Lactate Dehydrogenase Is an AU-rich Element-binding Protein That Directly Interacts with AUF1
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m204002200
ISSN1083-351X
AutoresPatricia A. Pioli, B. JoNell Hamilton, John E. Connolly, Gary Brewer, William F. C. Rigby,
Tópico(s)Viral Infections and Immunology Research
ResumoPost-transcriptional pathways provide a major means of regulating eukaryotic gene expression. Reiterations of the AU-rich element (ARE) within the 3′-untranslated region of many cytokine and proto-oncogene mRNAs serve as signals for rapid degradation and translational repression. The identification of thiscis-acting stability determinant has fueled the search for ARE-binding proteins (AUBP) that function as trans-acting factors that transduce this function. Previous work identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a major AUBP capable of binding the ARE of granulocyte-macrophage colony stimulating factor (GM-CSF) RNA in the context of a full-length mRNA. We report here that functional studies failed to indicate a role for hnRNP A1 in ARE-dependent mRNA turnover. In an effort to identify other functionally relevant AUBP, the major GM-CSF ARE-specific binding protein in cells lacking hnRNP A1 was purified from CB3 mouse erythroleukemia cells. Microsequencing identified this protein as the glycolytic enzyme lactate dehydrogenase (LDH) M. RNA binding by LDH was shown to occur in the NAD+-binding region (Rossmann fold). Polysome gradient analysis demonstrates that LDH is found in the translationally active fraction. Polysomal localization of LDH was dependent on RNA binding. Moreover, polysomal LDH exists in a complex with AUF1 and hsp-70, which has been implicated previously in the regulation of mRNA turnover. The interaction between LDH and AUF1 is direct as it can be demonstrated in vitro with purified proteins. Collectively these data implicate a role for LDH in the post-transcriptional regulation of gene expression. Post-transcriptional pathways provide a major means of regulating eukaryotic gene expression. Reiterations of the AU-rich element (ARE) within the 3′-untranslated region of many cytokine and proto-oncogene mRNAs serve as signals for rapid degradation and translational repression. The identification of thiscis-acting stability determinant has fueled the search for ARE-binding proteins (AUBP) that function as trans-acting factors that transduce this function. Previous work identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a major AUBP capable of binding the ARE of granulocyte-macrophage colony stimulating factor (GM-CSF) RNA in the context of a full-length mRNA. We report here that functional studies failed to indicate a role for hnRNP A1 in ARE-dependent mRNA turnover. In an effort to identify other functionally relevant AUBP, the major GM-CSF ARE-specific binding protein in cells lacking hnRNP A1 was purified from CB3 mouse erythroleukemia cells. Microsequencing identified this protein as the glycolytic enzyme lactate dehydrogenase (LDH) M. RNA binding by LDH was shown to occur in the NAD+-binding region (Rossmann fold). Polysome gradient analysis demonstrates that LDH is found in the translationally active fraction. Polysomal localization of LDH was dependent on RNA binding. Moreover, polysomal LDH exists in a complex with AUF1 and hsp-70, which has been implicated previously in the regulation of mRNA turnover. The interaction between LDH and AUF1 is direct as it can be demonstrated in vitro with purified proteins. Collectively these data implicate a role for LDH in the post-transcriptional regulation of gene expression. The regulation of mRNA turnover is a key mechanism of modulating eukaryotic gene expression. Labile cytokine, lymphokine, and proto-oncogene messages contain AU-rich sequences in their 3′-untranslated regions that are highly conserved across mammalian species. As demonstrated conclusively by Shaw and Kamen (1Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3107) Google Scholar), these AU-rich elements (ARE) 1The abbreviations used are: ARE, A + U-rich element; AUBP, AU-rich sequence-binding proteins; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDH, lactate dehydrogenase; UTR, untranslated