth modulating microtubule disassembly (Bondallaz system and essential in forming axons during elongation et al., 2006). Although most studies of MAP1B have (Gonzalez-Billault et al., 2004; Riederer, 2007). It controls focused on its interaction with microtubules, other roles directions of growth cone migration, while neurons that have been proposed independent of microtubule-associated lack MAP1B are characterized by increased terminal and function. MAP1B directly binds with glyceraldehyde-3- collateral branching and impaired turning of growth cones phosphate dehydrogenase (GAPDH), essential in the local (Bouquet et al., 2004; Garcia & Cleveland, 2001; energy provision of cytoskeletal structures, and help to keep this enzyme close to the cytoskeleton (Cueille et al., Jung-Woong Kim and So-Youn Lee contributed equally to this work as the first author . 2007). MAP1B also interacts with rho1 subunit of GABA receptor at neuronal synapses (Billups et al., 2000; Hanley * T o whom correspondence should be addressed. et al., 2000; Pattnaik et al., 2000). Anchoring to the T el: +82-2-820-5209; Fax: +82-2-824-7302 E-mail: khchoi@cau.ac.kr cytoskeleton lowers the sensitivity of GABA receptor in ANIMAL CELLS AND SYSTEMS Vo l. 12 No. 2 69 Jung-Woong K im, So-Youn Lee, Mi-H ee Jeong, Sang-Min Jang, Ki-Hyun Song, Chul-Hong Kim, You-Jin Kim and K yung-Hee Choi the inhibitory synapses and provides a functional overnight with 30 ml of fresh protein A/G bead in the modulation of the neuronal receptors. Recently, MAP1B presence of appropriate antibod ies. The beads were washed ligh t chain has been also identified as an interacting partner three times in PBS, resuspended in SDS sample buf fer , and of Pes1 which is required for ribosome biogenesis and cell boiled for 10 min. Samples were analyzed by Western proliferation (Lerch-Gaggl et al., 2007). The interaction blotting using the appropriate antibodies to detect protein with MAP1B-LC1 induces a cytoplasmic sequestration of expression. The MAP1B and p53 protein were detected by nucleolar Pes1, and that results in a reduction of cell each primary antibody (purchased from Santa-Cruz, 1:500 proliferation. Thus, MAP1B-LC1 acts as a negative dilution) and secondary antibody (HRP-anti-goat and -anti regulator of Pes1. Although MAP1B plays important roles rabbit, 1:5000 dilution). The polyclonal GFP, p21, Bax, in cytoskelectal stability and in other cellular functions, thePARP, α-tubulin and β-tubulin antibodies were from Santa exact underlying molecular mechanisms associated with Cruz Biotechnology. Western blot was detected by MAP1B remains unclear.chemoluminescence (ECL, Santa Cruz Biotechnology). The tumour suppressor protein p53 is involved in cell- cycle control, apoptosis, differentiation, DNA repair and Immunofluorescence Staining and Confocal recombination (Levine, 1997; Sengupta & Harris, 2005). In Microscopic Detection response to DNA damage, p53 accumulates in the nucleusHEK 293 cells were grown on a sterile coverslips in 60mm (Harris & Levine, 2005), where it transcriptionally activates dishes and treated with doxorubicin. After the doxorubicin the genes that encode p21, BAX, PUMA and MDM2, as treatment, cells were washed with PBS and fixed for 1 h in well as other genes that are involved in growth arrest and 4% paraformaldehyde. The cells were permeabilized with apoptosis (Kirsch & Kastan, 1998). In this study , we report 0.3% Triton X-100 for 20 min, incubated with a primary that MAP1B-LC1 binds with p53 and inhibits transcriptional antibody in blocking solution for 3 h at room temperature, activity of p53 through their interaction. Therefore the washed in blocking solution, and then incubated with the present study was undertaken to investigate the possible appropriate secondary antibody for 30 min. Primary effects of MAP1B-LC1 on doxorubicin induced apoptosisantibodies were used at 1:200 for HA (sc-805: Santa Cruz). in the HEK 293 cells. Our findings suggest the MAP1B- Cy3-conjugated goat anti-rabbit IgG (1:200, Amersham LC1 acts as a negative regulator of p53 transactivation and Biosciences) was used as secondary antibodies. Cells were doxorubicin induced apoptosis through their protein-visualized using a Radians 2000 confocal microscope (Bio- protein interaction.Rad). Luciferase Assay MA TERIALS AND METHODS HEK 293 cells were cultured in 60 mm dishes and Cell Culture and Transfection transfected with the firefly luciferase p21 reporter gene (0.1 HEK 293 cells were obtained from the ATCC (American µ g) and pCMV- β-galactosidase (0.1 µ g) together with type culture collection; Manassas, V A) and maintained in pEGFP-MAP1B-LC, pcDNA-H A-53 and MAP1B siRNA Dulbecco’s modified Eagle’s medium supplemented with (sc-35851: Santa Cruz) using Lipofectamine 2000. After 10% fetal bovine serum (Invitrogen; Carlsbad, CA) and24h of transfection, cells were lysed in reporter lysis buffer penicillin-streptomycin (50 units/ml). Doxorubucin was (Promega). Cell extracts were analyzed with the luciferase purchased from Sigma Aldrich (St. Louis, MO). Transient reporter assay system using a Lumat LB 9501 Berthold transfection was performed by Lipofectamine 2000 Luminometer. Luciferase activities of the p21-luciferase (Invitrogen) with different plasmid DNA according to the vector were normalized based on β-galactosidase activity manufacturer’s instructions.of the cotransfected vector. In Vivo Binding Assay and Western blotting Preparation of Subcellular Fractions HEK 293 cells were seeded in 100 mm plates at an initial HEK 293 cells were washed with ice-cold PBS, harvested density of 2 ×10 cells and allowed to grow for 12 h. The by centrifugation, and lysed in Buffer A (10 mM HEPES, cells were lysed in a buffer containing 1% Triton X-100, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.1% SDS, 1% 1mM DTT , and 1 mM phenylmethylsulfonyl fluoride) for Nonidet P-4 0, an d 1 mM PMSF . The cell susp ensions were 15 min. For cell lysis 10% (v/v) of Nonidet P-40 was incubated on ice for 20 min and centrifuged at 12,000 rpm added, and the cells were vortexed for 10 sec. After at 4 C for 20 min. For immunoprecipitation assays, we centrifugation at 5,000 rpm for 30 sec, the supernatant used previously described protocols (Kim WH et al., 2007).