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Structural Determinants for Post-transcriptional Stabilization of Lactate Dehydrogenase A mRNA by the Protein Kinase C Signal Pathway

2000; Elsevier BV; Volume: 275; Issue: 17 Linguagem: Inglês

10.1074/jbc.275.17.12963

ISSN

1083-351X

Autores

Sabine Short, Di Tian, Marc L. Short, Richard A. Jungmann,

Tópico(s)

Peroxisome Proliferator-Activated Receptors

Resumo

Activation of protein kinase C (PKC) and protein kinase A (PKA) in rat C6 glioma cells increases the half-life of short-lived lactate dehydrogenase (LDH)-A mRNA about 5- and 8-fold, respectively. PKA and PKC act synergistically and prolong LDH-A mRNA half-life more than 21-fold. Similar effects were observed after transfection and transcription of a globin/lactate dehydrogenase minigene consisting of a β-globin expression vector in which the 3′-untranslated region (UTR) of β-globin had been replaced with the LDH-A 3′-UTR. Synergism was only obtained by transcription of minigenes containing the entire 3′-UTR and did not occur when truncated 3′-UTR fragments were analyzed. Additional mutational analyses showed that a 20-nucleotide region, named PKC-stabilizing region (PCSR), is responsible for mediating the stabilizing effect of PKC. Previous studies (Tian, D., Huang, D., Short, S., Short, M. L., and Jungmann, R. A. (1998) J. Biol. Chem. 273, 24861–24866) have demonstrated the existence of a cAMP-stabilizing region in LDH-A 3′-UTR. Sequence analysis of PCSR identified a 13-nucleotide AU-rich region that is common to both cAMP-stabilizing region and PCSR. These studies identify a specific PKC-responsive stabilizing element and indicate that interaction of PKA and PKC results in a potentiating effect on LDH-A mRNA stabilization. Activation of protein kinase C (PKC) and protein kinase A (PKA) in rat C6 glioma cells increases the half-life of short-lived lactate dehydrogenase (LDH)-A mRNA about 5- and 8-fold, respectively. PKA and PKC act synergistically and prolong LDH-A mRNA half-life more than 21-fold. Similar effects were observed after transfection and transcription of a globin/lactate dehydrogenase minigene consisting of a β-globin expression vector in which the 3′-untranslated region (UTR) of β-globin had been replaced with the LDH-A 3′-UTR. Synergism was only obtained by transcription of minigenes containing the entire 3′-UTR and did not occur when truncated 3′-UTR fragments were analyzed. Additional mutational analyses showed that a 20-nucleotide region, named PKC-stabilizing region (PCSR), is responsible for mediating the stabilizing effect of PKC. Previous studies (Tian, D., Huang, D., Short, S., Short, M. L., and Jungmann, R. A. (1998) J. Biol. Chem. 273, 24861–24866) have demonstrated the existence of a cAMP-stabilizing region in LDH-A 3′-UTR. Sequence analysis of PCSR identified a 13-nucleotide AU-rich region that is common to both cAMP-stabilizing region and PCSR. These studies identify a specific PKC-responsive stabilizing element and indicate that interaction of PKA and PKC results in a potentiating effect on LDH-A mRNA stabilization. lactate dehydrogenase untranslated region cAMP-stabilizing region protein kinase C-stabilizing region nucleotide(s) adenosine 3′, 5′ cyclic monophosphorothioate 12–0-tetradecanoylphorbol-13-acetate dioctanoylglycerol protein kinase A protein kinase C 3-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(indol-3-yl)-maleimide glyceraldehyde-3-phosphate dehydrogenase During recent years we have established evidence for a bimodal mechanism of LDH-A1regulation involving transcriptional as well as post-transcriptional modulation of LDH-A expression (1.Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 2.Huang D. Jungmann R.A. Mol. Cell. Endocrinol. 1995; 108: 87-94Crossref PubMed Scopus (33) Google Scholar, 3.Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar, 4.Tian D. Huang D. Brown R.C. Jungmann R.A. J. Biol. Chem. 1998; 273: 28454-28460Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 6.Short 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 (42) Google Scholar). Both mechanisms are regulated by a number of agonists of intracellular signaling pathways. For instance, agonists of PKA and PKC can modify the metabolic functions of cells by inducing an altered program of LDH-A mRNA regulation. These mechanisms allow the cell to respond rapidly to changes in the physiologic environment of the cell and to cover a potential energy deficit through conversion of pyruvate to lactate. Clues to mechanisms underlying this dual mode of control were provided by the identification of cis-acting promoter elements instrumental in PKA- and PKC-mediated transcriptional regulation (2.Huang D. Jungmann R.A. Mol. Cell. Endocrinol. 1995; 108: 87-94Crossref PubMed Scopus (33) Google Scholar, 6.Short 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 (42) Google Scholar). However, less information is available about the mechanism of protein kinase-mediated LDH-A mRNA stability regulation resulting in higher levels of intracellular LDH-A mRNA (3.Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar). Attention has recently focused on the modulation of mRNA stability in response to a variety of physiological signals. For instance, it is known that activators of PKA and PKC are important effectors of mRNA stability regulation in a number of gene systems (1.Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 3.Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar, 5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar,7.Ross J. Microbiol. Rev. 1995; 59: 423-450Crossref PubMed Google Scholar, 8.Peng H. Lever J.E. J. Biol. Chem. 1995; 270: 23996-24003Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 9.Peng H. Lever J.E. J. Biol. Chem. 1995; 270: 20536-20542Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 10.Ahern S.M. Miyata T. Sadler J.E. J. Biol. Chem. 1993; 268: 2154-2159Abstract Full Text PDF PubMed Google Scholar, 11.Levine R.A. McCormack J.E. Buckler A. Sonesheim G.E. Mol. Cell. Biol. 1986; 6: 4112-4116Crossref PubMed Scopus (47) Google Scholar, 12.Mitchell R.L. Zokas L. Schreiber R.D. Verma I.M. Cell. 1985; 40: 209-217Abstract Full Text PDF PubMed Scopus (258) Google Scholar, 13.Chen M. Schnermann J. Smart A.M. Brosius F.C. Killen P.D. Briggs J.P. J. Biol. Chem. 1993; 268: 24138-24144Abstract Full Text PDF PubMed Google Scholar, 14.Hod Y. Hanson R.W. J. Biol. Chem. 1988; 263: 7747-7752Abstract Full Text PDF PubMed Google Scholar). Our own studies identified a synergistic interaction between PKA and PKC in regulating the stability of LDH-A mRNA (3.Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar). Whereas the molecular basis for the synergistic effect remains unknown, we have recently identified a cAMP-stabilizing region (CSR) within the 3′-UTR of LDH-A mRNA (5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) that in combination with specific CSR-binding proteins (4.Tian D. Huang D. Brown R.C. Jungmann R.A. J. Biol. Chem. 1998; 273: 28454-28460Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) is required to achieve LDH-A mRNA stabilization in response to PKA activation. The binding activity of the proteins to the CSR and the effect on LDH-A mRNA stabilization are regulated through a phosphorylation/dephosphorylation mechanism by PKA and as yet unknown protein phosphatases. Thus, it is now clear that the CSR, in concert with CSR-binding proteins, is absolutely required to achieve increased LDH-A mRNA stability in response to PKA activation. However, the molecular mechanism of mRNA stabilization by PKC is unknown. The present paper describes the identification of acis-regulatory element within the 3′-UTR of LDH-A mRNA that is required for PKC-mediated mRNA stability regulation. The identification was accomplished using a strategy previously developed for the identification of the cAMP-stabilizing region (5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Using