FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2140699300> ?p ?o ?g. }

• W2140699300 endingPage "33540" @default.
• W2140699300 startingPage "33537" @default.
• W2140699300 abstract "The δ-isoform of Ca2+/calmodulin-activated protein kinase II (CaMK II) is abundantly expressed in vascular smooth muscle, but relatively little is known about its regulation or its potential cellular substrates. There are few, if any, known substrates of CaMK II that are physiologically relevant in vascular smooth muscle cells. Studies presented earlier (Mishra-Gorur, K., Singer, H. A., and Castellot, J. J., Jr. (2002) Am. J. Pathol., in press) by our laboratory show an inhibitory effect of heparin on CaMK II phosphorylation and activity. During these studies we observed the specific co-immunoprecipitation of a 20-kDa protein with CaMK II. Purification and sequence analysis indicate that this protein is the S18 protein of the 40 S ribosome. S18 was found to be abundantly phosphorylated in response to serum treatment, and this effect was strongly inhibited by heparin. In addition, KN-93, a specific CaMK II inhibitor, blocks S18 phosphorylation in vascular smooth muscle cells; a concomitant 24% reduction in protein synthesis was observed. Taken together these data support the idea that S18 could be a novel substrate for CaMK II, thus providing a potential link between Ca2+-mobilizing agents and protein translation. The δ-isoform of Ca2+/calmodulin-activated protein kinase II (CaMK II) is abundantly expressed in vascular smooth muscle, but relatively little is known about its regulation or its potential cellular substrates. There are few, if any, known substrates of CaMK II that are physiologically relevant in vascular smooth muscle cells. Studies presented earlier (Mishra-Gorur, K., Singer, H. A., and Castellot, J. J., Jr. (2002) Am. J. Pathol., in press) by our laboratory show an inhibitory effect of heparin on CaMK II phosphorylation and activity. During these studies we observed the specific co-immunoprecipitation of a 20-kDa protein with CaMK II. Purification and sequence analysis indicate that this protein is the S18 protein of the 40 S ribosome. S18 was found to be abundantly phosphorylated in response to serum treatment, and this effect was strongly inhibited by heparin. In addition, KN-93, a specific CaMK II inhibitor, blocks S18 phosphorylation in vascular smooth muscle cells; a concomitant 24% reduction in protein synthesis was observed. Taken together these data support the idea that S18 could be a novel substrate for CaMK II, thus providing a potential link between Ca2+-mobilizing agents and protein translation. Ca2+/calmodulin-activated protein kinase II vascular smooth muscle cell fetal calf serum 4-morpholinepropanesulfonic acid CaMK II1 is found in virtually all tissues examined and is thought to play a role in diverse cellular processes such as neurotransmitter release (1Sumi M. Kiuchi K. Ishikawa T. Ishii A. Hagiwara M. Nagatsu T. Hidaka H. Biochem. Biophys. Res. Commun. 1991; 181: 968-975Crossref PubMed Scopus (461) Google Scholar, 2Benfenati F. Valtorta F. Rubenstein J.L. Gorelick F.S. Greengard P. Czernik A.J. Nature. 1992; 359: 417-420Crossref PubMed Scopus (238) Google Scholar), vascular smooth muscle cell (VSMC) migration (3Pauly R.R. Bilato C. Sollott S.J. Monticone R. Kelly P.T. Lakatta E.G. Crow M.T. Circulation. 1995; 91: 1107-1115Crossref PubMed Scopus (102) Google Scholar), contraction (4Tansey M.G. Word R.A. Hidaka H. Singer H.A. Schworer C.M. Kamm K.E. Stull J.T. J. Biol. Chem. 1992; 267: 12511-12516Abstract Full Text PDF PubMed Google Scholar), and transcription of genes (5Wegner M. Cao Z. Rosenfeld M.G. Science. 