The Pro-hypertrophic Basic Helix-Loop-Helix Protein p8 Is Degraded by the Ubiquitin/Proteasome System in a Protein Kinase B/Akt- and Glycogen Synthase Kinase-3-dependent Manner, whereas Endothelin Induction of p8 mRNA and Renal Mesangial Cell Hypertrophy Require NFAT4
2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês
10.1074/jbc.m312401200
ISSN1083-351X
AutoresSandro Goruppi, John Kyriakis,
Tópico(s)RNA regulation and disease
ResumoRenal disease is a common complication of diabetes. The initiating events in diabetic nephropathy are triggered by hyperglycemia and, possibly, advanced glycation end products. Subsequently, excess levels of vasoactive peptides (especially endothelin-1 (ET-1)) accumulate in the diabetic kidney, and there is evidence that these peptides mediate many of the pathophysiological changes associated with diabetic renal disease. These changes include an excess deposition of extracellular matrix proteins into the glomerular basement membrane and renal mesangial cell hypertrophy. Our transcriptional profiling studies have revealed that the p8 gene, which encodes a putative basic helix-loop-helix protein, is strongly induced in ET-1-treated renal mesangial cells and in an animal model of diabetic nephropathy. RNA interference experiments indicated that the p8 gene is required for ET-1-induced mesangial cell hypertrophy. Here, we show that the p8 polypeptide is a phosphoprotein subject to constitutive degradation by the ubiquitin/proteasome system. This degradation is mediated by phosphatidylinositol 3-kinase and protein kinase B/Akt. By contrast, stabilization of the p8 protein requires glycogen synthase kinase-3. Finally, short interfering RNA-mediated RNA interference experiments indicated that ET-1-stimulated mesangial cell hypertrophy and p8 mRNA induction require the NFAT4 transcription factor. Thus, p8 levels in the cell are likely maintained by a balance between signal-dependent transcriptional induction and proteolysis. Renal disease is a common complication of diabetes. The initiating events in diabetic nephropathy are triggered by hyperglycemia and, possibly, advanced glycation end products. Subsequently, excess levels of vasoactive peptides (especially endothelin-1 (ET-1)) accumulate in the diabetic kidney, and there is evidence that these peptides mediate many of the pathophysiological changes associated with diabetic renal disease. These changes include an excess deposition of extracellular matrix proteins into the glomerular basement membrane and renal mesangial cell hypertrophy. Our transcriptional profiling studies have revealed that the p8 gene, which encodes a putative basic helix-loop-helix protein, is strongly induced in ET-1-treated renal mesangial cells and in an animal model of diabetic nephropathy. RNA interference experiments indicated that the p8 gene is required for ET-1-induced mesangial cell hypertrophy. Here, we show that the p8 polypeptide is a phosphoprotein subject to constitutive degradation by the ubiquitin/proteasome system. This degradation is mediated by phosphatidylinositol 3-kinase and protein kinase B/Akt. By contrast, stabilization of the p8 protein requires glycogen synthase kinase-3. Finally, short interfering RNA-mediated RNA interference experiments indicated that ET-1-stimulated mesangial cell hypertrophy and p8 mRNA induction require the NFAT4 transcription factor. Thus, p8 levels in the cell are likely maintained by a balance between signal-dependent transcriptional induction and proteolysis. Diabetic nephropathy is a common complication of both type I and II diabetes and represents the major cause of end stage renal failure in the Western World (1Fine L.G. Norman J.T. Kujubu D.A. Knecht A. Seldin D.W. Geibisch G. The Kidney, Physiology and Pathophysiology. Raven Press, Ltd., New York1992: 3113-3134Google Scholar). The renal disease characteristic of diabetes mellitus arises relatively quickly with the onset of frank hyperglycemia and is marked by elevations in circulating vasoactive peptides, glomerular mesangial cell hypertrophy, and proliferation as well as the excess deposition of extracellular matrix proteins into the glomerular basement membrane (2Bonventre J.V. Force T. Curr. Opin. Nephrol. Hypertens. 