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• W2124921234 abstract "The balance of lipid flux in adipocytes is controlled by the opposing actions of lipolysis and lipogenesis, which are controlled primarily by hormone-sensitive lipase and lipoprotein lipase (LPL), respectively. Catecholamines stimulate adipocyte lipolysis through reversible phosphorylation of hormone-sensitive lipase, and simultaneously inhibit LPL activity. However, LPL regulation is complex and previous studies have described translational regulation of LPL in response to catecholamines because of an RNA-binding protein that interacts with the 3′-untranslated region of LPL mRNA. In this study, we identified several protein components of an LPL RNA binding complex. Using an LPL RNA affinity column, we identified two of the RNA-binding proteins as the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), and A kinase anchoring protein (AKAP) 121/149, one of the PKA anchoring proteins, which has known RNA binding activity. To determine whether the C subunit was involved in LPL translation inhibition, the C subunit was depleted from the cytoplasmic extract of epinephrine-stimulated adipocytes by immunoprecipitation. This resulted in the loss of LPL translation inhibition activity of the extract, along with decreased RNA binding activity in a gel shift assay. To demonstrate the importance of the AKAPs, inhibition of PKA-AKAP binding with a peptide competitor (HT31) prevented epinephrine-mediated inhibition of LPL translation. C subunit kinase activity was necessary for LPL RNA binding and translation inhibition, suggesting that the phosphorylation of AKAP121/149 or other proteins was an important part of RNA binding complex formation. The hormonal activation of PKA results in the reversible phosphorylation of hormone-sensitive lipase, which is the primary mediator of adipocyte lipolysis. These studies demonstrate a dual role for PKA to simultaneously inhibit LPL-mediated lipogenesis through inhibition of LPL translation. The balance of lipid flux in adipocytes is controlled by the opposing actions of lipolysis and lipogenesis, which are controlled primarily by hormone-sensitive lipase and lipoprotein lipase (LPL), respectively. Catecholamines stimulate adipocyte lipolysis through reversible phosphorylation of hormone-sensitive lipase, and simultaneously inhibit LPL activity. However, LPL regulation is complex and previous studies have described translational regulation of LPL in response to catecholamines because of an RNA-binding protein that interacts with the 3′-untranslated region of LPL mRNA. In this study, we identified several protein components of an LPL RNA binding complex. Using an LPL RNA affinity column, we identified two of the RNA-binding proteins as the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), and A kinase anchoring protein (AKAP) 121/149, one of the PKA anchoring proteins, which has known RNA binding activity. To determine whether the C subunit was involved in LPL translation inhibition, the C subunit was depleted from the cytoplasmic extract of epinephrine-stimulated adipocytes by immunoprecipitation. This resulted in the loss of LPL translation inhibition activity of the extract, along with decreased RNA binding activity in a gel shift assay. To demonstrate the importance of the AKAPs, inhibition of PKA-AKAP binding with a peptide competitor (HT31) prevented epinephrine-mediated inhibition of LPL translation. C subunit kinase activity was necessary for LPL RNA binding and translation inhibition, suggesting that the phosphorylation of AKAP121/149 or other proteins was an important part of RNA binding complex formation. The hormonal activation of PKA results in the reversible phosphorylation of hormone-sensitive lipase, which is the primary mediator of adipocyte lipolysis. These studies demonstrate a dual role for PKA to simultaneously inhibit LPL-mediated lipogenesis through inhibition of LPL translation. lipoprotein lipase protein kinase A hormone-sensitive lipase untranslated region A kinase anchoring protein adrenocorticotropic hormone Lipoprotein lipase (LPL)1 is a central enzyme in lipid metabolism and hydrolyzes the core of triglyceride-rich plasma lipoproteins into nonesterified fatty acids and monoacylglycerol (1Goldberg I.J. J. Lipid Res. 1996; 37: 693-707Abstract Full Text PDF PubMed Google Scholar). In adipose tissue and muscle, LPL is localized to the capillary endothelium, and contributes to the rapid removal of triglyceride-rich lipoproteins and their remnants. Catecholamines are of considerable physiologic importance in the mobilization of adipose tissue lipid in response to fasting and exercise. Hormones that cause elevated cAMP (β-adrenergic agonists, ACTH, and glucagon) result in the activation of cAMP-dependent protein kinase A (PKA), which then activates hormone-sensitive lipase (HSL) (2Stralfors P. Bjorgell P. Belfrage P. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3317-3321Crossref PubMed Scopus (198) Google Scholar, 3Holm C. Langin D. Manganiello V. Belfrage P. Degerman E. Methods Enzymol. 1997; 286: 45-67Crossref PubMed Scopus (51) Google Scholar). HSL is the primary mediator of adipocyte lipolysis (4Osuga J. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoiri F. Yahagi N. Kraemer F.B. Tsutsumi O. Yamada N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 787-792Crossref PubMed Scopus (502) Google Scholar), and the release of nonesterified fatty acids from adipocytes play a central role in obesity and insulin resistance (5Kelley D.E. Simoneau J.A. J. Clin. Invest. 1994; 94: 2349-2356Crossref PubMed Scopus (409) Google Scholar, 6Unger R.H. Diabetes. 1995; 44: 863-870Crossref PubMed Google Scholar). On the other hand, LPL hydrolyzes lipoproteins at the capillary endothelium generating nonesterified fatty acids for triglyceride storage. LPL and HSL serve opposing functions in adipose tissue, and they respond in an opposite fashion in response to hormonal regulation. In adipocytes, insulin and the fed state result in an increase in LPL activity along with a decrease in HSL activity, whereas hormones that are elevated during the fasting state, such as epinephrine and glucagon, inhibit LPL activity and stimulate HSL-mediated lipolysis (7Eckel R.H. Borensztajn J. Lipoprotein Lipase. Evener, Chicago1987: 79-132Google Scholar, 8Londos C. Brasaemle D.L. Schultz C.J. Adler-Wailes D.C. Levin D.M. Kimmel A.R. Rondinone C.M. Ann. N. Y. Acad. Sci. 1999; 892: 155-168Crossref PubMed Scopus (220) Google Scholar, 9Ramsay T.G. Endocrinol. Metab. Clin. North Am. 1996; 25: 847-870Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Although the decrease in LPL activity by catecholamines has been described previously (7Eckel R.H. Borensztajn J. Lipoprotein Lipase. Evener, Chicago1987: 79-132Google Scholar), the cellular mechanisms controlling LPL inhibition are complex. In rat adipocytes, we found that the LPL synthetic rate was inhibited more than 5-fold within 30 min of addition of epinephrine to the medium, with no change in LPL mRNA levels (10Ong J.M. Saffari B. Simsolo R.B. Kern P.A. Mol. Endocrinol. 1992; 6: 61-69Crossref PubMed Scopus (35) Google Scholar). Studies of 3T3 adipocytes demonstrated that the inhibition of LPL translation by epinephrine involved an RNA-binding protein that interacted with the proximal 3′-untranslated region (UTR) of the LPL mRNA (11Yukht A. Davis R.C. Ong J.M. Ranganathan G. Kern P.A. J. Clin. Invest. 1995; 96: 2438-2444Crossref PubMed Scopus (35) Google Scholar). Subsequent studies found the first 24 nucleotides of the LPL 3′-UTR essential for translational regulation, and a 30-kDa RNA-binding protein was identified by cross-linking as an important component of LPL translational regulation (12Ranganathan G., Vu, D. Kern P.A. J. Biol. Chem. 1997; 272: 2515-2519Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This study was intended to identify the components involved in the translational regulation of LPL following cAMP elevation. As described below, we have identified the catalytic (C) subunit of PKA as the important 30-kDa protein involved in LPL translational regulation. However, the C subunit of PKA is likely part of an RNA binding complex, which also involves A kinase anchoring protein (AKAP) 121/149, which is involved in binding the PKA holoenzyme, and which contains a known RNA binding domain (13Feliciello A. Gottesman M.E. Avvedimento E.V. J. Mol. Biol. 2001; 308: 99-114Crossref PubMed Scopus (240) Google Scholar). Approximately 100 T-75 flasks of 3T3-F442A adipocytes, representing ∼109cells, were induced to differentiate with insulin, and after 8 days of differentiation they were treated with epinephrine (10−5m). LPL translation was inhibited in these cells, and a cytoplasmic extract was prepared as described previously (11Yukht A. Davis R.C. Ong J.M. Ranganathan G. Kern P.A. J. Clin. Invest. 