deletion, mutation, and replacement analysis, we constructed chimeric β-globin/ldh 3′-UTR minigenes that were stably transfected and expressed in rat C6 glioma cells. Applying ribonuclease protection assays, we studied the effects of PKC activation on the rate of decay of chimeric β-globin/ldh 3′-UTR mRNAs. Using this methodology, we demonstrated the presence of an uridine-rich region within the LDH-A 3′-UTR, named PKC-stabilizing region (PCSR), capable of stabilizing LDH-A mRNA in response PKC activation. It is of particular interest that the sequences of CSR and PCSR overlap and possess a common 13-nucleotide region. Nucleic acid-modifying enzymes, acrylamide, and nucleoside triphosphates were from Roche Molecular Biochemicals. Radioisotopes were purchased from NEN Life Science Products. Other reagents were of molecular biology grade and purchased from Sigma. Cell culture products were purchased from Life Technologies, Inc. Synthesis and processing of synthetic DNA oligonucleotides and their ligation into respective plasmid vectors were performed as described before (5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Rat C6 glioma cells (American Type Culture Collection CCL 107) were maintained as monolayers in Ham's F-10 nutrient medium supplemented with 10% dialyzed fetal calf serum, 50 units/ml of penicillin, and 50 mg of streptomycin as described by us (5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The LDH-A 3′-UTR was derived from plasmid pLDH-2 (kindly provided by Dr. Richard Breathnach) containing a full-length rat fibroblast LDH-A cDNA insert. The mRNA consists of a 103-nucleotide 5′-untranslated region and a 510-nucleotide 3′-untranslated region (corresponding to nucleotides 1103–1610) (50.Matrisian L.M. Rautmann G. Magun B.E. Breathnach R. Nucleic Acids Res. 1985; 13: 711-726Crossref PubMed Scopus (102) Google Scholar). The 3′-UTR contains the classic polyadenylation signal AAUAAA 18 nucleotides before the poly(A) sequence. AHinfI/BamHI fragment containing the entire LDH-A 3′-UTR (with 28-base pair 5′ coding sequence and 100-base pair pLDH-2 vector sequence) was inserted into pGEM3Zf(−) at the BamHI site resulting in plasmid pLDH-5. To eliminate the 28-base pairldh-a coding and 100-base pair pLDH-2 vector sequences contained in pLDH-5, the complete 510-base pair 3′-UTR of LDH-A with 5′BamHI and 3′ HindIII sites was amplified by polymerase chain reaction. The fragment was cloned into theBamHI-HindIII sites of pBluescript II KS+ (Stratagene) resulting in pLDH-6 from which the various 3′-UTR fragments were prepared. The rabbit β-globin expression vector pRc/FBB (see Fig. 1) was constructed as described previously (5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) by Dr. D. Chagnovich (Northwestern University) in two steps from plasmids pRc/CMS (Invitrogen) and pBBB (kindly provided by Dr. M. E. Greenberg) (15.Shyu A.-B. Greenberg M.E. Belasco J.G. Genes Dev. 1989; 3: 60-72Crossref PubMed Scopus (452) Google Scholar). Plasmid pRc/FBB encodes a transcription unit consisting of β-globin coding region flanked by the β-globin 5′- and 3′-untranslated regions fused to the c-fos promoter. To construct the chimeric globin/ldh expression vectors, various LDH-A 3′-UTR fragments were inserted into the BglII site or, alternatively, they replaced the BglII/Hind III fragment (β-globin 3′-UTR) of pRc/FBB. The fragments were constructed as follows. To construct the pRc/FBB expression vector containing the full-length LDH-A 3′-UTR, the 510-base pair 3′-UTR was polymerase chain reaction-amplified from pLDH-6 using 5′- and 3′-oligonucleotide primers with BglII and HindIII restriction sites, respectively. The polymerase chain reaction product as well as pRc/FBB were digested with BglII and HindIII and ligated to generate expression vector pRc/FBB/LDH. In this vector, the β-globin 3′-UTR had been deleted from pRc/FBB and replaced with the LDH-A 3′-UTR. Truncated 3′-UTR fragments