1998; 7: 425-433Crossref PubMed Scopus (27) Google Scholar, 3Wolf G. J. Am. Soc. Nephrol. 2002; 13: 2611-2613PubMed Google Scholar). Mesangial cell hypertrophy is characterized at the cellular level as an increase in overall protein synthesis in the absence of a concomitant increase in overall DNA synthesis. Multiple extracellular stimuli are thought to be involved in the pathophysiology of diabetic mesangial cell hypertrophy. Prominent among these are vasoactive peptides such as endothelin-1 (ET-1), 1The abbreviations used are: ET-1, endothelin-1; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; Ub, ubiquitin; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; NFAT, nuclear factor of activated T cells; RNAi, RNA interference; GSK3, glycogen synthase kinase-3; FBS, fetal bovine serum; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; Mops, 4-morpholinepropanesulfonic acid; siRNA, small interfering RNA. vasopressin, angiotensin II, and transforming growth factor-β (2Bonventre J.V. Force T. Curr. Opin. Nephrol. Hypertens. 1998; 7: 425-433Crossref PubMed Scopus (27) Google Scholar, 4Sorokin A. Kohan D.E. Am. J. Physiol. 2003; 285: F579-F589Crossref PubMed Scopus (113) Google Scholar, 5Wang Y. Su B. Sah V.P. Brown J.H. Han J. Chien K.R. J. Biol. Chem. 1998; 273: 5423-5426Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 6Wang Y. Huang S. Sah V.P. Ross Jr., J. Brown J.H. Han J. Chien K.R. J. Biol. Chem. 1998; 273: 2161-2168Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar). Our recent findings indicate that renal mesangial cell hypertrophy is controlled by mechanisms similar to those governing cardiomyocyte hypertrophy (9Goruppi S. Bonventre J.V. Kyriakis J.M. EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar). In particular, in addition to the recruitment of MAPKs, mesangial cell hypertrophy requires activation of the phosphatidylinositol 3-kinase (PI3K) pathway (9Goruppi S. Bonventre J.V. Kyriakis J.M. EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar). Our studies have been aimed at the identification of mechanisms coupling signaling pathways with more distal elements that trigger renal mesangial cell hypertrophy. Using a combined pharmacological and transcriptional profiling approach, we have identified genes induced by ET-1 after the period of treatment needed to trigger hypertrophy. Seven genes were identified; of these, one transcript, encoding the small basic helix-loop-helix protein p8, was strikingly and stably induced by ET-1 (9Goruppi S. Bonventre J.V. Kyriakis J.M. EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar). The p8 gene has been conserved evolutionarily, with homologs found in man, rat, mouse, and Xenopus. p8 was initially isolated as a gene strongly induced during acute pancreatitis (10Mallo G.V. Fiedler F. Calvo E.L. Ortiz E.M. Vasseur S. Keim V. Morisset J. Iovanna J.L. J. Biol. Chem. 1997; 272: 32360-32369Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 11Vasseur S. Mallo G.V. Fiedler F. Bodeker H. Canepa E. Moreno S. Iovanna J.L. Eur. J. Biochem. 1999; 259: 670-675Crossref PubMed Scopus (97) Google Scholar, 12Igarashi T. Kuroda H. Takahashi S. Asashima M. Dev. Growth Differ. 2001; 43: 693-698Crossref PubMed Scopus (25) Google Scholar). In several cell lines, the p8 gene is transiently induced in response to stress and thus may be part of a general defense mechanism protecting against cellular injury (10Mallo G.V. Fiedler F. Calvo E.L. Ortiz E.M. Vasseur S. Keim V. Morisset J. Iovanna J.L. J. Biol. Chem. 1997; 272: 32360-32369Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 11Vasseur S. Mallo G.V. Fiedler F. Bodeker H. Canepa E. Moreno S. Iovanna J.L. Eur. J. Biochem. 1999; 259: 670-675Crossref PubMed Scopus (97) Google Scholar). Consistent with this, targeted disruption of the murine p8 gene, although not lethal, does result in higher mortality in the lipopolysaccharide injection model of sepsis (13Vasseur S. Hoffmeister A. Garcia-Montero A. Barthet M. Saint-Michel L. Berthezene P. Fiedler F. Closa D. Dagorn J.C. Iovanna J.L. BMC Gastroenterol. 