1995; 96: 2438-2444Crossref PubMed Scopus (35) Google Scholar). In brief, adipocytes were scraped from the plate and the cell pellet was resuspended in 10× the pellet volume of lysis buffer (50 mm Tris-HCl, pH 7.4, 250 mm sucrose, 35 mm KCl, 10 mm MgCl2, 0.5 mm EDTA, 7 mm β-mercaptoethanol), and homogenized with 10 strokes of a glass homogenizer. Homogenates were centrifuged at 10,000 × g for 15 min at 4 °C. The postnuclear extract was used to prepare a high speed supernatant fraction (S-100) by centrifugation at 100,000 × g for 2 h at 4 °C. Solid ammonium sulfate was added to the cytosolic fraction to 60% saturation and precipitated for ½ h on ice. Precipitated proteins were collected by centrifugation at 6,000 ×g for 10 min at 0 °C, redissolved and dialyzed against Buffer A (20 mm Tris-HCl, pH 7.4, 20 mm KCl, 7 mm β-mercaptoethanol, 0.1 mm EDTA, and 2 mm phenylmethylsulfonyl fluoride). The sample was then diluted to 25 ml and passed through a DEAE-cellulose column equilibrated in 20 mm Tris-HCl, pH 7.4. After washing the column with the same buffer, proteins were eluted with 5 ml of buffer containing 400 mm KCl. The DEAE fraction that passed unbound through the column demonstrated LPL RNA binding properties, as demonstrated by a gel shift experiment, whereas the 400 mmKCl eluted fraction had no RNA binding activity. Therefore, the flow-through was dialyzed against the initial column buffer, and fractionated on a LPL 3′ UTR-oligo(dT)-Sepharose column. To prepare the RNA binding column, poly(A) RNA transcripts were generated containing the C-terminal 50 nucleotides of coding sequence, and the first 100 nucleotides of the LPL 3′-UTR (nucleotides 1512 to 1635). A tracer amount of [32P]UTP was also added during transcription, to follow the binding and quality of the RNA. The poly(A) RNA was incubated with presoaked poly(T)-Sepharose beads for 60 min, and packed into a column. The column was washed with low salt buffer (20 mm Tris-HCl, pH 7.4, 20 mm KCl) to remove the unbound excess RNA. For the initial binding reaction, the epinephrine-treated 3T3-F442A adipocyte extracts were added in low salt (20 mm KCl) buffer containing heparin and yeast tRNA to prevent degradation and inhibit nonspecific binding. After washing extensively, bound proteins were eluted with a salt gradient varying from 0.1 to 0.5 mKCl in 20 mm Hepes, pH 7.5. Fractions were dialyzed against 40 mm KCl buffer, and analyzed by SDS-PAGE with colloidal blue staining (Novex), as described under “Results.” To obtain sequence information, pooled column fractions from the LPL 3′-UTR column were run on a preparatory 10% polyacrylamide gel. Parallel lanes were stained for identification, and a discrete band at 30 kDa was cut from a wet gel and sent to the Harvard Microchemistry facility (Cambridge, MA) for sequencing. The sequence analysis was performed on tryptic fragments by microcapillary reverse-phase high performance liquid chromatography nanoelectrospray tandem mass spectrometry on a Finnigan LCQ quadrupole ion trap mass spectrometer. In vitro translation of RNA transcripts was performed as described previously (11Yukht A. Davis R.C. Ong J.M. Ranganathan G. Kern P.A. J. Clin. Invest. 1995; 96: 2438-2444Crossref PubMed Scopus (35) Google Scholar). RNA transcripts were made from an LPL cDNA construct (LPL 35 of Wion et al. (14Wion K.L. Kirchgessner T.G. Lusis A.J. Schotz M.C. Lawn R.M. Science. 1987; 235: 1638-1641Crossref PubMed Scopus (350) Google Scholar)). Equal quantities of RNA transcripts (0.1 μg) were translated in a rabbit reticulocyte lysate system (Promega) in the presence of [35S]methionine, and the translation products were analyzed by SDS-PAGE and autoradiography. The intensity of the images was quantitated with the Eagle SightTM 3.0 image capture and analysis software (Stratagene 2, La Jolla, CA). We previously demonstrated that cell extracts from epinephrine-treated cells inhibited LPL translation in vitro (11Yukht A. Davis R.C. Ong J.M. Ranganathan G. Kern P.A. J. Clin. Invest. 