region; PBS, phosphate-buffered saline; NEPHGE, non-equilibrium pH-gradient electrophoresis; hnRNP, heterogeneous nuclear ribonucleoprotein; Pipes, 1,4-piperazinediethanesulfonic acid; MEL, murine erythroleukemia; IL, interleukin; CAPS, 3-(cyclohexylamino)propanesulfonic acid. 1The abbreviations used are: ARE, A + U-rich element; AUBP, AU-rich sequence-binding proteins; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDH, lactate dehydrogenase; UTR, untranslated region; PBS, phosphate-buffered saline; NEPHGE, non-equilibrium pH-gradient electrophoresis; hnRNP, heterogeneous nuclear ribonucleoprotein; Pipes, 1,4-piperazinediethanesulfonic acid; MEL, murine erythroleukemia; IL, interleukin; CAPS, 3-(cyclohexylamino)propanesulfonic acid. compose a major class of cis-acting stability determinants. These ARE consist of reiterations of the pentamer AUUUA or oligo(U) sequences in an AU-rich context (1Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3107) Google Scholar, 2Jones T.R. Cole M.D. Mol. Cell. Biol. 1987; 7: 4513-4521Crossref PubMed Scopus (213) Google Scholar, 3Shyu A.B. Greenberg M.E. Belasco J.G. Genes Dev. 1989; 3: 60-72Crossref PubMed Scopus (448) Google Scholar). Significantly, cytoplasmic and nuclear proteins have been identified that bind specifically to the ARE; these AU-binding proteins (AUBP) are thought to regulate mRNA stability in trans (reviewed in Ref. 4Jarzembowski J.A. Malter J.S. Prog. Mol. Subcell. Biol. 1997; 18: 141-172Crossref PubMed Scopus (16) Google Scholar). Although a variety of these AUBP has been described, functional roles in ARE-dependent turnover have been attributed to a comparatively small number of proteins, including HuR and AUF1 (5Ma W.J. Cheng S. Campbell C. Wright A. Furneaux H. J. Biol. Chem. 1996; 271: 8144-8151Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 6Myer V.E. Fan X.C. Steitz J.A. EMBO J. 1997; 16: 2130-2139Crossref PubMed Scopus (279) Google Scholar, 7Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (743) Google Scholar, 8Levy N.S. Chung S. Furneaux H. Levy A.P. J. Biol. Chem. 1998; 273: 6417-6423Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 9Brewer G. Ross J. Mol. Cell. Biol. 1989; 9: 1996-2006Crossref PubMed Scopus (130) Google Scholar, 10Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (399) Google Scholar, 11Kiledjian M. DeMaria C.T. Brewer G. Novick K. Mol. Cell. Biol. 1997; 17: 4870-4876Crossref PubMed Google Scholar, 12Dempsey L.A. Sun H. Hanakahi L.A. Maizels N. J. Biol. Chem. 1999; 274: 1066-1071Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar).HuR, a ubiquitously expressed member of the Elav family of RNA-binding proteins, interacts in vitro with the ARE of c-fos and IL-3 mRNA (5Ma W.J. Cheng S. Campbell C. Wright A. Furneaux H. J. Biol. Chem. 1996; 271: 8144-8151Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar), as well as with synthetic mRNA-destabilizing ARE (6Myer V.E. Fan X.C. Steitz J.A. EMBO J. 1997; 16: 2130-2139Crossref PubMed Scopus (279) Google Scholar). Several studies (7Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (743) Google Scholar, 8Levy N.S. Chung S. Furneaux H. Levy A.P. J. Biol. Chem. 1998; 273: 6417-6423Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar) have demonstrated that overexpression of HuR results in stabilization of reporter transcripts containing the ARE of GM-CSF, c-fos, and vascular endothelial growth factor. These studies did not demonstrate altered protein production as a consequence of increased mRNA accumulation (5Ma W.J. Cheng S. Campbell C. Wright A. Furneaux H. J. Biol. Chem. 1996; 271: 8144-8151Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 6Myer V.E. Fan X.C. Steitz J.A. EMBO J. 1997; 16: 2130-2139Crossref PubMed Scopus (279) Google Scholar, 7Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (743) Google Scholar, 8Levy N.S. Chung S. Furneaux H. Levy A.P. J. Biol. Chem. 1998; 273: 6417-6423Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar).Similarly, in vitro ARE binding activity has been demonstrated for AUF; this protein was initially purified because of its ability to accelerate the degradation of c-myc mRNA in anin vitro decay system (9Brewer G. Ross J. Mol. Cell. Biol. 1989; 9: 1996-2006Crossref