1992; 256: 370-373Crossref PubMed Scopus (308) Google Scholar, 6Enslen H. Soderling T.R. J. Biol. Chem. 1994; 269: 20872-20877Abstract Full Text PDF PubMed Google Scholar). Recent evidence also suggests that CaMK II regulates the induction of VSMC contraction by regulating the phosphorylation of myosin light chain kinase. Caldesmon and calponin, two other proteins involved in contraction, are also postulated to be substrates for CaMK II. Both these proteins can inhibit actin-activated myosin ATPase; however, when phosphorylated by CaMK II they lose their ability to inhibit actomyosin ATPase (7Bronstein J.M. Farber D.B. Wasterlain C.G. Brain Res. Rev. 1993; 18: 135-147Crossref PubMed Scopus (63) Google Scholar). Despite the potentially important role of CaMK II in VSMC contraction, there is minimal information available about its regulation in vivo. Furthermore, the relative abundance of this enzyme implies a role in other cellular functions, but the lack of other physiologically relevant substrates (besides calponin and caldesmon) for CaMK II in VSMCs has blunted the analysis of other putative functions. Here we report that the S18 protein of the 40 S ribosome is a potential substrate of CaMK II. While almost nothing is known about the function of S18 in animal cells, it is a highly basic protein showing high homology to the Escherichia coli ribosomal protein rpS13. rpS13 plays important roles in both initiation and elongation during protein translation and is thought to help regulate translational efficiency. Hence, the identification of S18 as a putative substrate for CaMK II may provide a novel link between Ca2+-mobilizing agents and protein translation control. All tissue culture plasticware was obtained from Costar (Cambridge, MA) and Falcon (BD PharMingen). Fetal calf serum was purchased from Hyclone (Logan, UT). RPMI base medium, trypsin-EDTA, glutamine, and penicillin-streptomycin were purchased from Invitrogen. Heparin, obtained from Glycomed (Alameda, CA), was derived from porcine mucosa (sodium salt) (molecular mass, 12–18 kDa). Chondroitin sulfate and reagents for buffers, cell extraction, and SDS-gel electrophoresis were from Sigma. Platelet-derived growth factor was from R&D Systems (Minneapolis, MN), and lysophosphatidic acid was from Sigma. Acrylamide solution (Protogel) was from National Diagnostics (Atlanta, GA). Horseradish peroxidase-conjugated goat anti-mouse IgG was from Invitrogen; horseradish peroxidase-conjugated sheep anti-rabbit IgG was obtained from Roche Molecular Biochemicals. Sodium orthovanadate was from Sigma; okadaic acid, calyculin, tautomycin, and KN-93 were from Calbiochem. [3H]Leucine was from PerkinElmer Life Sciences. Rat aortic smooth muscle cells from Sprague-Dawley rats (Charles River Breeding Laboratories, Inc.) were isolated, cultured, and characterized as described previously (8Castellot J.J., Jr. Choay J. Lormeau J.C. Petitou M. Sache E. Karnovsky M.J. J. Cell Biol. 1986; 102: 1979-1984Crossref PubMed Scopus (134) Google Scholar). Briefly, the abdominal segment of the aorta was removed, and the fascia was cleaned away under a dissecting microscope. The aorta was cut longitudinally, and small pieces of the media were carefully stripped from the vessel wall. Two or three such strips were placed in 60-mm dishes. Within 1–2 weeks, VSMCs migrated from the explants; they were capable of being passaged approximately 1 week after the first appearance of cells. They were identified as smooth muscle cells by their “hill and valley” growth pattern and indirect immunofluorescence staining for VSMC-specific α-actin. VSMC cultures were maintained in RPMI 1640, 10% fetal calf serum at 37 °C in a 5% CO2, 95% air incubator. Primary cultures were used at or before passage 9. Cells were counted using a Coulter Counter (Coulter Counter Corp., Hialeah, Fl). Cells were routinely grown in RPMI 1640, 10% fetal calf serum (FCS). SV40 large T antigen-transformed heparin-resistant and -sensitive cells (9Caleb B.L. Hardenbrook M. Cherington P.V. Castellot J.J., Jr. J. Cell. Physiol. 