1995; 96: 2438-2444Crossref PubMed Scopus (35) Google Scholar, 12Ranganathan G., Vu, D. Kern P.A. J. Biol. Chem. 1997; 272: 2515-2519Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The cell extracts (S-100 fractions) from control and epinephrine-treated adipocytes were prepared as described above followed by ammonium sulfate precipitation and dialysis against buffer A. Protein concentration in the cell extract was determined with a Bio-Rad protein assay, with bovine serum albumin as a standard. Equal quantities of the cell extract (0.1 μg) were used in the rabbit reticulocyte lysate reaction and the reaction was carried out for 60 min. To assess the role of PKA C subunit, a specific antibody to the Cα subunit (polyclonal antibody to the C terminus, Santa Cruz Biotechnology) was added to the cell extract 30 min prior to addition to the in vitro translation reaction. As a control, extracts were treated with antibodies to β-actin (Calbiochem). To assess the binding of the epinephrine cell extract to LPL RNA sequences, a [32P]RNA sequence corresponding to the proximal 3′-UTR of LPL (nucleotides 1512 to 1635) was synthesized. This 32P-labeled transcript (50,000 cpm) was incubated for 20 min in buffer A containing 10 μg/ml yeast tRNA, 10 units/ml heparin sulfate, along with 5 μg of cytoplasmic extract from control or epinephrine-treated 3T3-F442A adipocytes, and the products were analyzed on a 5% nondenaturing polyacrylamide gel. In some experiments, PKA C subunit was removed from the cell extract prior to incubation with the [32P]RNA. To eliminate PKA Cα from the extracts, 0.1 μg of anti-Cα antibody was incubated with the cell extract followed by 5 μl of 1:1 diluted protein A-agarose beads. The extract was centrifuged at 1500 ×g, and the Cα-depleted supernatant was then added to the32P-transcript and gel shift analysis was performed as described above. The effect of the anti-Cα antibodies was compared with irrelevant antibodies (β-actin). In additional experiments, PKA Cα subunit (0.5 Units, Calbiochem) was added back after immunoprecipitation of Cα. To inhibit PKA activity, cells were pretreated for 15 min with H89 (10 mm, Sigma), which is a specific inhibitor of the Cα ATP binding site (15Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka S. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar), followed by epinephrine treatment as described above. The gel shift was then performed as described above. RNA was extracted from adipocytes (16Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63130) Google Scholar), and equal amounts of total RNA were resolved by electrophoresis in 2.2 m formaldehyde, 1% agarose gels. Northern blots were probed using [32P]dCTP-labeled cDNA probes to AKAP149 and glyceraldehyde-3-phosphate dehydrogenase, which have been reported previously (17Furusawa M. Ohnishi T. Taira T. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2001; 276: 36647-36651Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 18Tso J.Y. Sun X.H. Kao T.H. Reece K.S. Wu R. Nucleic Acids Res. 1985; 13: 2485-2502Crossref PubMed Scopus (1759) Google Scholar). Antibodies to AKAP149 (Santa Cruz Biotechnology) were directed against the C-terminal region of the molecule, which is homologous with AKAP121, but not with other AKAPs (13Feliciello A. Gottesman M.E. Avvedimento E.V. J. Mol. Biol. 2001; 308: 99-114Crossref PubMed Scopus (240) Google Scholar, 19Chen Q. Lin R.Y. Rubin C.S. J. Biol. Chem. 1997; 272: 15247-15257Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Antiphosphoserine antibodies were obtained from Zymed Laboratories, San Francisco, CA. Western blotting was performed as described previously (20Ranganathan G. Kaakaji R. Kern P.A. J. Biol. Chem. 1999; 274: 9122-9127Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Samples containing 15 μg of total protein were fractionated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were treated with 20 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.2% Tween 20, and 5% nonfat dry milk overnight at 4 °C. Secondary antibodies were antispecies-specific peroxidase-labeled IgG (Sigma). Ligand blotting with [32P]PKA RII subunit was performed as described previously (21Hausken Z.E. Coghlan V.M. Scott J.D. Clegg R.A. Protein Targeting Protocols. Humana Press, Totowa, NJ1997: 47-64Google Scholar). To determine whether the PKA C subunit was associated with AKAP149/121, co-precipitation experiments were performed as described previously (22Xie G. Raufman J.P. Am. J. Physiol. 