PubMed Scopus (130) Google Scholar, 10Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (399) Google Scholar). However, recent work (11Kiledjian M. DeMaria C.T. Brewer G. Novick K. Mol. Cell. Biol. 1997; 17: 4870-4876Crossref PubMed Google Scholar, 12Dempsey L.A. Sun H. Hanakahi L.A. Maizels N. J. Biol. Chem. 1999; 274: 1066-1071Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) has suggested AUF1 may play multiple roles in the regulation of mRNA turnover. For example, Kiledjian et al. (11Kiledjian M. DeMaria C.T. Brewer G. Novick K. Mol. Cell. Biol. 1997; 17: 4870-4876Crossref PubMed Google Scholar) have demonstrated that AUF1 is a component of the α-globin mRNA stabilization complex, whereas other studies (12Dempsey L.A. Sun H. Hanakahi L.A. Maizels N. J. Biol. Chem. 1999; 274: 1066-1071Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) have implicated AUF1 in B-cell transcriptional activation. The diversity of functions attributed to AUF1 suggests its activity may be regulated by protein-protein interactions.Our laboratory has been examining potential trans-acting factors involved in mediating ARE-dependent mRNA turnover. Initial studies centered on investigating the role of hnRNP A1, prompted by its discovery as the major cytoplasmic protein capable of binding the GM-CSF ARE in activated T lymphocytes (13Hamilton B.J. Nagy E. Malter J.S. Arrick B.A. Rigby W.F. J. Biol. Chem. 1993; 268: 8881-8887Abstract Full Text PDF PubMed Google Scholar, 14Hamilton B.J. Burns C.M. Nichols R.C. Rigby W.F.C. J. Biol. Chem. 1997; 272: 28732-28741Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In addition, IL-2 overproduction by a retrovirally infected T cell line, MLA-144, was shown to be due to increased mRNA stability (15Henics T. Sanfridson A. Hamilton B.J. Nagy E. Rigby W.F. J. Biol. Chem. 1994; 269: 5377-5383Abstract Full Text PDF PubMed Google Scholar). Stabilization of IL-2 mRNA correlated with a proviral insertion in its 3′-UTR which enhances the binding of hnRNP A1 to its ARE relative to that of native IL-2 (15Henics T. Sanfridson A. Hamilton B.J. Nagy E. Rigby W.F. J. Biol. Chem. 1994; 269: 5377-5383Abstract Full Text PDF PubMed Google Scholar).To examine the role of hnRNP A1 in ARE-dependent mRNA turnover, we utilized the DP28-9, CB7, and CB3 murine erythroleukemia (MEL) cell lines, which vary in their expression of hnRNP A1 (16Ben-David Y. Bani M.R. Chabot B., De Koven A. Bernstein A. Mol. Cell. Biol. 1992; 12: 4449-4455Crossref PubMed Scopus (76) Google Scholar). DP28-9 MEL cells derive from DBA/2 adult mice infected with a polycythemia-inducing strain of the Friend leukemia virus (FV-P) and contain two functional hnRNP A1 genes (16Ben-David Y. Bani M.R. Chabot B., De Koven A. Bernstein A. Mol. Cell. Biol. 1992; 12: 4449-4455Crossref PubMed Scopus (76) Google Scholar). In contrast, the CB7 MEL cell line has only one active hnRNP A1 gene, due to silencing of one allele by a downstream insertion of F-murine leukemia virus helper virus at the Fli-2 locus. In CB3 MEL cells, proviral integration occurred at both Fli-2 loci, resulting in the absence of hnRNP A1 mRNA and protein (16Ben-David Y. Bani M.R. Chabot B., De Koven A. Bernstein A. Mol. Cell. Biol. 1992; 12: 4449-4455Crossref PubMed Scopus (76) Google Scholar). Transient as well as stable transfection experiments of CB3 MEL cells with hnRNP A1 failed to indicate an effect of hnRNP A1 on ARE-dependent mRNA turnover or expression of reporter gene constructs. Similarly, overexpression of hnRNP A1 in human Jurkat T cells did not result in alteration of ARE-dependent gene expression.As we were unable to detect an effect of hnRNP A1 on ARE-dependent gene regulation, we examined the AUBP profiles of the three cell lines. The major cytosolic protein capable of binding the ARE of GM-CSF in the context of its full-length RNA in CB3 erythroleukemia cells was identified as l-lactate dehydrogenase (LDH) isozyme M. Lactate dehydrogenase, which catalyzes the reversible conversion of pyruvate to lactate, is a tetrameric enzyme that may exist in several enzyme forms (17Lehninger A.L. Biochemistry. Worth Publishers, Inc., New York1975: 244-247Google Scholar). Each LDH subunit is one of two types, designated H (for heart) and