were similarly generated using the appropriate oligonucleotide primers. Fragments for insertion into the BglII site of pRc/FBB were blunt-end ligated, and fragments replacing the globin 3′-UTR contained BglII andHindIII ends. LDH-A 3′-UTR sequences with base deletions were generated by splicing upstream and downstream polymerase chain reaction fragments that lacked the appropriate base sequence as indicated in Fig. 3. The sequence and correct orientation of all inserts were confirmed by restriction and DNA sequence analyses. Sequencing was carried out in both directions by the dideoxynucleotide chain terminator method with specific synthetic oligonucleotides as primers. These experimental methods were described in detail in a previous publication (5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). All experiments were carried out at about 90% confluence. Serum was withdrawn 24–28 h prior to addition of fetal bovine serum (final concentration, 15%) together with various agents at concentrations indicated in the text. One hour after the addition (taken as 0 h time point), RNA was isolated at subsequent time points up to 12 h and analyzed. These methods were previously described in detail (5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Messenger RNA decay was assessed by ribonuclease protection assay. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control, because GAPDH mRNA was stable under the conditions of the experiments regardless of the absence or presence of effector agents. Nuclear run-off experiments were carried out as described by us (1.Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 3.Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar) using a LDH-A 3′-UTR cDNA inserted in Bluescript II KS+ as hybridization probe. Previous studies in our laboratory demonstrated that intracellular steady-state levels of LDH-A mRNA are regulated, in part, through modulation of mRNA stability. For instance, after treatment of rat glioma cells with activators of PKA or PKC, a dramatic but transient increase of LDH-A mRNA levels takes place (1.Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 3.Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar). The induced mRNA exhibits a markedly increased half-life as compared with the relatively short half-life of LDH-A mRNA in noninduced cells. This indicates that the LDH-A transcript in noninduced cells is targeted for rapid degradation through processes that can be modulated by effector agents capable of activating the PKA or PKC signal transduction pathways. To investigate the molecular basis of the protein kinase-stabilizing effect, initial studies were carried out to determine whether or not the decay of wild-type LDH-A mRNA and chimeric globin/ldh mRNA followed similar patterns, justifying the use of chimeric vectors for subsequent stability studies. By choosing an expression vector (pRc/FBB) with a serum-induciblec-fos promoter (15.Shyu A.-B. Greenberg M.E. Belasco J.G. Genes Dev. 1989; 3: 60-72Crossref PubMed Scopus (452) Google Scholar, 16.Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3124) Google Scholar, 17.Greenberg M.E. Ziff E.B. Nature. 1984; 311: 433-438Crossref PubMed Scopus (2009) Google Scholar), we also avoided artifacts that potentially occur when commonly used transcriptional inhibitors (18.Harrold S. Genovese C. Kobrin B. Morrison S.L. Milcarek C. Anal. Biochem. 1991; 198: 19-29Crossref PubMed Scopus (104) Google Scholar,19.Scott W.A. Tomkins G.M. Methods Enzymol. 