(cytosolic extracts) was transferred to a new tube. The The supernatants were pre-cleaned with 20 µ l of protein A/ pellet was added to ice-cold Buffer C (20 mM HEPES, pH G agarose bead (50% slurry) and then incubated at 4 C 7.9, 0.4 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM 70 ANIMAL CELLS AND SYSTEMS Vol. 12 No. 2 MA P1B -LC1 Represses T ranscriptional Activity of p53 DTT , and 1 mM phenylmethylsulfonyl fluoride), incubated proteins was significantly increased (Fig. 1A, lane 2) for 15min at 4 C, and centrifuged at 14,000rpm for 5min. compared to that in the absence of MAP1B-LC1 (Fig. 1A, The supernatant (nuclear extracts) was transferred to new lane 1). Conversely, HA-MAP1B-LC1 was also markedly tubes and kept frozen at −70C until use.immunoprecipitated with GFP-p53 (Fig. 1B, lane 2) but not with control GFP-empty vector (Fig. 1B, lane 1). For the Apoptosis assays control an almost even expression level of GFP-p53 and To analyze cellular apoptosis, HEK 293 cells were grown HA-MAP1B-LC1 in whole cell lysates was detected from to 60-70% confluence in complete media then treated with each experimental condition, respectively (Fig. 1; total cell 2 µ M d oxorub icin in the presence or absen c e of a MAP1B- lysate). Taken together, these results demonstrate that p53 LC expression plasmid. Apoptotic cells were identified by ph y s i call y in terac ts wi th MAP1B-LC 1 in cu ltu red m a m m a l ian their rounded morphology, compared to the spread-outcells in vivo. mo r phology of non- apoptoti c c ells . Chromati n conde nsati on as an apoptotic marker was visualized by Hoechst 33342 p53 binds with MAP1B-LC1 in the nucleus (Sigma-Aldrich, St. Louis, MO) staining. To determine the localization of the HA-tagged p53 and GFP-fused MAP1B-LC1, we immuno-stained HEK 293 RESUL TS cells, which were co-transfected with HA-p53 and GFP- MAP1B-LC1 expression plasmids, using anti-HA antibodies. p53 interacts with MAP1B-LC1 in HEK 293 cells As shown in Fig. 2A, p53 (red) and MAP1B-LC1 (green) Using yeast two-hybrid assay, we have previously found were co-localized in the nucleus under confocal microscopy . that p73b, p53 homolog protein, binds with MAP1B lightFor further confirmation of the interaction between p53 and chain (unpublished our data). To examine whether MAP1B-LC1 in the nucleus, we examined the nuclear co- MAP1B-LC1 can also interacts with p53, we co-tran sfected localization and the interaction of the p53 and MAP1B- HEK293 cells with combined exp ression p lasmids o f GFP- LC1 by Western blot analysis and immunoprecipitation. fused-p53 with a HA-tagged MAP1B-LC1 expression After HEK 293 cells were co-transfected with HA-tagged vector, the whole cell lysates were immunoprecipitated p53, GFP-fused-MAP1B-LC1 expression plasmids, both with anti-HA antibodies. The immunoprecipitated proteins nuclear and cytoplasmic sub-fractions were separated and were then analyzed by W estern blotting using specific anti- verified by W estern blot analysis using antibodies for each GFP and anti-HA antibodies to determine p53 and marker protein, which were P ARP for nucleus and β-tubulin MAP1B-LC1, respectively (Fig. 1A). In the presence offor cytoplasm (Fig. 2B; bottom panel). The nuclear extracts MAP1B-LC1, the amount of co-immunoprecipitated p53 were then immunoprecipitated with anti-HA antibodies, Fig. 1. MAP1B-LC interacts with p53 in vivo. (A) HEK 293 cells were transfected with expression plasmids of GFP-p53 together with eithe r pcDNA-HA empty (lane 1), pcDNA-HA-MAP1BLC1 (lane 2). After whole cell lysates were immunoprecipitated with anti-HA antibody, western blot was performed using indicated antibodies. (B) HEK 293 cells were transfected with expression plasmids of pcDNA-HA-MAP1B-LC 1 together with either pEGFP empty (lane 1), pEGFP-p53 (lane 2). After whole cell lysates were immunoprecipitated with anti-GFP antibody, western blot was performed using indicated antibodies. ANIMAL CELLS AND SYSTEMS Vo l. 12 No. 2 71 Jung-Woong K im, So-Youn Lee, Mi-H ee Jeong, Sang-Min Jang, Ki-Hyun Song, Chul-Hong Kim, You-Jin Kim and K yung-Hee Choi 293 cells were transiently co-transfected with a p53 expression plasmid plus a luciferase reporter plasmid containing the p53-responsive element from the p21 promoter. As indicated in Fig. 3A, enhanced luciferase activities by p53 transactivation (lane 4) were repressed by MAP1B-LC1 expression in a dose dependent manner (lane 5, 6). Indeed, transfection of MAP1B siRNA into HEK 293 cells decreases repressive effect of both overexpressed GFP-MAP1B (Fig. 3B, lane 4) and endogenous MAP1B (Fig. 3B, lane 5) expression. Taken together, these results coherently suggest that MAP1B-LC1 could negatively regulate the transcriptional activity of p53. The interaction betw een MAP1B-LC1 and p53 inhibit s doxorubicin-induced apoptosis Because MAP1B-LC1 inhibits transcriptional activity of p53, these results raise the possibility that MAP1B light chain may inhibit the doxorubicin-induced apoptosis. To investigate whether MAP1B-LC1 reduce doxorubicin- induced p53 activities, HE K 293 cells we re tran sfected with MAP1B-LC1 and treated with 2 mM of doxorubicin. Then we examined the expression level of endogenous p21 and Bax, which is tar get genes of p53, by immunoblot analysis. As shown in Fig. 4A, the expression level of p21 and Bax