1996; 167: 185-195Crossref PubMed Scopus (14) Google Scholar) were grown and maintained under similar conditions. Cells were routinely plated at 5–6 × 105/100-cm2 dish, washed with RPMI, and placed in RPMI containing 0.2–0.4% FCS for 72 h (10Castellot J.J., Jr. Pukac L.A. Caleb B.L. Wright T.C., Jr. Karnovsky M.J. J. Cell Biol. 1989; 109: 3147-3155Crossref PubMed Scopus (148) Google Scholar). Flow microfluorometry and determination of [3H]thymidine-labeled nuclei indicated that greater than 95% of the cells were arrested in G0(G1). Cells were released from quiescence by replacing the low serum medium with normal growth medium (i.e. RPMI 1640 containing 10% FCS). The cells were ∼40–60% confluent at the time of harvest. Quiescent cells were treated with 10% FCS, RPMI with or without the indicated concentrations of heparin or chondroitin sulfate for various time intervals. The proteins were then harvested as follows with all procedures performed at 4 °C. Cells were rinsed twice with cold TBS (20 mm Tris, pH 8, 137 mm NaCl) and lysed with 100 μl of lysis buffer (TBS + 1% Nonidet P-40, 10% glycerol, 100 mm sodium fluoride, 2 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 1 μg/ml leupeptin, 0.5 mm sodium orthovanadate). The extracts were rocked for 20 min and centrifuged at 12,000 rpm (Eppendorf microcentrifuge) for 10 min to remove insoluble material. The supernatant was stored at −20 °C until use. Protein estimations were done by the Pierce BCA method adapted for microtiter plates. Extracts containing 50 μg of protein were boiled with 1× SDS sample loading buffer, resolved by SDS-PAGE (11Ausebel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley and Sons, New York1996: 286-289Google Scholar), and blotted onto nitrocellulose membrane (Schleicher & Schu¨ll) in Towbin buffer (25 mm Trizma (Tris base), 192 mmglycine, 20% methanol) at 250 mA overnight. The blots were stained with Ponceau stain to confirm equal loading and transfer of proteins to the membrane. The membranes were blocked with 5% milk in 1× TBS (20 mm Tris base, 137 mm NaCl, pH 7.6), and Western blots were performed using anti-phosphotyrosine or anti-phosphoserine antibodies (1:1000) and horseradish peroxidase-conjugated anti-mouse IgG (1:10,000) in 1× TBST (TBS + 0.2% Tween 20). The proteins were visualized using the DuPont Renaissance Enhanced ChemiLuminescence detection reagents and autoradiography as described by the manufacturer. Prestained protein standard markers (Invitrogen) were used as molecular weight markers. Densitometric analysis on the autoradiograms was performed using the Stratagene (La Jolla, CA) Eagle Eye II system and the Scanalytics (Billerica, MA) ONE D-scan software. Protein synthesis was measured as described previously for smooth muscle cells (12Cochran D.L. Castellot J.J., Jr. Karnovsky M.J. J. Cell. Physiol. 1985; 124: 29-36Crossref PubMed Scopus (41) Google Scholar). Briefly, 2 × 104 VSMCs were plated into 24-well microplates. 2 days later, 2 μCi/ml [3H]leucine was added to the culture medium in the presence or absence of 30 μm KN-93, a specific inhibitor of CaMK II (1Sumi M. Kiuchi K. Ishikawa T. Ishii A. Hagiwara M. Nagatsu T. Hidaka H. Biochem. Biophys. Res. Commun. 1991; 181: 968-975Crossref PubMed Scopus (461) Google Scholar). After 2 h, cells were washed five times with ice-cold phosphate-buffered saline, and 1.0 ml of ice-cold 10% trichloroacetic acid (w/v) was added to each well. The cells were incubated on ice for 1 h and washed five times with ice-cold 10% trichloroacetic acid. 1.0 ml of 1.0 m NaOH was added to each well overnight at 37 °C, pH was neutralized with 10 m HCL, and a small portion was counted using liquid scintillation spectroscopy. Replicate wells within the same multiwell plate were harvested for both protein determination and cell counting (12Cochran D.L. Castellot J.J., Jr. Karnovsky M.J. J. Cell. Physiol. 