2001; 281: G1051-G1058Google Scholar). Epinephrine-treated cells were lysed in phosphate-buffered saline containing 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitors and immunoprecipitated with either anti-PKA Cα antibodies or anti-AKAP149/121 antibodies. These immunoprecipitated products were analyzed on SDS-PAGE, followed by ligand blotting with the [32P]PKA RII subunit, as described above. The LPL synthetic rate was measured in cultured 3T3-F442A cells as described previously (23Ong J.M. Kirchgessner T.G. Schotz M.C. Kern P.A. J. Biol. Chem. 1988; 263: 12933-12938Abstract Full Text PDF PubMed Google Scholar). Cells were incubated in methionine-free medium for 2 h prior to the addition of 50 μCi of [35S]methionine for 30 min. The cells were lysed and immunoprecipitated with anti-LPL antibodies (24Goers J.F. Petersen M.E. Kern P.A. Ong J. Schotz M.C. Anal. Biochem. 1987; 166: 27-35Crossref PubMed Scopus (59) Google Scholar), followed by analysis of the samples on a 10% polyacrylamide-SDS gel, followed by autoradiography. Within each experiment, an aliquot of cell lysate was precipitated with trichloroacetic acid and counted, and the amount of lysate taken for immunoprecipitation was adjusted to give equal trichloroacetic acid counts. To study the effects of AKAP-PKA disruption (25Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar), 10 μg of myristylated HT31 (Promega) was added to the cells for 15 min prior to epinephrine treatment. When adipocytes were treated with epinephrine, LPL translation was inhibited (12Ranganathan G., Vu, D. Kern P.A. J. Biol. Chem. 1997; 272: 2515-2519Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and the epinephrine-treated cell extract caused a gel shift when added to a [32P]RNA fragment corresponding to LPL mRNA nucleotides 1512 to 1635 (Fig.1 A). To purify the RNA-binding protein, our methodology was designed to take advantage of the affinity of the RNA-binding protein for the 3′-UTR of LPL. When the epinephrine-treated cell extract was applied to a DEAE column, as described under “Materials and Methods,” the RNA binding properties of the extract were predominantly found in the unbound fraction from the column. This material was then dialyzed against a low salt buffer (see “Materials and Methods”), and applied to a poly(U)-Sepharose column containing the relevant binding region of the 3′-UTR of the LPL mRNA. After washing the column extensively with the initial column buffer, increasing salt concentrations were applied, and the gradual elution of proteins was monitored by SDS-PAGE and colloidal blue staining. Fig. 1 shows the stained gel of the column elution fractions from the 3′-UTR column. With progressive salt elution, we observed the appearance of a predominant protein that migrated at 30–35 kDa. Other less prominent bands were also apparent, mostly at higher molecular weights. A cytoplasmic extract from control cells, which had been through the same column procedures, demonstrated essentially no proteins eluting from the RNA binding column except for a faint 30–35-kDa band (Fig. 1, lanes 5–8). In addition, no proteins were eluted when an epinephrine-treated cell extract was passed through a column containing irrelevant RNA (data not shown). Because of our previous identification of a cross-linked band at about 30–35 kDa (12Ranganathan G., Vu, D. Kern P.A. J. Biol. Chem. 1997; 272: 2515-2519Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), the prominent 35-kDa band was cut from the wet gels, and subjected to sequence analysis of proteolytic peptides from this band. The results demonstrated the presence of several proteolytic fragments belonging to the C subunit of PKA. Peptides to aldolase and cyclophilin were also identified inconsistently. Because these are abundant cellular proteins and do not fit a known mechanism for LPL regulation, further studies were not pursued. There were no unassigned peptides. As a result of the elution of PKA C subunit from the LPL RNA affinity column, we sought additional evidence that this subunit was involved in the inhibition of LPL translation. To