was increased by doxorubicin treatment. However the increased p21 and Bax levels were reduced by MAP1B- LC1 overexpression (lane 3). These results suggest that Fi g. 2 . p53 b i nd s wi t h MAP1B- LC1 in the nucl e u s . ( A ) HEK 293 c e ll s MAP1B-LC1 represses doxorubicin-induced p53 activities were co-transfected with GFP-MAP1B-LC1 and pcDNA-HA-p53 expression plasmids. The cells were then immunostained and by interaction of MAP1B-LC1 and endogenous p53. To visualized green (GFP-MAP1B-LC1), red (HA-p53) under a confocal determine whether MAP1B-LC1 inhibits doxorubicin- mic r os c opic s yst em as desc r ibed in the “Materi a ls and Methods” . ( B ) induced apoptosis, we examined the changes of cellular HEK 293 cells were transfected with expression plasmids of GFP- MAP1B- LC1 together wi th either pc DNA -H A empty ( l ane 1), pc DN A- morphology by GFP empty vector transfection and nuclear HA-p53 (lane 2). Their nuclear and cytoplasmic fractions were condensation by Hoechst staining (Fig. 4B). After separately prepared as described in the “Materials and Methods”. doxorubicin treatment for 24 h, the HEK 293 cells show After nuclear fractions were immunoprecipitated with anti-H A anti b o d i e s , W e s t ern blot analys is wa s per formed usi ng anti - G F P an d characteristics of apoptotic morphology and chromatin anti-HA antibodies. Their whole cell lysates served as controls. Eithe r condensation. However, doxorubicin treated cells expressing cytoplasmic or nuclear extracts were immunoblotted with anti- β- MAP1B light chain showed a normal morphology that tubulin or anti-PARP antibodies, respectively, to ensure prope r subcellular fractions. diffusely stained intact nuclei (Fig. 4B, bottom panel). Taken together, these results indicate that MAP1B-LC1 interaction with p53 suppresses transcriptional activity of and Western blot analysis was performed using anti-GFP p53 and doxorubicin-induced apoptosis in HEK 293 cells and anti-HA antibodies. As indicated in Fig. 2B, GFP-fusedin vivo. MAP1B-LC1 was specifically immunoprecipitated with HA-tagged p53 in the nucleus fraction (Fig. 2B, lane 2) DISCUSSION compared to HA-empty vector transfected group (Fig. 2B, lane1) even though all proteins were markedly detected in In this study, we have shown that MAP1B light chain the total cell lysates. Therefore, we show that p53 interacts with tumor suppressor p53, and it specifically specifically localizes and interacts with MAP1B-LC1 in the binds with transactivation- and oligomerization-domain of nucleus. p53 (unpublished our data). W e also demonstrate that both p53 and MAP1B-LC1 co-localized in the nucleus in HEK MAP 1B-LC1 r epr esses tr anscr iptional activity of p53 293 cells. Indeed, we demonstrate that the MAP1B-LC1 To examine whether MAP1B-LC1 could affect p53-dependent negatively regulates p53-dependent transcriptional activity transcriptional activation through their interaction, HEK of a reporter containing the p21 promoter. Consequently, 72 ANIMAL CELLS AND SYSTEMS Vol. 12 No. 2 MA P1B -LC1 Represses T ranscriptional Activity of p53 Fig. 3. MAP1B-LC represses transcriptional activity of p53. (A) HEK 293 cells were transiently co-transfected with GFP-empty, HA-p53 with inc r easi n g amount s of the G F P-MAP1B- LC1 expr es s i on pl as mid ( 0 .2, 0. 4 µ g). Luc iferas e ac ti vi ty wa s meas ured 36 h af ter trans fec t i on. All dat a were normalized to β-galactosidase activity . The data are expressed in relative fold increase of luciferase units (RLU). (B) HEK 293 cells were c o - t rans fec t ed wi t h HA -t agged p53 and GFP - MAP1B- LC1 expr ess i on plas mi ds together wi th MAP1B s i RN A. Luci f eras e ac t i v i ty wa s measured 36 h after transfection. All data were normalized to β-galactosidase activity . The data are expressed in relative fold increase of luciferase units (RLU). All data are representative of three independent experiments, and statistical analysis was represented by ±S.E.M (standa rd error meaning). Fig. 4. MAP1B-LC1 inhibits doxorubicin-induced apoptosis in HEK 293 cells in vivo . (A) HEK 293 cells were transfected with GFP-tagged MAP1B light chain, and were treated with 2 µ M doxorubicin for 24 h. Immunoblot analysis of protein levels of p53, p21 and Bax in cells were performed. β-tubulin levels are shown as loading control. (B) HEK 293 cells were transfected with GFP-MAP1B light chain, and treated with 2 µ M doxorubicin for 24 h. And then cells were stained with Hoechst 33342 for detection of apoptotic cells. MAP1B light chain binds with p53 and their interaction nucleolar Pes1, and that results in a reduction of cell leads to the inhibition of doxorubicin-induced apoptosis in p r oli f e r a tio n. Th us , MA P 1 B - L C 1 a c t s as a nega ti ve r e g ula tor HEK 2 93 cells.of Pes1. Previous studies have demonstrated that MAP1B not Using yeast two-hybrid screening, we have identified a only form cross-links between individual microtubules forvariety of p73 β, p53 homolog, -interacting proteins, such as ras stabilizing microtubule structure but can interact with non- Amphiphysin IIb-1 (Kim et al., 2001) and p19 (Jeong et micr o tub ule - a s s oci at ed pr o t e ins th at a l s o r e gula te micr o tub ule al., 2006) that are involved in p73 β-mediated transactivity stability (Riederer, 2007). It has been also reported that regulation and apoptosis. W e also found the MAP1B-LC1 MAP1B has a novel another function (Lerch-Gaggl et al.,as a binding partner of p73 β and confirmed their interaction 2007). As a novel function of MAP1B-LC1, it interacts in vivo and in vitro (unpublished our data). In addition, with Pes1 and induces a cytoplasmic sequestration of MAP1B-LC1 also can interact with p53 (Fig. 1) and ANIMAL CELLS AND SYSTEMS Vo l. 12 No. 2 73 Jung-Woong K