2003; http://www.biomedcentral.com/1471-230X/3/25PubMed Google Scholar). Other studies have implicated p8 gene expression with metastatic potential (14Ree A.H. Tvermyr M. Engebraaten O. Rooman M. Rosok O. Hovig E. Meza-Zepeda L.A. Bruland O.S. Fodstad O. Cancer Res. 1999; 59: 4675-4680PubMed Google Scholar), and the p8 gene may also be involved in the control of cell proliferation, although the precise function of p8 in cell proliferation is unclear. Thus, disruption of the p8 gene suppresses transformation associated with ectopic expression of ras and adenoviral E1A, blunting the growth of these transformed cells in soft agar and preventing the formation of tumors in mice injected with ras/E1A-transformed p8–/– cells (15Vasseur S. Hoffmeister A. Garcia S. Bagnis C. Dagorn J.C. Iovanna J.L. EMBO Rep. 2002; 3: 165-170Crossref PubMed Scopus (69) Google Scholar). By contrast, p8–/– cells proliferate more rapidly in culture and are resistant to chemical stress-induced apoptosis (16Vasseur S. Hoffmeister A. Garcia-Montero A. Mallo G.V. Fieil R. Kühbander S. Dagorn J.C. Iovanna J.L. Oncogene. 2002; 21: 1685-1694Crossref PubMed Scopus (81) Google Scholar). The product of the human p8 gene is an 82-amino acid protein with no significant homology to any other known eukaryotic protein. Analysis of the primary structure of p8 revealed a canonical bipartite nuclear localization signal and a potential DNA interaction motif (see Fig. 1A) (13Vasseur S. Hoffmeister A. Garcia-Montero A. Barthet M. Saint-Michel L. Berthezene P. Fiedler F. Closa D. Dagorn J.C. Iovanna J.L. BMC Gastroenterol. 2003; http://www.biomedcentral.com/1471-230X/3/25PubMed Google Scholar). Accordingly, recombinant p8 has been shown to bind in vitro to DNA, an interaction enhanced in vitro by phosphorylation of the recombinant protein with protein kinase A (17Encinar J.A. Mallo G.V. Mizyrycki C. Giono L. Gonzalez-Ros J.M. Rico M. Canepa E. Moreno S. Neira J.L. Iovanna J.L. J. Biol. Chem. 2001; 276: 2742-2751Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Consistent with the possibility that p8 is regulated by phosphorylation, 17% of the amino acid composition of p8 is composed of phosphorylatable residues (17Encinar J.A. Mallo G.V. Mizyrycki C. Giono L. Gonzalez-Ros J.M. Rico M. Canepa E. Moreno S. Neira J.L. Iovanna J.L. J. Biol. Chem. 2001; 276: 2742-2751Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In addition, the p8 protein contains an N-terminal PEST (Pro/Glu/Ser/Thr-rich) region, suggesting regulation of p8 levels by the ubiquitin (Ub)/proteasome system (see Fig. 1A) (18Rechsteiner M. Rogers S.W. Trends Biochem. Sci. 1996; 21: 267-271Abstract Full Text PDF PubMed Scopus (1413) Google Scholar). We have previously dissected the rat renal mesangial cell molecular signaling events activated by ET-1 that are required for hypertrophy and found that hypertrophy requires activation of the ERK and JNK MAPKs as well as PI3K-dependent signaling, possibly through NFAT proteins (9Goruppi S. Bonventre J.V. Kyriakis J.M. EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar). ET-1 induction of the p8 gene requires activation of these same signaling pathways. The p8 gene is also strongly induced in the kidneys of rats made diabetic with streptozotocin. Experiments using RNA interference (RNAi) to specifically silence mesangial cell p8 mRNA identified the p8 gene as a necessary component in the ET-1-stimulated transcriptional program that triggers mesangial cell hypertrophy (9Goruppi S. Bonventre J.V. Kyriakis J.M. EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar). Although there is a substantial understanding of the regulation of p8 gene expression and even of the putative biological function(s) of the p8 gene product, little or nothing is known about the regulation of the p8 polypeptide itself or of the regulation p8 protein levels in the cell. In our initial studies of the endogenous mesangial cell p8 polypeptide, we were intrigued to find that p8 was almost undetectable unless cells were preincubated with inhibitors of the Ub/proteasome system. Under these circumstances, we could detect a modest ET-1-stimulated increase in the p8 protein; however, the significance of this increase was unclear given the conditions necessary to visualize it, and we were compelled to ascertain whether or not p8 protein stability, as with p8 mRNA, is regulated by extracellular signaling pathways. Here, we