1985; 124: 29-36Crossref PubMed Scopus (41) Google Scholar). To ensure that intracellular and extracellular leucine pools were completely equilibrated and that the specific radioactivity of leucyl-tRNA was unchanged by KN-93, the methods outlined by Gulves and Dice were followed (13Gulves E.A. Dice J.F. Biochem. J. 1989; 260: 377-387Crossref PubMed Scopus (85) Google Scholar). Under the conditions used in our experiments, these parameters were found not to contribute to the changes in protein synthesis rates observed in the presence of KN-93. Quiescent cells were rinsed in phosphate-free RPMI and incubated in this medium for 2 h in the presence of 200 μCi/ml [γ-32P]ATP (14Hardie D.G. Campbell D.G. Caudwell F.B. Haystead T.A.J. Hardie D.G. Protein Phosphorylation. A Practical Approach. IRL Press, Oxford1993: 113-118Google Scholar). Cells were then treated with 10% FCS, RPMI + heparin or with 1 μm ionomycin, RPMI + heparin. Proteins were harvested in Buffer A (50 mm MOPS, pH 7.4, 2 mm EGTA, 100 mm NaF, 100 mm sodium pyrophosphate, 2 mm dithiothreitol, 0.2 mmphenylmethylsulfonyl fluoride, 1% Nonidet P-40, and 0.4 unit/ml aprotinin) and allowed to sit on ice for 5 min, and the lysates were cleared by centrifugation at 10,000 rpm for 10 min at 4 °C. The supernatant was used for immunoprecipitation with anti-CaMK II antibodies (90 min at 4 °C) and protein A + G-agarose beads (Oncogene Science) (60 min at 4 °C). The immunoprecipitates were resolved by SDS-PAGE and blotted onto nitrocellulose, and differential phosphorylation was analyzed after overnight exposure on a PhosphorImager. Quiescent VSMCs were stimulated with 10% FCS, RPMI, and the cell lysates were used for immunoprecipitation with the anti-CaMK II δ-isoform-specific antibodies as described above. The immunoprecipitates were resolved by SDS-PAGE, and the proteins were visualized by Colloidal Coomassie Blue staining. The p20 band was excised from the gel and submitted for sequence analysis to the Harvard microsequencing facility. Quiescent VSMCs were radioactively labeled with [γ-32P]ATP as described above. 90 min into the incubation, KN-93 was added to a final concentration of 30 μm. After 30 min the cells were stimulated with 10% FCS for 5 min. The proteins were then harvested and used for immunoprecipitation as described above. We have reported that heparin inhibits CaMK II phosphorylation and activation (15Mishra-Gorur K. Castellot J.J. Mol. Biol. Cell. 1997; 8 (abstr.): 401aGoogle Scholar, 34Mishra-Gorur, K., Singer, H. A., and Castellot, J. J., Jr. (2002)Am. J. Pathol., in pressGoogle Scholar). In these experiments, growth-arrested VSMCs were radioactively labeled with [γ-32P]ATP followed by stimulation with 10% FCS, RPMI + heparin for 5 min. The cell lysates were then used for immunoprecipitation with anti-CaMK II δ-isoform-specific antibodies. This antibody was chosen because the δ-isoform is the predominant form found in VSMCs (16Schworer C.M. Rothblum L.I. Thekkumkara T.J. Singer H.A. J. Biol. Chem. 1993; 268: 14443-14449Abstract Full Text PDF PubMed Google Scholar), and the antibody is highly specific as it is made against the NFSGGTSLWQNI peptide unique to the C terminus of the δ2-isoform (16Schworer C.M. Rothblum L.I. Thekkumkara T.J. Singer H.A. J. Biol. Chem. 1993; 268: 14443-14449Abstract Full Text PDF PubMed Google Scholar, 17Abraham S.T. Benscoter H.A. Schworer C.M. Singer H.A. Circ. Res. 1997; 81: 575-584Crossref PubMed Scopus (100) Google Scholar). A single protein (Mr ∼ 20,000; p20) was found to co-immunoprecipitate with CaMK II (Fig.1). In addition, p20 