M (for skeletal muscle). The five tetrameric isozymes of LDH are five different combinations of these subunits (17Lehninger A.L. Biochemistry. Worth Publishers, Inc., New York1975: 244-247Google Scholar). The isozyme of LDH identified in these studies is LDH-M, which consists of five M polypeptide subunits. (For purposes of ease of reference, the LDH-M will be referred to as simply LDH throughout the text.). LDH was shown to bind specifically to the ARE of GM-CSF RNA using both UV cross-linking and filter binding assays. LDH is polysomally associated and coimmunoprecipitates with AUF1 and hsp-70. These finding suggest that LDH may serve multiple roles in RNA metabolism beyond its role in glycolysis. Indeed, non-overlapping roles of LDH are suggested by the fact that the binding of the ARE by LDH is competed by NAD+, indicating that each utilizes the Rossmann fold. Similar roles have been well defined for the iron-response element-binding protein and its dual role as an aconitase (18Hentze M.W. Curr. Stud. Hematol. Blood. Transfus. 1991; 58: 115-126Crossref Google Scholar, 19Melefors O. Hentze M.W. Bioessays. 1993; 15: 85-90Crossref PubMed Scopus (106) Google Scholar). Collectively, these data implicate LDH as a functionally relevant AUBP and prompt consideration of an expanded role of this enzyme in the post-transcriptional regulation of gene expression.DISCUSSIONAlthough identified as a major AUBP in human and gibbon T lymphocytes as well as mouse erythroleukemia cells (13Hamilton B.J. Nagy E. Malter J.S. Arrick B.A. Rigby W.F. J. Biol. Chem. 1993; 268: 8881-8887Abstract Full Text PDF PubMed Google Scholar, 14Hamilton B.J. Burns C.M. Nichols R.C. Rigby W.F.C. J. Biol. Chem. 1997; 272: 28732-28741Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), a variety of experiments failed to demonstrate a role for hnRNP A1 on ARE-dependent turnover or translation of reporter gene constructs or GM-CSF mRNA. We concluded therefore that another AUBP besides hnRNP A1 mediates ARE-dependent gene regulation. Experiments to identify the trans-acting factor responsible for mediating the rapid turnover of GM-CSF message were undertaken. Despite the absence of hnRNP A1, CB3 cytosols contain a 36-kDa protein that binds specifically to the ARE of GM-CSF RNA. All previously characterized AUBP of comparable size (GAPDH, HuR, and AUF1) were excluded using immunoprecipitation and two-dimensional NEPHGE immunoblotting approaches.Matrix-assisted laser desorption/ionization-mass spectrometry identified the p36 AUBP as LDH. This finding was confirmed by immunoprecipitation of LDH-GM-CSF RNA complexes from CB3 cytosols as well as by in vitro binding (UV cross-linking and filter binding) assays with purified LDH. In addition, incubation of either LDH or CB3 cytosol (data not shown) with increasing concentrations of NAD+ inhibited p36 AUBP activity. This latter finding is consistent with the interpretation that the Rossmann fold serves as the RNA binding domain of LDH, as has been reported previously (20Nagy E. Rigby W.F. J. Biol. Chem. 1995; 270: 2755-2763Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar) for other glycolytic enzymes. LDH localized to the polysomes in multiple cell types besides CB3 cells. Thus, the polysomal location of LDH did not occur due to the absence of hnRNP A1. Moreover, in all cell types examined, LDH localized to the bottom of the sucrose gradients, consistent with its association with mRNA being actively translated. In addition, direct association of LDH with AUF1 was demonstrated.The role of LDH in intermediary metabolism has been well documented. LDH is a tetrameric enzyme with five isoforms, each consisting of combinations of two subunits, LDH-M and LDH-H (17Lehninger A.L. Biochemistry. Worth Publishers, Inc., New York1975: 244-247Google Scholar). The M subunit catalyzes the conversion of pyruvate to lactate under anaerobic conditions, whereas the H subunit kinetically favors the conversion of lactate to pyruvate and predominates in aerobic tissue, such as heart muscle (17Lehninger A.L. Biochemistry. Worth Publishers, Inc., New York1975: 244-247Google Scholar). The biosynthesis of each subunit and thus the amount of each LDH isozyme in a given tissue type is