1975; 40: 273-293Crossref PubMed Scopus (22) Google Scholar) are used to stop ongoing transcription. We modified pRc/FBB, which is under the control of a serum-inducible c-fos promoter, by replacing the BglII/HindIII fragment (Fig.1), containing the globin 3′-UTR, with the entire LDH-A 3′-UTR. The resulting chimeric minigene (pRc/FBB/LDH) was stably transfected into rat C6 glioma cells. After serum deprivation of cells for 25–30 h, the c-fos promoter was pulse-induced with fetal calf serum resulting in a brief pulse of transcription of chimeric β-globin/ldh mRNA. Nuclear run-off assays indicated a rapid induction of nuclear chimeric globin/ldh transcripts at 15 min (TableI). After 1 h the level of transcription had already decreased to levels seen before serum stimulation. Similar transient kinetics were observed without or with added DG or (S p)-cAMPS. Because nuclear transcription had essentially ceased after 1 h, RNA was subsequently isolated at various time points, and the rate of decay and the half-life of chimeric β-globin/ldh mRNA were determined using quantitative ribonuclease protection assays. As expected from our previous studies (5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), wild-type β-globin mRNA was remarkably stable (Fig. 2 A, wt Globin) and decayed with a half-life of about 21 h (extrapolated from Fig. 2 B). In marked contrast, chimeric β-globin/ldh mRNA in unstimulated cells (Fig.2 A, Control) decayed at a much faster rate (t 12 ≈ 65 min) (Fig. 2 B) similar to the half-life of wild-type LDH-A mRNA rate (t 12≈ 55 min) in glioma cells (1.Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar, 3.Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar).Table INuclear run-off analysis of chimeric globin/ldh mRNA transcription rates after serum stimulation of transfected rat C6 glioma cellsTreatmentRate of chimeric globin/ldh mRNA synthesisaRate of mRNA synthesis (expressed in parts per million) = [(cpm globin/ldh mRNA − cpm pBluescript)/cpm total 32P-RNA input] × 100/efficiency) × 1710/510. 1710 is the length of the chimeric globin/ldhmRNA, and 510 is the length of the LDH-A 3′-UTR in nucleotides.ppmNone 0 min448 ± 65 15 min1077 ± 91 30 min1166 ± 88 45 min667 ± 48 60 min456 ± 53DG (200 nm) 0 min677 ± 88 15 min1277 ± 94 30 min1987 ± 123 45 min1132 ± 88 60 min510 ± 69(Sp)-cAMPS (100 μm) 0 min645 ± 58 15 min1524 ± 132 30 min1166 ± 210 45 min955 ± 76 60 min480 ± 41C6 glioma cells were stably transfected with pRc/FBB/LDH. Serum was withdrawn for 24–28 h, after which serum and effector agents were added. At the indicated times after addition, nuclei were isolated and allowed to incorporate [α-32P]UTP. RNA was isolated, purified, and hybridized to 2-aminophenylthioether filters carrying immobilized globin cDNA in Bluescript or wild-type pBluescript (1.Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar). Hybridized radioactivity was eluted from the filters and determined by liquid scintillation counting. Nonspecific hybridization to wild-type pBluescript filters was subtracted from the counts/min hybridized to globin filters. RNA synthesis results are given as the means ± S.E. determined from four separate experiments.a Rate of mRNA synthesis (expressed in parts per million) = [(cpm globin/ldh mRNA − cpm pBluescript)/cpm total 32P-RNA input] × 100/efficiency) × 1710/510. 1710 is the length of the chimeric globin/ldhmRNA, and 510 is the length of the LDH-A 3′-UTR in nucleotides. Open table in a new tab C6 glioma cells were stably transfected with pRc/FBB/LDH. Serum was withdrawn for 24–28 h, after which serum and effector agents were added. At the indicated times after addition, nuclei were isolated and allowed to incorporate [α-32P]UTP. RNA was isolated, purified, and hybridized to 2-aminophenylthioether filters carrying immobilized globin cDNA in Bluescript or wild-type pBluescript (1.Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar). Hybridized radioactivity was eluted from the filters and determined by liquid scintillation counting. Nonspecific hybridization to wild-type pBluescript filters was subtracted from the counts/min hybridized to globin filters. RNA synthesis results are given as the means ± S.E. determined from four separate experiments. To examine the effect of protein kinase activation on the rate of decay of β-globin/ldh mRNA, we used theS p-isomeric form of adenosine 3′, 5′ cyclic monophosphorothioate ((S p)-cAMPS), a potent activator of PKA, and DG, a membrane-permeable diacylglycerol analog that activates PKC (20.Berridge M.J. Annu. Rev. Biochem. 