was abundantly phosphorylated upon serum stimulation, and this effect was potently inhibited by heparin. Control immunoprecipitations with the agarose beads alone were performed to ensure that p20 was not simply interacting with the beads. Based on the observations that 1) the phosphorylation of both CaMK II and p20 was inhibited by heparin and 2) p20 co-immunoprecipitated with CaMK II, we considered the possibility that p20 might be a novel substrate of CaMK II. To identify this protein, we performed a large scale immunoprecipitation with anti-CaMK II antibodies on serum-treated VSMC lysates. The immunoprecipitates were resolved by SDS-PAGE and stained with Colloidal Coomassie Blue stain. The p20 protein band was excised from the gel and submitted for sequence analysis. Two peptides were sequenced, and both peptides exhibited 100% homology to the corresponding sequence of the rat ribosomal protein S18 of the 40 S ribosome (Fig. 2). To test whether cellular S18 was phosphorylated by CaMK II, we used KN-93, a specific inhibitor of CaMK II (1Sumi M. Kiuchi K. Ishikawa T. Ishii A. Hagiwara M. Nagatsu T. Hidaka H. Biochem. Biophys. Res. Commun. 1991; 181: 968-975Crossref PubMed Scopus (461) Google Scholar). Quiescent VSMCs were radioactively labeled for 2 h with [γ-32P]ATP. 90 minutes into the incubation, KN-93 was added to the medium at a final concentration of 30 μm. After 30 min of incubation with KN-93 the cells were treated with 10% FCS or 1 μm ionomycin for 5 min. Cells were harvested, and the lysates were used for immunoprecipitation with anti-CaMK II δ-isoform-specific antibodies. Quiescent cells and cells treated with KN-93 alone were used as controls. Immunoprecipitation with beads alone was done to confirm the specificity of the co-immunoprecipitation. KN-93 treatment resulted in a reduction of CaMK II phosphorylation due to either FCS or ionomycin stimulation (Fig. 3). A concomitant reduction in the phosphorylation of the S18 protein was observed, suggesting that S18 is phosphorylated by CaMK II (Fig. 3). If S18 phosphorylation by CaMK II plays a role in regulating translational efficiency, then inhibiting S18 phosphorylation with KN-93 should reduce protein synthesis rates in VSMCs. To test this hypothesis, we added [3H]leucine to exponentially growing VSMCs in the presence or absence of 30 μm KN-93 (Table I), a concentration that reduces S18 phosphorylation by ∼74% (Fig. 3). Using two independently isolated VMSC cultures, KN-93 reduced protein synthesis rates by an average of 24%. Control experiments (12Cochran D.L. Castellot J.J., Jr. Karnovsky M.J. J. Cell. Physiol. 1985; 124: 29-36Crossref PubMed Scopus (41) Google Scholar, 13Gulves E.A. Dice J.F. Biochem. J. 1989; 260: 377-387Crossref PubMed Scopus (85) Google Scholar) indicate that the intracellular leucine pool is fully equilibrated, that the leucyl-tRNA fraction is unchanged in the presence of KN-93, and that >95% of the cell protein is collected by the methods used (data not shown).Table IS18 phosphorylation affects protein synthesis rates in VSMCsCulturedpm/105cells% ChangeVSMC-3A − KN-9323,656 ± 1,922 + KN-9318,869 ± 840−21VSMC-8A − KN-9319,813 ± 2,047 + KN-9314,462 ± 1,115−27Two independently isolated VSMC cultures (designated 3A and 8A) were used to measure protein synthesis rates as described under “Experimental Procedures.” Cells in quadruplicate wells were exposed to [3H]leucine for 2 h in the presence or absence of 30 μm KN-93, a concentration that blocks S18 phosphorylation by 74% (Fig. 3). At the end of the incubation period, radiolabeled protein was precipitated with trichloroacetic acid and processed for scintillation spectroscopy. dpm ± S.D. is shown. Statistical analysis using Student’s t test indicates that the inhibition of protein synthesis is statistically