subject to genetic regulation. Although each isozyme catalyzes the same reaction, they have markedly different K m values for pyruvate (17Lehninger A.L. Biochemistry. Worth Publishers, Inc., New York1975: 244-247Google Scholar). The isozyme identified in these studies consists of four identical M subunits and is therefore designated M4. This isozyme predominates in skeletal muscle and has a low K m for pyruvate; hence it readily transfers electrons from lactate to NAD+, yielding pyruvate and NADH (17Lehninger A.L. Biochemistry. Worth Publishers, Inc., New York1975: 244-247Google Scholar). This reaction is dependent on the binding of NAD+ to LDH in the Rossmann fold (17Lehninger A.L. Biochemistry. Worth Publishers, Inc., New York1975: 244-247Google Scholar).Other enzymatic proteins have been identified that bind RNA; these RNA binding activities appear distinct from their functions in metabolism (reviewed in Ref. 27Hentze M.W. Trends Biochem. Sci. 1994; 19: 101-108Abstract Full Text PDF PubMed Scopus (123) Google Scholar). In previous work from this laboratory (20Nagy E. Rigby W.F. J. Biol. Chem. 1995; 270: 2755-2763Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), GAPDH was purified as a polysomal p36 AUBP from human spleen. A number of features of LDH led us to conclude that LDH and GAPDH play differing roles in post-transcriptional gene regulation. First, GAPDH appeared to be less specific in its interaction with ARE than LDH, as it was shown to bind a variety of ARE, including those lacking reiterated AUUUA pentamers (IFN-γ and c-Myc) in their 3′-UTR (20Nagy E. Rigby W.F. J. Biol. Chem. 1995; 270: 2755-2763Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Second, nitrocellulose filter binding experiments performed with purified protein revealed the K d of GAPDH for GM-CSF RNA is approximately twice that of LDH for GM-CSF RNA (data not shown). These data indicate that although GAPDH and LDH are capable of binding ARE, LDH binds the 3′-UTR of GM-CSF RNA with greater affinity than GAPDH when analyzed as a purified protein. The interaction of LDH in vivo with other proteins may further enhance this difference in affinity, as we were unable to detect any GM-CSF RNA binding by GAPDH in CB3 cytosols, despite operating under conditions of probe excess. In this regard, immunoprecipitation studies demonstrate that LDH, but not GAPDH, interacts with AUF1 in vivo (data not shown). These data reflect the specificity of the LDH/AUF1 association, as well as suggest that LDH and GAPDH may play different roles in ARE-dependent gene regulation, with distinct substrate specificity and function.The localization of LDH to the polysomes of human lymphoid and monocytic cells, which express GM-CSF, supports a functional role for LDH in the regulation of gene expression in vivo. The functional relevance of LDH AUBP activity is supported by its specific interaction with mRNA undergoing active translation. As levels of LDH on polysomes were approximately one-tenth those in the S130 fraction across cell types and species (data not shown), levels of polysomal and cytosolic LDH appear to be tightly regulated. Interestingly, the NAD+ concentration (1 μm) required to significantly inhibit RNA binding by purified LDH is lower than cytosolic NAD+ levels (30–70 μm) (32Micheli V. Sestini S. Methods Enzymol. 1997; 280: 211-221Crossref PubMed Scopus (23) Google Scholar,33Zhang Q. Piston D.W. Goodman R.H. Science. 2002; 295: 1895-1897PubMed Google Scholar). Displacement of LDH from the polysome compartment required much higher NAD+ concentrations (100 μm) than those necessary to inhibit RNA binding by purified enzyme. These data suggest that polysomal LDH may have an enhanced affinity for RNA or a decreased affinity for NAD+, perhaps mediated through post-translational modification or protein-protein interactions.In this regard, we have shown that polysomal LDH exists in a large protein complex in the absence of RNA binding. By immunoprecipitation, we demonstrated AUF1 and LDH exist as a complex in vivo. This finding is consistent with the sedimentation of LDH in the most dense gradient of the polysomes, indicating active translation, because AUF1 exists in a complex including heat shock proteins hsp-70, translation initiation factor eIF4G, and poly(A)-binding protein (31Laroia G. Cuesta R. Brewer G. Schneider R.J. Science. 