show that the p8 protein is heavily phosphorylated in vivo. We found that the regulation of p8 polypeptide levels runs counter to the regulation of p8 mRNA. p8 is a predominantly nuclear protein that is constitutively degraded through a proteasome-dependent mechanism, and this degradation is regulated by the Akt pathway. Accordingly, p8 stability requires in vivo GSK3, the activity of which is inhibited by Akt. GSK3 also directly phosphorylates p8 in vitro. Overexpression of a dominant-negative Akt mutant or the addition of LY294002, a PI3K inhibitor, stabilized the p8 protein and confirmed that destabilization of p8 is mediated by the PI3K/Akt pathway. The proteasome inhibitor lactacystin substantially enhanced the levels of endogenous and recombinant p8. LiCl, a GSK3 inhibitor, destabilized p8 by a process that could be reversed with proteasome inhibitors (lactacystin). Finally, our RNAi experiments indicated that ET-1-induced mesangial cell hypertrophy and p8 mRNA induction require NFAT4, a transcription factor that is itself inhibited by GSK3 and activated by Akt-mediated GSK3 inhibition. Cell Culture—Primary rat renal mesangial cells were isolated as described (9Goruppi S. Bonventre J.V. Kyriakis J.M. EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar). Mesangial cells were cultured in RPMI 1640 medium and 10% fetal bovine serum (FBS). Human embryonic 293 cells were cultured in Dulbecco's modified Eagle's medium and 10% FBS. ET-1, lactacystin, LY294002, and SB203580 were from Calbiochem. Calyculin A was from Cell Signaling Technology. Immunofluorescence studies of NIH3T3 and HeLa cells were performed as described (19Goruppi S. Ruaro E. Varnum B. Schneider C. Mol. Cell. Biol. 1997; 17: 4442-4453Crossref PubMed Scopus (171) Google Scholar). DNA and Vectors—pcDNA-p8-His5 was obtained by mutating by PCR the stop codon of the full-length human p8 cDNA and introducing in-frame a short sequence coding for five histidines, followed by a stop codon. The construct was subcloned into the vector pcDNA3.1/hygro. pEGFP-p8 was obtained by cloning full-length human p8 in-frame at the C terminus of the enhanced green fluorescent protein (EGFP). Vectors encoding wild-type and kinase-dead GSK3β have been described (20Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1868) Google Scholar). pUSEamp vectors expressing dominant-negative and membrane-targeted, constitutively active Akt1 were from Upstate Biotechnology, Inc. Transfections, Kinase Assays, and Western Blot Analysis—293 cells were transfected using LipofectAMINE (Invitrogen). Cells at 70–80% confluency were transfected as suggested by the manufacturer. Cells were used after 24 h. Drugs (LY294002 (20 μm), SB203580 (20 μm), lactacystin (10 μm), and calyculin A (0.1 μm)) were added to the medium 3 h prior to lysing the cells. In cotransfection experiments, cells were lysed in 400 μl of Laemmli denaturing loading buffer. Equal amounts of total protein were separated on 15% SDS-polyacrylamide gels. Immunoblotting was performed with either our rabbit anti-p8 polyclonal antibody or commercial antibody against histidine (G-18), NFAT4 (F-1), NFAT1 (7A6), Ub (FL-76), or c-Myc (9E10) (Santa Cruz Biotechnology). Polypeptides were detected with the enhanced chemiluminescence system. Mesangial cells at 70% confluency were serum-starved for 48 h in Dulbecco's modified Eagle's medium and 0.5% FBS and then stimulated for 24 h with vehicle or 100 nm ET-1 in the presence or absence of vehicle or 10 μm lactacystin. Cells were lysed in SDS-PAGE loading buffer, and 60 μg of total proteins were subjected to SDS-PAGE and immunoblotting with rabbit anti-p8 polyclonal antibody. To detect endogenous p8, the membrane was developed using the SuperSignal system (Pierce). For treatment of the recombinant p8 protein with λ-phosphatase, 293 cells were transfected with pcDNA-p8-His5 as described above. After 24 h, the cells were lysed in 900 μl of 8 m urea and 100 mm Tris (pH 8) containing 10 mm phenylmethylsulfonyl fluoride. The recombinant p8 protein was immobilized for 3 h at 4 °C on 60 μl of nitrilotriacetic acid-agarose resin (QIAGEN Inc.). The beads were washed twice with lysis buffer, twice with 150 mm NaCl and 50 mm Tris (pH 8), and twice with λ-phosphatase