im, So-Youn Lee, Mi-H ee Jeong, Sang-Min Jang, Ki-Hyun Song, Chul-Hong Kim, You-Jin Kim and K yung-Hee Choi and Nothias F (2004) Microtubule-associated protein 1B specifically b in ds with transactivation- and oligo merization controls directionality of growth cone migration and axonal domain of p53 (data not shown). Until now, there are b ra nching in regene ration of adult dorsal ro ot gang lia ne urons. several reports demonstrating that p53 has a potential of J N e ur os c i 24: 7204-7213. interaction with microtubule cytoskeleton (Giannakakou et Cueille N, Blanc CT, Riederer IM, and Riederer BM (2007) al., 2000; T rostel et al., 2006). p53 associates preferentially Microtubule-associated protein 1B binds glyceraldehyde-3- with the polymerized form of tubulin (microtubules), andphosphate dehydrogenase. J Proteome Res 6: 2640-2647. transported to the nucleus by the dynein motor complex. Dashiell SM, Tanner SL, Pant HC, and Quarles RH (2002) Myelin-associated glycoprotein modulates expression and This interaction is lost after treatment with microtubule phosphorylation of neuronal cytoskeletal elements and their depolymerizing drugs. In turn, the association of p53 to associated kinases. J N e ur oc h e m 81: 1263-1272. microtubule allows for transport to the peri-nuclear region Ding J, Liu JJ, Kowal AS, Nardine T , Bhattachary a P , Lee A, and on microtubules, allowing for nuclear import. Yang Y (2002) Microtubule-associated protein 1B: a neuronal Furthermore, we tested the physiological significance of b i nd ing p a r t ner f o r g i ga xoni n. J Cell Biol 158: 427-433. binding between MAP1B light chain and p53 by assessing Foo RS, Nam YJ, Ostreicher MJ, Metzl MD, Whelan RS, Peng modulations in the transcriptional activity of p53. MAP1B- C F , A s h t on A W , Fu W , Ma ni K , C h in S F , Pro v e nz a n o E, El li s I, Figg N, Pinder S, Bennett MR, Caldas C, and Kitsis RN LC1 significantly decreased the activity of p53 (Fig. 3). (2007) Regulation of p53 tetramerization and nuclear export Perhaps, MAP1B-LC1 might repress transcriptional activity by ARC. Proc Natl Acad Sci USA 104: 20826-20831. of p53 through specific interactions with transactivation- Franzen R, T anner SL, Dashiell SM, Rottkamp CA, Hammer JA, and oligomerization domain of p53. MAP1B-LC1 binding and Quarles RH (2001) Microtubule-associated protein 1B: a to transactivation domain could prevent the association of neuronal binding partner for myelin-associated glycoprotein. co-activators, such as p300/CBP and PCAF to p53 and J C e ll Bi o l 155: 893-898. binding to oligomerization domain also inhibit the formationGarcia ML and Cleveland DW (2001) Going new places using an old MAP: tau, microtubules and human neurodegenerative of activated p53-tetramer (Foo et al., 2007). Thus, our d i sease. Curr Opin Cell Biol 13: 41-48. finding provide a molecular and functional linkage between Giannakakou P , Sackett DL, W ard Y , W ebster KR, Blagosklonny microtubule-associated protein 1B (MAP1B) and p53 in MV, and Fojo T (2000) p53 is associated with cellular cellular signaling. microtubules and is transported to the nucleus by dynein. Nat Despite intensive f unctional studies, the ef fects of MAP1B Cell Biol 2: 709-717. on cellular physiology are most focused on microtubule Gonzalez-Billault C, Engelke M, Jimenez-Mateos EM, W andosell related mechanisms and the other functions remain to be F, Caceres A, and Avila J (2002) Participation of structural microtubule-associated proteins (MAPs) in the development fully elucidated. In this aspect it is important to note that of neuronal polarity. J N e ur osci Re s 6 7 : 71 3- 719 . our findings contribute to a functional linkage between Gonzalez-Billault C, Jimenez-Mateos EM, Caceres A, Diaz-Nido MAP1B and p53 in the biological network, showing that J, Wandosell F, and Avila J (2004) Microtubule-associated MAP1B-LC1 associates with and regulates p53 as a protein 1B function during normal development, regeneration, negative regulator in the HEK 293 cells. Furthermore, these and pathological conditions in the nervous system. J Neurobiol 58: 48-59. examinations might be taken into consideration when Hanley JG , Jones EM, and Moss SJ (2000) GABA receptor rho1 knock-down of MAP1B-LC1 is used as a cancer subunit interacts with a novel splice variant of the glycine therapeutic strategy to enhance p53’s apoptotic activity in transporter, GLYT-1. J Biol Chem 275: 840-846. chemotherapy. Harris SL and Levine AJ (2005) The p53 pathway: positive and n e gat i ve f eed back l oops. Onc ogen e 24: 2899-2908. ACKNOWLEDGMENTS Jeong MH, Bae J, Ki m W H , Y oo S M, Kim JW , Son g PI , a nd Choi This work was supported by grants from Chung-Ang University. KH (2006) p19ras interacts with and activates p73 by involving the MDM2 protein. J Biol Chem 281: 8707-8715. 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http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.pngAnimal Cells and SystemsTaylor & Francishttp://www.deepdyve.com/lp/taylor-francis/interaction-of-microtubule-associated-protein-1b-light-chain-map1b-lc1-nmkqJ9n8g9
Interaction of microtubule‐associated protein 1B light chain (MAP1B‐LC1) and p53 represses transcriptional activity of p53
Interaction of microtubule‐associated protein 1B light chain (MAP1B‐LC1) and p53 represses transcriptional activity of p53
Abstract
Abstract The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and can trigger apoptosis in many cell types including neurons. In this study, we have shown that Microtubule‐associated protein 1B (MAP1B) light chain interacts with tumor suppressor p53. MAP1B is one of the major cytoskeletal proteins in the developing nervous system and essential in forming axons during elongation. We also demonstrate that both p53 and MAP1B‐LC1 interact...