significant (p < 0.05). Background dpm, 68 ± 30. “Zero time” control (cell exposed momentarily to radiolabel at 4 °C), 1,213 ± 49. Open table in a new tab Two independently isolated VSMC cultures (designated 3A and 8A) were used to measure protein synthesis rates as described under “Experimental Procedures.” Cells in quadruplicate wells were exposed to [3H]leucine for 2 h in the presence or absence of 30 μm KN-93, a concentration that blocks S18 phosphorylation by 74% (Fig. 3). At the end of the incubation period, radiolabeled protein was precipitated with trichloroacetic acid and processed for scintillation spectroscopy. dpm ± S.D. is shown. Statistical analysis using Student’s t test indicates that the inhibition of protein synthesis is statistically significant (p < 0.05). Background dpm, 68 ± 30. “Zero time” control (cell exposed momentarily to radiolabel at 4 °C), 1,213 ± 49. This study provides evidence that the S18 protein of the 40 S ribosome can be phosphorylated by CaMK II, thus identifying a putative substrate for CaMK II in VSMCs. The evidence includes 1) a physical association of S18 with CaMK II as determined by co-immunoprecipitation of the two proteins and 2) pharmacologic suppression of S18 phosphorylation by two inhibitors of CaMK II, heparin and KN-93. Furthermore, inhibiting phosphorylation of S18 with KN-93 also produced a reduction in protein translation rates. Although the initial observation of a putative CaMK II/S18 interaction was made using heparin, work in our laboratory (10Castellot J.J., Jr. Pukac L.A. Caleb B.L. Wright T.C., Jr. Karnovsky M.J. J. Cell Biol. 1989; 109: 3147-3155Crossref PubMed Scopus (148) Google Scholar, 15Mishra-Gorur K. Castellot J.J. Mol. Biol. Cell. 1997; 8 (abstr.): 401aGoogle Scholar, 34Mishra-Gorur, K., Singer, H. A., and Castellot, J. J., Jr. (2002)Am. J. Pathol., in pressGoogle Scholar) and by others (18Pukac L.A. Carter J.E. Ottlinger M.E. Karnovsky M.J. J. Cell. Physiol. 1997; 172: 69-78Crossref PubMed Scopus (60) Google Scholar, 19Herbert J.M. Clowes M. Lea H.J. Pascal M. Clowes A.W. J. Biol. Chem. 1996; 271: 25928-25935Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20Ottlinger M.E. Pukac L.A. Karnovsky M.J. J. Biol. Chem. 1993; 268: 19173-19176Abstract Full Text PDF PubMed Google Scholar) clearly indicates that heparin inhibits kinases other than CaMK II. We therefore used the highly specific CaMK II inhibitor KN-93 to confirm this interaction. KN-93 is a more hydrophilic analogue of the isoquinolinyl sulfonamide KN-62. Both these inhibitors prevent the activation of CaMK II by interacting with the calmodulin binding domain of the kinase (1Sumi M. Kiuchi K. Ishikawa T. Ishii A. Hagiwara M. Nagatsu T. Hidaka H. Biochem. Biophys. Res. Commun. 1991; 181: 968-975Crossref PubMed Scopus (461) Google Scholar). KN-93 has no significant effects on myosin light chain kinase, protein kinase C, or protein kinase A at the concentrations used in the current study (17Abraham S.T. Benscoter H.A. Schworer C.M. Singer H.A. Circ. Res. 1997; 81: 575-584Crossref PubMed Scopus (100) Google Scholar). KN-93 pretreatment resulted in an attenuation of CaMK II phosphorylation upon FCS stimulation. A concomitant reduction in S18 phosphorylation as well as the reproducible co-immunoprecipitation of S18 with CaMK II may provide a link between calcium-mobilizing agents and protein translation. This hypothesis is supported by the observation that the inhibition of S18 phosphorylation is accompanied by a 24% reduction in protein synthesis. Little is known about the eukaryotic S18 protein except for its sequence. However, its prokaryotic homolog, the E. colirpS13, has been studied more extensively, and the available data may provide insights into the role of S18 protein in eukaryotes, especially since there are many conserved domains between the two. Both S18 and rpS13 are very basic proteins (21Chan Y.