buffer. The immobilized p8 protein was distributed into three aliquots. One was left untreated. The others were treated at 30 °C for 40 min with 10 units/μl λ-phosphatase in the presence or absence of 2 mm MnCl2, which is required for λ-phosphatase activity. Phosphatase reactions were stopped with 25 mm NaF. The samples were then subjected to SDS-PAGE and immunoblotting with anti-His antibody. In experiments of p8 ubiquitination, p8-His5 was transfected alone or together with Myc-tagged Ub and purified from the total cellular lysates as described above. The immunoblots were decorated using anti-Ub or anti-Myc antibody and ECL solutions. For the kinase assays, 293 cells were transfected with plasmids encoding Myc-tagged wild-type or kinase-dead GSK3β. The next day, cells were washed with cold phosphate-buffered saline and then lysed in 900 μl of ice-cold radioimmune precipitation assay buffer containing protease and phosphatase inhibitors. Equal amounts of total protein extracts were incubated with 2 μg of antibody 9E10 for 2 h at 4 °C with 60 μl of protein G-agarose (50%). The immunocomplexes were washed three times with lysis buffer and twice with kinase buffer (20 mm Mops (pH 7.2), 2 mm EGTA, 10 mm MgCl2, 1 mm dithiothreitol, and 0.1% Triton X-100) and then incubated for 20 min at 30 °C with 1 μg of bacterially expressed p8 protein, 100 μm ATP, and 2 μCi of [32P]ATP. Reactions were stopped by the addition of 2× SDS-PAGE loading buffer, separated by 15% SDS-PAGE, and subjected to autoradiography. In Vivo Labeling with 32PO4 and [35S]Methionine—293 cells transfected as described above were incubated for 3 h in 3 ml of phosphate-free medium (Dulbecco's modified Eagle's medium) containing 1 mCi/ml carrier-free 32PO4. Cells were washed with cold phosphate-buffered saline and lysed as described above in 900 μl of ice-cold radioimmune precipitation assay buffer containing protease and phosphatase inhibitors. Equal cpm of labeled extract were incubated with 4 μg of either anti-His or anti-GFP antibody (Santa Cruz Biotechnology) for 3 h at 4 °C with 60 μl of protein G-agarose (50%), and the immunocomplexes were separated by SDS-PAGE and subjected to autoradiography. Mesangial cells were serum-starved for 48 h in a 6-cm Petri dish, stimulated with 100 nm ET-1, and then labeled for 4 h with methionine-free Dulbecco's modified Eagle's medium supplemented with [35S]methionine (100 μCi/ml) and 0.5% FBS. Cells were lysed in 800 μl of ice-cold radioimmune precipitation assay buffer and immunoprecipitated with 10 μl of either preimmune serum or anti-p8 antiserum as described above. Northern Blotting—Northern blotting was performed as described (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Small Interfering RNA (siRNA)-mediated Mammalian Cell RNAi— Complementary RNA oligonucleotides derived from the rat nfat4 target sequence (AAGGGAATATTTGGAAAGGCCTT) were from Dharmacon. The method for rat p8 RNAi has been reported previously (9Goruppi S. Bonventre J.V. Kyriakis J.M. EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar). Deprotection and annealing of the oligonucleotides were performed according to the manufacturer's instructions. Procedures for RNAi were essentially as described by Elbashir et al. (22Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8186) Google Scholar). Double-stranded RNAs were introduced into mesangial cells at concentrations of 1, 5, and 10 μg of oligonucleotide/6-cm dish. After an overnight incubation with the siRNA, mesangial cells were serum-starved for 48 h in RPMI 1640 medium and 0.5% FBS before proceeding. Analysis of ET-1-induced mesangial cell hypertrophy has been described previously (9Goruppi S. Bonventre J.V. Kyriakis J.M. EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar). Regulation of p8 Protein Levels by the Ub/Proteasome System—We have recently shown that p8 mRNA expression is rapidly induced by ET-1 during the onset of mesangial cell hypertrophy (9Goruppi S. Bonventre J.V. Kyriakis J.M. EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar). Little is known, however, about how this induction is reflected in changes, if any, in the in vivo levels of p8 protein. To our surprise, we were unable to detect p8 protein in lysates of