1999; 284: 499-502Crossref PubMed Scopus (346) Google Scholar). In support of this model is our finding that hsp-70 coimmunoprecipitates with AUF1 and LDH in THP-1 monocytic cells (Fig.7 A).These data are especially intriguing in light of the diverse cellular functions which have been described for AUF1, which range from telomere maintenance to transcription, as well as mRNA turnover (9Brewer G. Ross J. Mol. Cell. Biol. 1989; 9: 1996-2006Crossref PubMed Scopus (130) Google Scholar, 10Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (399) Google Scholar, 11Kiledjian M. DeMaria C.T. Brewer G. Novick K. Mol. Cell. Biol. 1997; 17: 4870-4876Crossref PubMed Google Scholar, 12Dempsey L.A. Sun H. Hanakahi L.A. Maizels N. J. Biol. Chem. 1999; 274: 1066-1071Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 34Eversole A. Maizels N. Mol. Cell. Biol. 2000; 20: 5425-5432Crossref PubMed Scopus (79) Google Scholar). In the K562 erythroleukemia cell line, distinct effects on mRNA stability have been noted. Kiledjian et al. (11Kiledjian M. DeMaria C.T. Brewer G. Novick K. Mol. Cell. Biol. 1997; 17: 4870-4876Crossref PubMed Google Scholar) demonstrated that AUF1 is a component of the α-globin mRNA stabilization complex. Similar to our findings with hnRNP A1, overexpression of AUF1 in the K562 erythroleukemia cell line had no effect on ARE-mediated mRNA turnover (35Loflin P. Chen C.Y. Shyu A.B. Genes Dev. 1999; 13: 1884-1897Crossref PubMed Scopus (262) Google Scholar). However, when induced by hemin to undergo erythroid differentiation, ARE-mediated turnover in K562 erythroleukemia cells was inhibited. Overexpression of AUF1 led to a restoration of ARE-mediated turnover (35Loflin P. Chen C.Y. Shyu A.B. Genes Dev. 1999; 13: 1884-1897Crossref PubMed Scopus (262) Google Scholar). It is unclear if the changes in ARE-dependent turnover induced by hemin treatment or AUF1 were due to effects on translation and loading onto polysomes. This observation is potentially important as ARE-mediated turnover has been reported to be dependent on either translation or ribosomal transit (36Curatola A.M. Nadal M.S. Schneider R.J. Mol. Cell. Biol. 1995; 15: 6331-6340Crossref PubMed Scopus (69) Google Scholar). Thus, it is possible that overexpression of AUF1 restored ARE-dependent mRNA turnover by permitting polysomal loading of these mRNA.These studies are central to consideration of the functional role of the ARE, as protein-protein interactions may influence the role(s) AUF1 serves in DNA and RNA metabolism. In this regard, our data indicate that LDH is associated with AUF1 on mRNA undergoing active translation. Nevertheless, LDH was initially characterized because of its potential role in mediating rapid ARE-dependent turnover in erythroleukemia cells that lack hnRNP A1. One potential model for LDH function is that it provides further specificity for the translation and turnover of certain ARE-containing mRNA through its direct interaction with both the ARE and AUF1 protein. The importance of such a role by LDH is suggested by the finding that recombinant AUF1 binds an array of RNA ligands, including those lacking ARE (11Kiledjian M. DeMaria C.T. Brewer G. Novick K. Mol. Cell. Biol. 1997; 17: 4870-4876Crossref PubMed Google Scholar, 12Dempsey L.A. Sun H. Hanakahi L.A. Maizels N. J. Biol. Chem. 1999; 274: 1066-1071Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 34Eversole A. Maizels N. Mol. Cell. Biol. 2000; 20: 5425-5432Crossref PubMed Scopus (79) Google Scholar,37Bhattacharya S. Giordano T. Brewer G. Malter J.S. Nucleic Acids Res. 1999; 27: 1464-1472Crossref PubMed Scopus (39) Google Scholar). Alternatively, LDH may influence, through its direct interaction with AUF1, the makeup of this protein complex or the functional nature of its interaction with ARE. For example, a model has been proposed in which AUF1 mediates ARE-dependent turnover through proteasomal targeting and degradation of the RNP complex (31Laroia G. Cuesta R. Brewer G. Schneider R.J. Science. 