1987; 56: 159-193Crossref PubMed Scopus (2455) Google Scholar) and mimicks the effect of endogenous diacylglycerol on PKC (21.Boynton A.L. Whitfield J.F. Kleine L.P. Biochem. Biophys. Res. Commun. 1983; 115: 383-386Crossref PubMed Scopus (22) Google Scholar, 22.Lapetina E.G. Reep B. Ganon B.R. Bell R.M. J. Biol. Chem. 1985; 260: 1358-1361Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 2 A, DG as well as (S p)-cAMPS achieved a marked stabilization of chimeric β-globin/ldh mRNA (Fig.2 A, compare Control with DG and(S p )-cAMPS). Activation of PKC increased the half-life of globin/ldh mRNA from about 65 min in untreated to 4.2 h in TPA- and 3.9 h in DG-treated cells (Fig. 2 B). Activation of PKA with (S p)-cAMPS achieved an approximate 7-fold increase of the half-life of β-globin/ldh mRNA from 65 min to 8 h (Fig. 2 B). In view of our previous identification of a synergistic action of PKA and PKC on LDH-A mRNA stability (3.Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar), we examined whether such an effect also occurred when the chimeric β-globin/ldh 3′-UTR minigene was the transcribed template. Our data show that synergism was, indeed, demonstrable when a combination of DG + (S p)-cAMPS was used as activators of the protein kinase pathways (Fig. 2 A, DG + (S p )-cAMPS). The half-life of β-globin/ldh mRNA increased 18-fold to a half-life of about 21 h (extrapolated from Fig. 2 B). We conclude from the data that the stability of β-globin/ldh mRNA in glioma cells is similar or identical to wild-type LDH-A mRNA. Furthermore, the decay patterns of chimeric β-globin/ldhmRNA in protein kinase-activated cells appear to follow mechanisms identical to wild-type LDH-A mRNA. To further test the pivotal role of protein kinase activation in regulation of β-globin/ldh mRNA stability, we used inhibitors that prevent activation of the respective protein kinase pathway. Exposure of transfected cells to various activators and inhibitors of PKA and PKC markedly modified the half-life of β-globin/ldh mRNA. As shown in TableII, the phorbol ester TPA, which binds to and activates PKC irreversibly (23.Nishizuka Y. Nature. 1984; 308: 693-698Crossref PubMed Scopus (5764) Google Scholar), caused an approximate 4-fold increase of β-globin/ldh mRNA half-life. Because TPA may possibly achieve this effect through mechanisms other than activation of PKC (24.Nishizuka Y. Science. 1986; 233: 305-312Crossref PubMed Scopus (4038) Google Scholar), we chose DG as activator. DG is a synthetic cell membrane-permeable analog of diacylglycerol, the endogenous activator of PKC, and mimicks the effect of endogenous diacylglycerol (21.Boynton A.L. Whitfield J.F. Kleine L.P. Biochem. Biophys. Res. Commun. 1983; 115: 383-386Crossref PubMed Scopus (22) Google Scholar, 22.Lapetina E.G. Reep B. Ganon B.R. Bell R.M. J. Biol. Chem. 1985; 260: 1358-1361Abstract Full Text PDF PubMed Google Scholar). Indeed, DG treatment of transfected glioma cells increased the half-life of β-globin/ldh mRNA about 4-fold. The α-isomeric form of phorbol 12β,13α-didecanoate, which is unable to activate PKC (25.Kreibich G. Hecker E. Z. Krebsforsch. 1970; 74: 448-456Crossref PubMed Scopus (48) Google Scholar, 26.Akiguchi I. Izumi M. Nagataki S. J. Endocrinol. 1993; 138: 379-389Crossref PubMed Scopus (12) Google Scholar), lacked the stabilizing effect of TPA on globin/ldh mRNA, strongly suggesting that TPA exerts its effect through activation of PKC. Furthermore, to prevent activation of PKC, we used the specific PKC inhibitor bisindolylmaleimide GF 109203X (BIM) (27.Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar). The use of BIM alone did not change the half-life of globin/ldh mRNA. In combination with DG or TPA, BIM prevented stabilization of the mRNA. We have already previously shown that the use of (R p)-cAMPS, which prevents activation of PKA, similarly failed to cause stabilization of chimeric globin/ldh mRNA (3.Huang D. Hubbard C.J. Jungmann R.A. Mol. Endocrinol. 