mesangial cells by immunoblotting, even after treatment with ET-1 (Fig. 1B, left two lanes). We were able to detect mesangial cell p8 only after immunoprecipitation from extracts of cells that had been metabolically labeled with [35S]methionine (Fig. 1C). In this instance, 35S-labeled p8 was immunoprecipitated only with anti-p8 antiserum, and not with preimmune serum. Of note, ET-1 treatment for 24 h, which is sufficient to induce maximally p8 mRNA (see Fig. 4C) (9Goruppi S. Bonventre J.V. Kyriakis J.M. EMBO J. 2002; 21: 5427-5436Crossref PubMed Scopus (67) Google Scholar), did not trigger increases in the p8 polypeptide (Fig. 1C). Thus, ET-1 stimulated a very large increase in p8 mRNA expression, but failed to trigger a concomitant increase in the p8 protein (compare Figs. 1C and 4C). Given this observation and the difficulty in detecting the endogenous p8 protein, we wondered whether p8 might be constitutively degraded. Examination of the primary sequence of p8 indicated the presence of a PEST sequence (Fig. 1A). PEST sequences are present in many polypeptides that are subject to rapid degradation by the Ub/proteasome system (18Rechsteiner M. Rogers S.W. Trends Biochem. Sci. 1996; 21: 267-271Abstract Full Text PDF PubMed Scopus (1413) Google Scholar). Thus, we examined the effects on p8 protein levels of the specific proteasome inhibitor lactacystin (23Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1504) Google Scholar). Preincubation of mesangial cells with lactacystin enables detection of endogenous p8 by immunoblotting. Under these circumstances, we observed a modest increase in the p8 protein upon subsequent treatment with ET-1 (Fig. 1B, right two lanes); however, given that this increase was apparent only upon examination of lactacystin-treated cells (Fig. 1, compare B and C), its physiological significance is unclear. Still, it is evident from Fig. 1B that endogenous p8 was destabilized by the Ub/proteasome system. Recombinant p8 expressed in 293 cells behaved in a similar manner. Thus, Fig. 1D shows that p8 immunoreactivity was present in extracts expressing pcDNA-p8, but not in mock-transfected 293 cells. Interestingly, even after ectopic overexpression, p8 protein levels remained extremely low. The addition of 10 μm lactacystin to the culture medium stimulated a significant accumulation of the p8 polypeptide (Fig. 1D), indicating regulation of p8 levels by the Ub/proteasome system. Ub is attached to target proteins via a covalent bond between a specific Lys residue in the Ub polypeptide (typically Lys48 for proteins destined for proteasomal degradation) and a Lys residue in the target protein. We found that p8 was ubiquitinated in vivo either in cells expressing p8 alone or in cells expressing p8 and Myc-tagged recombinant Ub (Fig. 1E). Of note, Fig. 1 (B–D), which examines total p8 protein levels, indicates that p8 migrated at ∼18 kDa. By contrast, Ub-p8 (Fig. 1E) migrated at ∼25 kDa, indicating an increase in size of ∼7 kDa, the predicted size of the Ub polypeptide. Thus, detectable Ub-p8 appears to be monoubiquitinated. We could not detect poly-Ub-p8; whether this is due to the overall extremely poor expression of endogenous or ectopically expressed p8 or to the fact that p8 is indeed monoubiquitinated remains to be determined. Nevertheless, taken together, these results strongly indicate that p8 stability is regulated by the Ub/proteasome system. p8 Is a Nuclear Protein—Analysis of the primary structure of p8 revealed a canonical bipartite nuclear localization signal and a potential DNA interaction motif (Fig. 1A) (13Vasseur S. Hoffmeister A. Garcia-Montero A. Barthet M. Saint-Michel L. Berthezene P. Fiedler F. Closa D. Dagorn J.C. Iovanna J.L. BMC Gastroenterol. 2003; http://www.biomedcentral.com/1471-230X/3/25PubMed Google Scholar). We thus investigated the protein localization by overexpressing pEGFP-p8 in different cell lines. (The N-terminal EGFP tag stabilized the recombinant p8 protein, rendering it resistant to degradation. 