1999; 284: 499-502Crossref PubMed Scopus (346) Google Scholar). In this regard, it is important to note that LDH did not copurify with proteasomes following EDTA treatment of polysomes gradients (data not shown). The possibility that LDH may serve a distinct role in mRNA translation and turnover independent of AUF1 is not excluded by this model.Additionally consistent with its association with both AUF1 and hsp-70in vivo, LDH may mediate effects on mRNA turnover as a component of eukaryotic degradation machinery, in a manner analogous to enolase in E. coli. The degradosome of E. coliconsists of a high molecular weight complex of proteins including RNase E, an endoribonuclease; polyribonucleotide nucleotidyltransferase (PNPase); an ATP-dependent helicase and 3′-5′-exoribonuclease; RhlB, a member of the DEAD box family; polyphosphate kinase; and enolase, a glycolytic enzyme (38Grunberg-Manago M. Annu. Rev. Genet. 1999; 33: 193-227Crossref PubMed Scopus (243) Google Scholar). Additional proteins are often associated with the degradosome, notably the heat shock chaperones GroEL and DnaK (38Grunberg-Manago M. Annu. Rev. Genet. 1999; 33: 193-227Crossref PubMed Scopus (243) Google Scholar). The metabolic function of enolase has been defined as the catalysis of 2-phosphoglycerate to phosphoenolpyruvate (17Lehninger A.L. Biochemistry. Worth Publishers, Inc., New York1975: 244-247Google Scholar), although its role in mRNA degradation is not yet clear (39Blum E. Carpousis A.J. Higgins C.F. J. Biol. Chem. 1999; 274: 4009-4016Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar).In conclusion, the discovery of LDH as an RNA-binding protein points to an expanded role for this protein in the regulation of gene expression. Its binding specificity, polysomal localization, and association with AUF1 collectively suggest a role in ARE-dependent mRNA turnover beyond its function in metabolism. The ability of LDH to serve as an AUBP may represent a global mechanism for regulating ARE-mediated decay, perhaps by modulating the effects of AUF1. Of particular note, c-Myc overexpression results in the up-regulation of LDH at the level of gene transcription (40Shim H. Dolde C. Lewis B.C., Wu, C.S. Dang G. Jungmann R.A. Dalla-Favera R. Dang C.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6658-6663Crossref PubMed Scopus (833) Google Scholar). Elevated levels of LDH are frequently detected in human cancers (41Chung K.C. Huang D. Chen Y. Short S. Short M.L. Zhang Z. Jungmann R.A. Mol. Cell. Biol. 1995; 15: 6139-6149Crossref PubMed Scopus (20) Google Scholar, 42Huang D. Jungmann R.A. Mol. Cell. Endocrinol. 1995; 108: 87-94Crossref PubMed Scopus (33) Google Scholar, 43Matrisian L.M. Rautmann G. Magun B.E. Breathnach R. Nucleic Acids Res. 1985; 13: 711-726Crossref PubMed Scopus (102) Google Scholar, 44Short M.L. Huang D. Milkowski D.M. Short S. Kunstman K. Soong C.J. Chung K.C. Jungmann R.A. Biochem. J. 1994; 304: 391-398Crossref PubMed Scopus (41) Google Scholar). The overexpression of LDH may thus confer neoplastic growth advantage, either through its enzymatic or gene regulatory function. Understanding the dual functions of this protein may lead to greater understanding of carcinogenesis. The regulation of mRNA turnover is a key mechanism of modulating eukaryotic gene expression. Labile cytokine, lymphokine, and proto-oncogene messages contain AU-rich sequences in their 3′-untranslated regions that are highly conserved across mammalian species. As demonstrated conclusively by Shaw and Kamen (1Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3107) Google Scholar), these AU-rich elements (ARE) 1The abbreviations used are: ARE, A + U-rich element; AUBP, AU-rich sequence-binding proteins; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colony-stimulating factor; LDH, lactate dehydrogenase; UTR, untranslated region; PBS, phosphate-buffered saline; NEPHGE, non-equilibrium pH-gradient electrophoresis; hnRNP, heterogeneous nuclear ribonucleoprotein; Pipes, 1,4-piperazinediethanesulfonic acid; MEL, murine erythroleukemia; IL, interleukin; CAPS, 3-(cyclohexylamino)propanesulfonic acid. 1The abbreviations used are: ARE, A + U-rich element; AUBP, AU-rich sequence-binding proteins; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM-CSF, granulocyte-ma
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