further characterize this interaction, and to obtain direct evidence for PKA C binding to the LPL mRNA, we performed a gel shift assay. A 32P-labeled transcript corresponding to nucleotides 1512 to 1635 of the LPL mRNA was incubated with the control and epinephrine-treated cell extracts. As shown in Fig. 2, the cell extract from epinephrine-treated cells resulted in a gel shift (lane 2) when compared with the control extract (lane 1). To confirm the role of PKA C subunit, we added anti-PKA Cα antibody to the epinephrine-treated cell extract, followed by protein A-agarose, to immunoprecipitate the PKA Cα subunit. This PKA Cα-depleted extract was then added to the [32P]RNA transcript in a gel shift reaction. As shown in Fig. 2 (lane 3), there was a greatly reduced intensity of the shifted band, and the addition of less anti-PKA Cα antibody resulted in a greater intensity of the gel-shifted band (lane 4). The addition of irrelevant antibodies did not reduce the intensity of the shifted band (data not shown). To determine whether we could then restore RNA binding, we added active PKA Cα (0.5 units) back to the PKA Cα-depleted cell extract. Addition of Cα subunit after the immunoprecipitation restored and augmented the mobility gel shift (Fig. 2, lane 5). However, the addition of the active PKA Cα protein to the [32P]RNA, in the absence of cell extract, did not cause a gel shift (lane 6), suggesting that the PKA Cα subunit is part of a binding complex that involves other proteins. The above experiments demonstrate the involvement of PKA Cα in RNA binding, but do not necessarily imply inhibition of translation. As described previously, the cell extract from control adipocytes inhibited LPL translation in vitro when compared with the addition of no extract, and epinephrine-treated cell extract yielded a much greater inhibition of LPL translation (11Yukht A. Davis R.C. Ong J.M. Ranganathan G. Kern P.A. J. Clin. Invest. 1995; 96: 2438-2444Crossref PubMed Scopus (35) Google Scholar, 12Ranganathan G., Vu, D. Kern P.A. J. Biol. Chem. 1997; 272: 2515-2519Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). To determine whether the PKA Cα subunit is involved in translation inhibition, antibodies to PKA Cα were added to the in vitrotranslation reaction containing the LPL mRNA and cell extracts. As shown in Fig. 3, the cytoplasmic extract from epinephrine-treated cells inhibited LPL translation in vitro (lane 2). However, when the PKA Cα subunit was immunoprecipitated from the epinephrine-treated cell extract, the inhibition of LPL translation was abolished (lane 4). Indeed, the in vitro translation of LPL was increased in both the control and epinephrine-treated cell extracts after depletion of PKA Cα, which likely reflected the constitutive presence of the PKA Cα subunit. Addition of irrelevant antibody had no effect on the proportional change in LPL translation because of control and epinephrine extracts, although there was a small nonspecific decrease in translation in both lanes 5 and 6 (Fig. 3). Thus, these data suggested that PKA Cα subunit was involved in LPL translation inhibition. These data indicated that the Cα subunit was not by itself sufficient to cause a gel shift or inhibit translation, and suggested the presence of other proteins as part of a complex. PKA regulatory subunit was not detected by Western blotting of the column eluate from the epinephrine-treated cell extract (data not shown). Indeed, the R subunit was tightly bound to the DEAE column, and was not present in either the flow-through or the high salt wash. However, one class of proteins that is known to anchor PKA through the regulatory subunit is the AKAPs. To determine whether AKAPs were involved in the RNA binding complex, we used the32P-labeled PKA regulatory subunit to perform ligand blotting of the elute from the LPL RNA affinity column described in Fig. 1. As shown in Fig. 4 B(lane 1), a ligand blot of the proteins eluted off the column at 500 mm KCl demonstrated two bands: the expected band at 30 kDa, which represented the C subunit, and a band that migrated with a molecular mass between the markers at 116 and 205 kDa. AKAP121/149 is a member of the AKAP family of PKA-binding proteins that are notable for a consensus KH domain (26Lin R.Y. Moss S.B. Rubin C.S. J. Biol. Chem. 1995; 270: 27804-27811Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 27Trendelenburg G. Hummel M. Riecken E.O. Hanski C. Biochem. Biophys. Res. Commun. 