2S. Goruppi, unpublished data. ) When overexpressed as a fusion with GFP, p8 was localized in the nuclear compartment of both HeLa and NIH3T3 cells (Fig. 2A, left panels), even in absence of external stimuli. The control EGFP protein was localized throughout the cell (Fig. 2A, right panels). This cellular localization was not significantly modified by stimulation of the cells with epidermal growth factor or after osmotic stress with sorbitol (data not shown). Taken together, these results thus strongly suggest that p8 functions in the nuclear compartment. p8 Is a Phosphoprotein: Regulation of Proteasomal Degradation by Protein Kinase Signaling Pathways—p8 is a highly charged protein with a large percentage of residues that are potential targets for protein kinases (Fig. 1A). Consistent with this, p8 has been reported to be an in vitro substrate for protein kinase A and casein kinase-2 (17Encinar J.A. Mallo G.V. Mizyrycki C. Giono L. Gonzalez-Ros J.M. Rico M. Canepa E. Moreno S. Neira J.L. Iovanna J.L. J. Biol. Chem. 2001; 276: 2742-2751Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). We thus asked if p8 is phosphorylated in vivo. Given the low abundance of endogenous p8, we decided to examine recombinant p8 expressed in 293 cells. Cells were separately transfected with empty vector, pcDNA-p8-His5, or pEGFP-p8 and then metabolically labeled with 32PO4. p8 was immunoprecipitated with either anti-GFP or anti-His antibody. Both the His5- and EGFP-tagged p8 proteins were heavily phosphorylated (Fig. 3A). Both p8-His5 and endogenous p8 migrated on SDS-polyacrylamide gels at an apparent molecular mass of 18-kDa, more than two times larger than the expected molecular mass based on amino acid composition. To determine whether this discrepancy was due to phosphorylation of the protein, we incubated the recombinant protein in vitro with λ-protein phosphatase. p8-His5 was purified from transfected 293 cells and either left untreated or incubated with λ-phosphatase in the presence or absence of MnCl2, a necessary cofactor for λ-phosphatase activity (24Cohen P.T.W. Cohen P. Biochem. J. 1989; 260 (P. T. W.): 931-934Crossref PubMed Scopus (96) Google Scholar). After the treatment, the reactions were stopped with NaF, and the samples were analyzed by anti-His immunoblotting. In the presence of MnCl2, phosphatase selectively enhanced the mobility of p8 on SDS gels. It should be noted, however, that the dephosphorylated p8 polypeptide still migrated on SDS gels more slowly than expected, indicating that the observed migration of p8 upon SDS-PAGE cannot be attributed solely to phosphorylation. Endogenous p8 is present at extremely low levels in vivo; and even after ectopic expression, it is difficult to detect the C-terminally tagged recombinant p8 protein in the absence of proteasome inhibitors. Many targets of the Ub/proteasome system are degraded in a manner stimulated by extracellular signaling pathways (25Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). Accordingly, we wished to determine whether the signaling pathways known to regulate p8 mRNA levels might also regulate the stability of the p8 protein in vivo. Thus, 293 cells were transfected with pcDNA-p8-His5 and treated for various times with calyculin A, a specific inhibitor of protein phosphatase-1 and -2A (26Resjo S. Oknianska A. Zolnierowicz S. Manganiello V. Degerman E. Biochem. J. 1999; 341: 839-845Crossref PubMed Scopus (55) Google Scholar). Calyculin A rapidly destabilized p8 to a point at which the overexpressed protein was nearly undetectable (Fig. 3C). This destabilization was reversed with lactacystin, indicating that inhibition of a calyculin-sensitive phosphatase triggers the proteasome-dependent degradation of p8. Bacterially expressed p8 has been reported to be a target in vitro for protein kinase A and casein kinase-2, but its regulation in vivo is still poorly understood (15Vasseur S. Hoffmeister A. Garcia S. Bagnis C. Dagorn J.C. Iovanna J.L. EMBO Rep. 2002; 3: 165-170Crossref PubMed Scopus (69) Google Scholar, 16Vasseur S. Hoffmeister A. Garcia-Montero A. Mallo G.V. Fieil R. Kühbander S. Dagorn J.C. Iovanna J.L. Oncogene. 2002; 21: 1685-1694Crossref PubMed Scopus (81) Google Scholar, 17Encinar J.A. Mallo G.V. Mizyrycki C. Giono L. Gonzalez-Ros J.M. Rico M. Canepa E. Moreno S. Neira J.L. Iovanna J.L. J. Biol. Chem. 2001; 276: 2742-2751Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Four re
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