Animal Cells and Systems 12: 69-75, 2008 Interaction of Microtubule-associated Protein 1B Light Chain (MAP1B-LC1) and p53 Represses T ranscriptional Activity of p53 † † Jung-W oong Kim , So-Y oun Lee , Mi-Hee Jeong, Sang-Min Jang, Ki-Hyun Song, Chul-Hong Kim, Y ou-Jin Kim and Kyung-Hee Choi* Laboratory of Molecular Biology , Department of Life Science, College of Natural Sciences, Chung-Ang University , Seoul 156- 756, Korea Abstract: The tumor suppressor and transcription factor Gonzalez-Billault et al., 2002; T akei et al., 2000). MAP1B p 53 i s a key mo dul ator o f cel lul ar str ess re sponse s, and can consists of 2464 amino acids and synthesized as a trigger apoptosis in many cell types including neurons. In polyprotein precursors which is rapidly cleaved to give rise this study, we have shown that Microtubule-associated to the respective heavy and light chains, termed MAP1B- protein 1B (MAP1B) light chain interacts with tumor HC and MAP1B-LC1 (Langkopf et al., 1992). The site of suppressor p53. MAP1B is one of the major cytoskeletal proteolytic cleavage of the MAP1B precursor has been proteins in the developing nervous system and essential in narrowed down to a region in the proximity of amino acid forming axons during elongation. We also demonstrate that both p53 and MAP1B-LC1 interact in the nucleus in HEK 2210 (T ogel et al., 199 9). However , its cleavage proteolytic 293 cells. Indeed, we show that the MAP1B-LC1 negatively enzyme does not yet identified. Both MAP1B heavy and regulates p53-dependent transcriptional activity of a reporter light chain has microtubule binding domain (MTB) and has containing the p21 promoter. Consequently, MAP1B light the capacity to cross-link microtubules directly interacting chain binds with p53 and their interaction leads to the with tubulin and actin (Riederer, 2007). inhibition of doxorubicin-induced apoptosis in HEK 293 Because MAP1B interacts with varieties of other cells. Furthermore, these examinations might be taken into consideration when knock-down of MAP1B-LC1 is used as proteins, there is increasing evidence that MAP1B not only a cancer therapeutic strategy to enhance p53’s apoptotic plays a crucial role in the stability of the cytoskeleton but activity in chemotherapy . also has other cellular functions. To maintain microtubule stability , MAP1B interacts with several other proteins such Key words: MAP1B-LC1, p53, transcriptional activity , apopto- sis, protein interaction as gigaxonin, a protein that links microtubules and intermediate filaments (Allen et al., 2005; Ding et al., 2002), myelin associated glycoprotein (MAG), typical for cytoskeleton formation of glial cells (Dashiell et al., 2002; Microtubule-associated protein 1B (MAP1B) is one of the Franzen et al., 2001) and SCG10, essential in neurite major cytoskeletal proteins in the developing nervous outgrowth modulating microtubule disassembly (Bondallaz system and essential in forming axons during elongation et al., 2006). Although most studies of MAP1B have (Gonzalez-Billault et al., 2004; Riederer, 2007). It controls focused on its interaction with microtubules, other roles directions of growth cone migration, while neurons that have been proposed independent of microtubule-associated lack MAP1B are characterized by increased terminal and function. MAP1B directly binds with glyceraldehyde-3- collateral branching and impaired turning of growth cones phosphate dehydrogenase (GAPDH), essential in the local (Bouquet et al., 2004; Garcia & Cleveland, 2001; energy provision of cytoskeletal structures, and help to keep this enzyme close to the cytoskeleton (Cueille et al., Jung-Woong Kim and So-Youn Lee contributed equally to this work as the first author . 2007). MAP1B also interacts with rho1 subunit of GABA receptor at neuronal synapses (Billups et al., 2000; Hanley * T o whom correspondence should be addressed. et al., 2000; Pattnaik et al., 2000). Anchoring to the T el: +82-2-820-5209; Fax: +82-2-824-7302 E-mail: khchoi@cau.ac.kr cytoskeleton lowers the sensitivity of GABA receptor in ANIMAL CELLS AND SYSTEMS Vo l. 12 No. 2 69 Jung-Woong K im, So-Youn Lee, Mi-H ee Jeong, Sang-Min Jang, Ki-Hyun Song, Chul-Hong Kim, You-Jin Kim and K yung-Hee Choi the inhibitory synapses and provides a functional overnight with 30 ml of fresh protein A/G bead in the modulation of the neuronal receptors. Recently, MAP1B presence of appropriate antibod ies. The beads were washed ligh t chain has been also identified as an interacting partner three times in PBS, resuspended in SDS sample buf fer , and of Pes1 which is required for ribosome biogenesis and cell boiled for 10 min. Samples were analyzed by Western proliferation (Lerch-Gaggl et al., 2007). The interaction blotting using the appropriate antibodies to detect protein with MAP1B-LC1 induces a cytoplasmic sequestration of expression. The MAP1B and p53 protein were detected by nucleolar Pes1, and that results in a reduction of cell each primary antibody (purchased from Santa-Cruz, 1:500 proliferation. Thus, MAP1B-LC1 acts as a negative dilution) and secondary antibody (HRP-anti-goat and -anti regulator of Pes1. Although MAP1B plays important roles rabbit, 1:5000 dilution). The polyclonal GFP, p21, Bax, in cytoskelectal stability and in other cellular functions, thePARP, α-tubulin and β-tubulin antibodies were from Santa exact underlying molecular mechanisms associated with Cruz Biotechnology. Western blot was detected by MAP1B remains unclear.chemoluminescence (ECL, Santa Cruz Biotechnology). The tumour suppressor protein p53 is involved in cell- cycle control, apoptosis, differentiation, DNA repair and Immunofluorescence Staining and Confocal recombination (Levine, 1997; Sengupta & Harris, 2005). In Microscopic Detection response to DNA damage, p53 accumulates in the nucleusHEK 293 cells were grown on a sterile coverslips in 60mm (Harris & Levine, 2005), where it transcriptionally activates dishes and treated with doxorubicin. After the doxorubicin the genes that encode p21, BAX, PUMA and MDM2, as treatment, cells were washed with PBS and fixed for 1 h in well as other genes that are involved in growth arrest and 4% paraformaldehyde. The cells were permeabilized with apoptosis (Kirsch & Kastan, 1998). In this study , we report 0.3% Triton X-100 for 20 min, incubated with a primary