1995; 9: 994-1004PubMed Google Scholar, 5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar).Table IIEffect of activators and inhibitors of protein kinases A and C on stability regulation of chimeric globin/ldh mRNATreatmentHalf-life (t 12)minNone69 ± 4TPA (100 nm)252 ± 15PD (100 nm)73 ± 6BIM (10−11m)75 ± 8TPA + BIM72 ± 5DG (200 nm)234 ± 30DG + BIM58 ± 3.5(S p)-cAMPS (100 μm)522 ± 75(R p)-cAMPS (100 μm)78 ± 4C6 glioma cells were stably transfected with pRc/FBB in which the globin 3′-UTR was replaced with the entire LDH 3′-UTR fragment. Experiments were carried out at about 90% confluence. Serum was withdrawn for 24–28 h, after which serum and effector agents were added. One hour after the addition (taken as 0 h time point), RNA was isolated at various time points up to 12 h. For further details see "Experimental Procedures." Results are expressed as the means and S.E. of four separate experiments. PD, phorbol 12β-13α-didecanoate. Open table in a new tab C6 glioma cells were stably transfected with pRc/FBB in which the globin 3′-UTR was replaced with the entire LDH 3′-UTR fragment. Experiments were carried out at about 90% confluence. Serum was withdrawn for 24–28 h, after which serum and effector agents were added. One hour after the addition (taken as 0 h time point), RNA was isolated at various time points up to 12 h. For further details see "Experimental Procedures." Results are expressed as the means and S.E. of four separate experiments. PD, phorbol 12β-13α-didecanoate. LDH-A mRNA is characterized by a moderately short half-life of approximately 55 min (1.Jungmann R.A. Kelley D.C. Miles M.F. Milkowski D.M. J. Biol. Chem. 1983; 258: 5312-5318Abstract Full Text PDF PubMed Google Scholar). Sequence analysis of its 3′-UTR identifies a 99-nucleotide domain (nt 1450–1549) that is relatively AU-rich when compared with the overall nucleotide composition of the 3′-UTR. Recently, we have shown that the 3′-UTR imparts a relatively short half-life to LDH-A mRNA because of the presence of three determinants of instability (5.Tian D. Huang D. Short S. Short M.L. Jungmann R.A. J. Biol. Chem. 1998; 273: 24861-24866Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Although two of the instability regions are not regulated, one of them, the 22-base 3′-UTR region comprised of nt 1478–1499, is subject to regulation by the PKA signal pathway. Its presence is an absolute requirement for cAMP-mediated stabilization of LDH-A mRNA. Using an experimentally similar approach, we now identified putative region(s) within the LDH-A 3′-UTR that are responsible for PKC-mediated stabilization of LDH-A mRNA. Our strategy consisted of the synthesis of two types of chimeric globin/ldh 3′-UTR vectors. First, we constructed a series of globin/ldh 3′-UTR vectors that contained (a) systematically truncated wild-type 3′-UTR fragments; (b) mutated 3′-UTR fragments; and (c) 3′-UTRs from which we had deleted short base regions. The deleted regions were of approximately similar size to prevent artifactual effects because of drastic variations in mRNA size. The fragments were inserted into the unique BglII site of pRc/FBB located at the junction of the β-globin translated and 3′-untranslated regions (Fig. 1) (28.van Ooyen A. van den Berg J. Mantei N. Weissmann C. Science. 1979; 206: 337-344Crossref PubMed Scopus (162) Google Scholar). In the second approach, the entire globin 3′-UTR was deleted by restriction at the BglII/HindIII sites (Fig. 1) and replaced with wild-type and various mutated LDH-A 3′-UTR fragments. Upon stable transfection and transcription of the appropriate vectors, unique chimeric globin/ldh mRNAs were produced whose stability was assayed by ribonuclease protection assay. Because in each vector the promoter (c-fos) and globin translation reg

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