1996; 225: 313-319Crossref PubMed Scopus (68) Google Scholar), which is found in many known RNA-binding proteins (28Ashley C.T.J. Wilkinson K.D. Reines D. Warren S.T. Science. 1996; 262: 563-566Crossref Scopus (616) Google Scholar). To determine whether the slower migrating band from the ligand blot was AKAP121/149, we performed Western blotting with specific antibodies to AKAP121/149. As shown in Fig. 4 A, the anti-AKAP121/149 antibodies identified the same protein that was identified in the ligand blot. In addition, the anti-AKAP antibodies did not detect any AKAP121/149 in the 3′-UTR column eluate from the control cell extract fraction, or from the lower salt eluate from the epinephrine-treated cells. A negative result was obtained when the blot was probed with antibodies to AKAP150, which has no C-terminal homology to AKAP121/149, but which has a similar migration (data not shown). Hence, AKAP121/149 and the PKA Cα subunit co-eluted from the 3′-UTR LPL mRNA column. The PKA kinase activity has numerous targets, and we performed Western blots with antiphosphoserine antibodies to determine whether any proteins from the 3′-UTR LPL mRNA column were phosphorylated. As shown in Fig.4 B, the same band identified as AKAP121/149 was also identified by the antiphosphoserine antibodies, suggesting that AKAP121/149 is phosphorylated. Antiphosphoserine antibodies identified no other proteins from the 3′-UTR column, and identified no proteins from the control cell extract column (data not shown). The Cα subunit was present, as described above, but was not detected with antiphosphoserine antibodies, suggesting that it became dephosphorylated during the purification. To determine whether AKAP is functionally involved with the inhibition of LPL translation, we used HT31 to inhibit cellular AKAP-PKA binding. HT31 is derived from the consensus peptide motif on AKAPs that bind to the R subunit of PKA (25Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar). LPL was immunoprecipitated from cells treated with myristylated HT31 with and without the presence of epinephrine, and labeled with [35S]methionine. As shown in Fig.5, epinephrine inhibited LPL synthesis, and this inhibition was disrupted by HT31. Indeed, the translation of LPL was up-regulated in both control and epinephrine-treated cells, suggesting that AKAPs are involved in a constitutive inhibition of LPL even in control cells. Hence, this experiment demonstrated a functional role of the AKAPs in LPL translation inhibition by epinephrine. To determine whether PKA Cα and AKAP149/121 were associated with each other as a complex, co-precipitation experiments were performed. Cell lysates from epinephrine-treated adipocytes were immunoprecipitated with either anti-Cα antibodies or anti-AKAP149/121 antibodies, followed by either Western blotting with the other antibody or ligand blotting with 32P-regulatory subunit. No association between AKAP121/149 and C subunit was detected using this method (data not shown). Thus, both PKA Cα and AKAP149/121 appeared to be binding to the LPL 3′-UTR, but were not associated with each other. The kinase activity of the PKA C subunit may be important in mediating LPL mRNA binding. To determine whether PKA Cα kinase activity was important, we treated cells with H89 (10 μm), which is a specific inhibitor of the Cα ATP binding site (15Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka S. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar). This treatment woul" @default.
• W2124921234 created "2016-06-24" @default.
• W2124921234 creator A5008096458 @default.
• W2124921234 creator A5009288566 @default.
• W2124921234 creator A5030591708 @default.
• W2124921234 creator A5047894137 @default.
• W2124921234 creator A5049866305 @default.
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• W2124921234 date "2002-11-01" @default.
• W2124921234 modified "2023-09-28" @default.
• W2124921234 title "The Translational Regulation of Lipoprotein Lipase by Epinephrine Involves an RNA Binding Complex Including the Catalytic Subunit of Protein Kinase A" @default.
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