that MAP1B-LC1 binds with p53 and inhibits transcriptional antibody in blocking solution for 3 h at room temperature, activity of p53 through their interaction. Therefore the washed in blocking solution, and then incubated with the present study was undertaken to investigate the possible appropriate secondary antibody for 30 min. Primary effects of MAP1B-LC1 on doxorubicin induced apoptosisantibodies were used at 1:200 for HA (sc-805: Santa Cruz). in the HEK 293 cells. Our findings suggest the MAP1B- Cy3-conjugated goat anti-rabbit IgG (1:200, Amersham LC1 acts as a negative regulator of p53 transactivation and Biosciences) was used as secondary antibodies. Cells were doxorubicin induced apoptosis through their protein-visualized using a Radians 2000 confocal microscope (Bio- protein interaction.Rad). Luciferase Assay MA TERIALS AND METHODS HEK 293 cells were cultured in 60 mm dishes and Cell Culture and Transfection transfected with the firefly luciferase p21 reporter gene (0.1 HEK 293 cells were obtained from the ATCC (American µ g) and pCMV- β-galactosidase (0.1 µ g) together with type culture collection; Manassas, V A) and maintained in pEGFP-MAP1B-LC, pcDNA-H A-53 and MAP1B siRNA Dulbecco’s modified Eagle’s medium supplemented with (sc-35851: Santa Cruz) using Lipofectamine 2000. After 10% fetal bovine serum (Invitrogen; Carlsbad, CA) and24h of transfection, cells were lysed in reporter lysis buffer penicillin-streptomycin (50 units/ml). Doxorubucin was (Promega). Cell extracts were analyzed with the luciferase purchased from Sigma Aldrich (St. Louis, MO). Transient reporter assay system using a Lumat LB 9501 Berthold transfection was performed by Lipofectamine 2000 Luminometer. Luciferase activities of the p21-luciferase (Invitrogen) with different plasmid DNA according to the vector were normalized based on β-galactosidase activity manufacturer’s instructions.of the cotransfected vector. In Vivo Binding Assay and Western blotting Preparation of Subcellular Fractions HEK 293 cells were seeded in 100 mm plates at an initial HEK 293 cells were washed with ice-cold PBS, harvested density of 2 ×10 cells and allowed to grow for 12 h. The by centrifugation, and lysed in Buffer A (10 mM HEPES, cells were lysed in a buffer containing 1% Triton X-100, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.1% SDS, 1% 1mM DTT , and 1 mM phenylmethylsulfonyl fluoride) for Nonidet P-4 0, an d 1 mM PMSF . The cell susp ensions were 15 min. For cell lysis 10% (v/v) of Nonidet P-40 was incubated on ice for 20 min and centrifuged at 12,000 rpm added, and the cells were vortexed for 10 sec. After at 4 C for 20 min. For immunoprecipitation assays, we centrifugation at 5,000 rpm for 30 sec, the supernatant used previously described protocols (Kim WH et al., 2007).(cytosolic extracts) was transferred to a new tube. The The supernatants were pre-cleaned with 20 µ l of protein A/ pellet was added to ice-cold Buffer C (20 mM HEPES, pH G agarose bead (50% slurry) and then incubated at 4 C 7.9, 0.4 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM 70 ANIMAL CELLS AND SYSTEMS Vol. 12 No. 2 MA P1B -LC1 Represses T ranscriptional Activity of p53 DTT , and 1 mM phenylmethylsulfonyl fluoride), incubated proteins was significantly increased (Fig. 1A, lane 2) for 15min at 4 C, and centrifuged at 14,000rpm for 5min. compared to that in the absence of MAP1B-LC1 (Fig. 1A, The supernatant (nuclear extracts) was transferred to new lane 1). Conversely, HA-MAP1B-LC1 was also markedly tubes and kept frozen at −70C until use.immunoprecipitated with GFP-p53 (Fig. 1B, lane 2) but not with control GFP-empty vector (Fig. 1B, lane 1). For the Apoptosis assays control an almost even expression level of GFP-p53 and To analyze cellular apoptosis, HEK 293 cells were grown HA-MAP1B-LC1 in whole cell lysates was detected from to 60-70% confluence in complete media then treated with each experimental condition, respectively (Fig. 1; total cell 2 µ M d oxorub icin in the presence or absen c e of a MAP1B- lysate). Taken together, these results demonstrate that p53 LC expression plasmid. Apoptotic cells were identified by ph y s i call y in terac ts wi th MAP1B-LC 1 in cu ltu red m a m m a l ian their rounded morphology, compared to the spread-outcells in vivo. mo r phology of non- apoptoti c c ells . Chromati n conde nsati on as an apoptotic marker was visualized by Hoechst 33342 p53 binds with MAP1B-LC1 in the nucleus (Sigma-Aldrich, St. Louis, MO) staining. To determine the localization of the HA-tagged p53 and GFP-fused MAP1B-LC1, we immuno-stained HEK 293 RESUL TS cells, which were co-transfected with HA-p53 and GFP- MAP1B-LC1 expression plasmids, using anti-HA antibodies. p53 interacts with MAP1B-LC1 in HEK 293 cells As shown in Fig. 2A, p53 (red) and MAP1B-LC1 (green) Using yeast two-hybrid assay, we have previously found were co-localized in the nucleus under confocal microscopy . that p73b, p53 homolog protein, binds with MAP1B lightFor further confirmation of the interaction between p53 and chain (unpublished our data). To examine whether MAP1B-LC1 in the nucleus, we examined the nuclear co- MAP1B-LC1 can also interacts with p53, we co-tran sfected localization and the interaction of the p53 and MAP1B- HEK293 cells with combined exp ression p lasmids o f GFP- LC1 by Western blot analysis and immunoprecipitation. fused-p53 with a HA-tagged MAP1B-LC1 expression After HEK 293 cells were co-transfected with HA-tagged vector, the whole cell lysates were immunoprecipitated p53, GFP-fused-MAP1B-LC1 expression plasmids, both with anti-HA antibodies. The immunoprecipitated proteins nuclear and cytoplasmic sub-fractions were separated and were then analyzed by W estern blotting using specific anti- verified by W estern blot analysis using antibodies for each GFP and anti-HA antibodies to determine p53 and marker protein, which were P ARP for nucleus and β-tubulin MAP1B-LC1, respectively (Fig. 1A). In the presence offor cytoplasm (Fig. 2B; bottom panel). The nuclear extracts MAP1B-LC1, the amount of co-immunoprecipitated p53 were then immunoprecipitated with anti-HA antibodies, Fig. 1. MAP1B-LC interacts with p53 in vivo. (A) HEK 293 cells were transfected with expression plasmids of GFP-p53 together with eithe r pcDNA-HA empty (lane 1), pcDNA-HA-MAP1BLC1 (lane 2). After whole cell lysates were immunoprecipitated with anti-HA antibody, western blot was performed using indicated antibodies. (B) HEK 293 cells were transfected with expression plasmids of pcDNA-HA-MAP1B-LC 1 together with either pEGFP empty (lane 1), pEGFP-p53 (lane 2). After whole cell lysates were immunoprecipitated with anti-GFP antibody, western blot was performed using indicated antibodies. ANIMAL CELLS AND SYSTEMS Vo l. 12 No. 2 71 Jung-Woong K im, So-Youn Lee, Mi-H ee Jeong, Sang-Min Jang, Ki-Hyun Song, Chul-Hong Kim, You-Jin Kim and K yung-Hee Choi 293 cells were transiently co-transfected with a p53 expression plasmid plus a luciferase reporter plasmid containing the p53-responsive element from the p21 promoter. As indicated in Fig. 3A, enhanced luciferase activities by p53 transactivation (lane 4) were repressed by MAP1B-LC1 expression in a dose dependent manner (lane 5, 6). Indeed, transfection of MAP1B siRNA into HEK 293 cells decreases repressive effect of both overexpressed GFP-MAP1B (Fig. 3B, lane 4) and endogenous MAP1B (Fig. 3B, lane 5) expression. Taken together, these results coherently suggest that MAP1B-LC1 could negatively regulate the transcriptional activity of p53. The interaction betw een MAP1B-LC1 and p53 inhibit s doxorubicin-induced apoptosis Because MAP1B-LC1 inhibits transcriptional activity of p53, these results raise the possibility that MAP1B light chain may inhibit the doxorubicin-induced apoptosis. To investigate whether MAP1B-LC1 reduce doxorubicin- induced p53 activities, HE K 293 cells we re tran sfected with MAP1B-LC1 and treated with 2 mM of doxorubicin. Then we examined the expression level of endogenous p21 and Bax, which is tar get genes of p53, by immunoblot analysis. As shown in Fig. 4A, the expression level of p21 and Bax was increased by doxorubicin treatment. However the increased p21 and Bax levels were reduced by MAP1B- LC1 overexpression (lane 3). These results suggest that Fi g. 2 . p53 b i nd s wi t h MAP1B- LC1 in the nucl e u s . ( A ) HEK 293 c e ll s MAP1B-LC1 represses doxorubicin-induced p53 activities were co-transfected with GFP-MAP1B-LC1 and pcDNA-HA-p53 expression plasmids. The cells were then immunostained and by interaction of MAP1B-LC1 and endogenous p53. To visualized green (GFP-MAP1B-LC1), red (HA-p53) under a confocal determine whether MAP1B-LC1 inhibits doxorubicin- mic r os c opic s yst em as desc r ibed in the “Materi a ls and Methods” . ( B ) induced apoptosis, we examined the changes of cellular HEK 293 cells were transfected with expression plasmids of GFP- MAP1B- LC1 together wi th either pc DNA -H A empty ( l ane 1), pc DN A- morphology by GFP empty vector transfection and nuclear HA-p53 (lane 2). Their nuclear and cytoplasmic fractions were condensation by Hoechst staining (Fig. 4B). After separately prepared as described in the “Materials and Methods”. doxorubicin treatment for 24 h, the HEK 293 cells show After nuclear fractions were immunoprecipitated with anti-H A anti b o d i e s , W e s t ern blot analys is wa s per formed usi ng anti - G F P an d characteristics of apoptotic morphology and chromatin anti-HA antibodies. Their whole cell lysates served as controls. Eithe r condensation. However, doxorubicin treated cells expressing cytoplasmic or nuclear extracts were immunoblotted with anti- β- MAP1B light chain showed a normal morphology that tubulin or anti-PARP antibodies, respectively, to ensure prope r subcellular fractions. diffusely stained intact nuclei (Fig. 4B, bottom panel). Taken together, these results indicate that MAP1B-LC1 interaction with p53 suppresses transcriptional activity of and Western blot analysis was performed using anti-GFP p53 and doxorubicin-induced apoptosis in HEK 293 cells and anti-HA antibodies. As indicated in Fig. 2B, GFP-fusedin vivo. MAP1B-LC1 was specifically immunoprecipitated with HA-tagged p53 in the nucleus fraction (Fig. 2B, lane 2) DISCUSSION compared to HA-empty vector transfected group (Fig. 2B, lane1) even though all proteins were markedly detected in In this study, we have shown that MAP1B light chain the total cell lysates. Therefore, we show that p53 interacts with tumor suppressor p53, and it specifically specifically localizes and interacts with MAP1B-LC1 in the binds with transactivation- and oligomerization-domain of nucleus. p53 (unpublished our data). W e also demonstrate that both p53 and MAP1B-LC1 co-localized in the nucleus in HEK MAP 1B-LC1 r epr esses tr anscr iptional activity of p53 293 cells. Indeed, we demonstrate that the MAP1B-LC1 To examine whether MAP1B-LC1 could affect p53-dependent negatively regulates p53-dependent transcriptional activity transcriptional activation through their interaction, HEK of a reporter containing the p21 promoter. Consequently, 72 ANIMAL CELLS AND SYSTEMS Vol. 12 No. 2 MA P1B -LC1 Represses T ranscriptional Activity of p53 Fig. 3. MAP1B-LC represses transcriptional activity of p53. (A) HEK 293 cells were transiently co-transfected with GFP-empty, HA-p53 with inc r easi n g amount s of the G F P-MAP1B- LC1 expr es s i on pl as mid ( 0 .2, 0. 4 µ g). Luc iferas e ac ti vi ty wa s meas ured 36 h af ter trans fec t i on. All dat a were normalized to β-galactosidase activity . The data are expressed in relative fold increase of luciferase units (RLU). (B) HEK 293 cells were c o - t rans fec t ed wi t h HA -t agged p53 and GFP - MAP1B- LC1 expr ess i on plas mi ds together wi th MAP1B s i RN A. Luci f eras e ac t i v i ty wa s measured 36 h after transfection. All data were normalized to β-galactosidase activity . The data are expressed in relative fold increase of luciferase units (RLU). All data are representative of three independent experiments, and statistical analysis was represented by ±S.E.M (standa rd error meaning). Fig. 4. MAP1B-LC1 inhibits doxorubicin-induced apoptosis in HEK 293 cells in vivo . (A) HEK 293 cells were transfected with GFP-tagged MAP1B light chain, and were treated with 2 µ M doxorubicin for 24 h. Immunoblot analysis of protein levels of p53, p21 and Bax in cells were performed. β-tubulin levels are shown as loading control. (B) HEK 293 cells were transfected with GFP-MAP1B light chain, and treated with 2 µ M doxorubicin for 24 h. And then cells were stained with Hoechst 33342 for detection of apoptotic cells. MAP1B light chain binds with p53 and their interaction nucleolar Pes1, and that results in a reduction of cell leads to the inhibition of doxorubicin-induced apoptosis in p r oli f e r a tio n. Th us , MA P 1 B - L C 1 a c t s as a nega ti ve r e g ula tor HEK 2 93 cells.of Pes1. Previous studies have demonstrated that MAP1B not Using yeast two-hybrid screening, we have identified a only form cross-links between individual microtubules forvariety of p73 β, p53 homolog, -interacting proteins, such as ras stabilizing microtubule structure but can interact with non- Amphiphysin IIb-1 (Kim et al., 2001) and p19 (Jeong et micr o tub ule - a s s oci at ed pr o t e ins th at a l s o r e gula te micr o tub ule al., 2006) that are involved in p73 β-mediated transactivity stability (Riederer, 2007). It has been also reported that regulation and apoptosis. W e also found the MAP1B-LC1 MAP1B has a novel another function (Lerch-Gaggl et al.,as a binding partner of p73 β and confirmed their interaction 2007). As a novel function of MAP1B-LC1, it interacts in vivo and in vitro (unpublished our data). In addition, with Pes1 and induces a cytoplasmic sequestration of MAP1B-LC1 also can interact with p53 (Fig. 1) and ANIMAL CELLS AND SYSTEMS Vo l. 12 No. 2 73 Jung-Woong K im, So-Youn Lee, Mi-H ee Jeong, Sang-Min Jang, Ki-Hyun Song, Chul-Hong Kim, You-Jin Kim and K yung-Hee Choi and Nothias F (2004) Microtubule-associated protein 1B specifically b in ds with transactivation- and oligo merization controls directionality of growth cone migration and axonal domain of p53 (data not shown). Until now, there are b ra nching in regene ration of adult dorsal ro ot gang lia ne urons. several reports demonstrating that p53 has a potential of J N e ur os c i 24: 7204-7213. interaction with microtubule cytoskeleton (Giannakakou et Cueille N, Blanc CT, Riederer IM, and Riederer BM (2007) al., 2000; T rostel et al., 2006). p53 associates preferentially Microtubule-associated protein 1B binds glyceraldehyde-3- with the polymerized form of tubulin (microtubules), andphosphate dehydrogenase. J Proteome Res 6: 2640-2647. transported to the nucleus by the dynein motor complex. Dashiell SM, Tanner SL, Pant HC, and Quarles RH (2002) Myelin-associated glycoprotein modulates expression and This interaction is lost after treatment with microtubule phosphorylation of neuronal cytoskeletal elements and their depolymerizing drugs. In turn, the association of p53 to associated kinases. J N e ur oc h e m 81: 1263-1272. microtubule allows for transport to the peri-nuclear region Ding J, Liu JJ, Kowal AS, Nardine T , Bhattachary a P , Lee A, and on microtubules, allowing for nuclear import. Yang Y (2002) Microtubule-associated protein 1B: a neuronal Furthermore, we tested the physiological significance of b i nd ing p a r t ner f o r g i ga xoni n. J Cell Biol 158: 427-433. binding between MAP1B light chain and p53 by assessing Foo RS, Nam YJ, Ostreicher MJ, Metzl MD, Whelan RS, Peng modulations in the transcriptional activity of p53. MAP1B- C F , A s h t on A W , Fu W , Ma ni K , C h in S F , Pro v e nz a n o E, El li s I, Figg N, Pinder S, Bennett MR, Caldas C, and Kitsis RN LC1 significantly decreased the activity of p53 (Fig. 3). (2007) Regulation of p53 tetramerization and nuclear export Perhaps, MAP1B-LC1 might repress transcriptional activity by ARC. Proc Natl Acad Sci USA 104: 20826-20831. of p53 through specific interactions with transactivation- Franzen R, T anner SL, Dashiell SM, Rottkamp CA, Hammer JA, and oligomerization domain of p53. MAP1B-LC1 binding and Quarles RH (2001) Microtubule-associated protein 1B: a to transactivation domain could prevent the association of neuronal binding partner for myelin-associated glycoprotein. co-activators, such as p300/CBP and PCAF to p53 and J C e ll Bi o l 155: 893-898. binding to oligomerization domain also inhibit the formationGarcia ML and Cleveland DW (2001) Going new places using an old MAP: tau, microtubules and human neurodegenerative of activated p53-tetramer (Foo et al., 2007). Thus, our d i sease. Curr Opin Cell Biol 13: 41-48. finding provide a molecular and functional linkage between Giannakakou P , Sackett DL, W ard Y , W ebster KR, Blagosklonny microtubule-associated protein 1B (MAP1B) and p53 in MV, and Fojo T (2000) p53 is associated with cellular cellular signaling. microtubules and is transported to the nucleus by dynein. Nat Despite intensive f unctional studies, the ef fects of MAP1B Cell Biol 2: 709-717. on cellular physiology are most focused on microtubule Gonzalez-Billault C, Engelke M, Jimenez-Mateos EM, W andosell related mechanisms and the other functions remain to be F, Caceres A, and Avila J (2002) Participation of structural microtubule-associated proteins (MAPs) in the development fully elucidated. In this aspect it is important to note that of neuronal polarity. J N e ur osci Re s 6 7 : 71 3- 719 . our findings contribute to a functional linkage between Gonzalez-Billault C, Jimenez-Mateos EM, Caceres A, Diaz-Nido MAP1B and p53 in the biological network, showing that J, Wandosell F, and Avila J (2004) Microtubule-associated MAP1B-LC1 associates with and regulates p53 as a protein 1B function during normal development, regeneration, negative regulator in the HEK 293 cells. Furthermore, these and pathological conditions in the nervous system. J Neurobiol 58: 48-59. examinations might be taken into consideration when Hanley JG , Jones EM, and Moss SJ (2000) GABA receptor rho1 knock-down of MAP1B-LC1 is used as a cancer subunit interacts with a novel splice variant of the glycine therapeutic strategy to enhance p53’s apoptotic activity in transporter, GLYT-1. J Biol Chem 275: 840-846. chemotherapy. Harris SL and Levine AJ (2005) The p53 pathway: positive and n e gat i ve f eed back l oops. Onc ogen e 24: 2899-2908. ACKNOWLEDGMENTS Jeong MH, Bae J, Ki m W H , Y oo S M, Kim JW , Son g PI , a nd Choi This work was supported by grants from Chung-Ang University. KH (2006) p19ras interacts with and activates p73 by involving the MDM2 protein. J Biol Chem 281: 8707-8715. 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Journal
Animal Cells and Systems
– Taylor & Francis
Published: Jan 1, 2008
Keywords: MAP1B‐LC1; p53; transcriptional activity; apoptosis; protein interaction
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