-L. Paz V. Wool I.G. Biochem. Biophys. Res. Commun. 1991; 178: 1212-1218Crossref PubMed Scopus (13) Google Scholar, 22Garwood J. Lepesant J.-A. Gene (Amst.). 1994; 141: 231-235Crossref PubMed Scopus (9) Google Scholar). The E. coli rpS13 is thought to be involved in the initiation of translation as it is a surface protein at the interface of the ribosomal subunits that cross-links to all three initiation factors (23Boileau G. Butler P. Hershey J.W. Traut R.R. Biochemistry. 1983; 22: 3162-3170Crossref PubMed Scopus (65) Google Scholar). It interacts strongly with the 20 S rRNA and has been cross-linked to the 3′ major domain of the 16 S rRNA (24Powers T. Stern S. Changchien L.M. Noller H.F. J. Mol. Biol. 1988; 201: 697-716Crossref PubMed Scopus (42) Google Scholar, 25Osswald M. Doring T. Brimacombe R. Nucleic Acids Res. 1995; 23: 4635-4641Crossref PubMed Scopus (45) Google Scholar, 26Heilik G.M. Noller H.F. RNA. 1996; 2: 597-602PubMed Google Scholar), to ribosomal protein S19 (27Pohl T. Wittman-Liebold B. J. Biol. Chem. 1988; 263: 4293-4301Abstract Full Text PDF PubMed Google Scholar), and to tRNA in both the P and A sites (25Osswald M. Doring T. Brimacombe R. Nucleic Acids Res. 1995; 23: 4635-4641Crossref PubMed Scopus (45) Google Scholar, 28Wower J. Malloy T.A. Hixson S.S. Zimmermann R.A. Biochim. Biophys. Acta. 1990; 1050: 38-44Crossref PubMed Scopus (20) Google Scholar). These studies indicate that rpS13 is important for both translation initiation and elongation. rpS13 can be phosphorylated by a eukaryotic protein kinase (29Issinger O.G. Kiefer M.C. Traut R.R. Eur. J. Biochem. 1975; 59: 137-143Crossref PubMed Scopus (7) Google Scholar), and there are a number of phosphorylation sites in the eukaryotic S18 protein for phosphorylation by casein kinase II, protein kinase C, and a tyrosine kinase. Based on the high degree of homology between S18 and rpS13, it is possible that S18 plays an important role in ribosome assembly as well as in translational efficiency. In support of this idea, a mutant of the E. coli rpS13 protein lacking the C-terminal 19 amino acids shows a 20–30% reduction in translation rate (30Faxen M.A. Granberg W. Isaksson L.A. Biochim. Biophys. Acta. 1994; 1218: 27-34Crossref PubMed Scopus (6) Google Scholar) in agreement with the 24% drop in protein synthesis rates observed when S18 phosphorylation is blocked in VSMCs. Interestingly the C-terminal portion is required for 16 S rRNA recognition (31Schwarzbauer J. Craven G.R. Nucleic Acids Res. 1985; 13: 6767-6786Crossref PubMed Scopus (17) Google Scholar), and the consensus motif for CaMK II phosphorylation is present within the C-terminal 19 residues of the protein sequence of S18. The notion that phosphorylation of S18 regulates the rate of translation becomes even more interesting in light of the ability of heparin to inhibit both CaMK II and protein kinase C, both of which can phosphorylate the S18 protein (22Garwood J. Lepesant J.-A. Gene (Amst.). 1994; 141: 231-235Crossref PubMed Scopus (9) Google Scholar), implying that heparin may alter translation rates. Although previous studies have indicated that heparin blocks proliferation of VSMC, this glycosaminoglycan alters the overall rate of protein synthesis in VSMCs by 20% or less (12Cochran D.L. Castellot J.J., Jr. Karnovsky M.J. J. Cell. Physiol. 1985; 124: 29-36Crossref PubMed Scopus (41) Google Scholar). When one considers that growth regulatory proteins are very labile and comprise a very small percentage of the total protein in a cell while housekeeping proteins are generally much more stable, even large fluctuations in the levels of growth regulatory proteins would not be detected when measuring overall protein synthesis. In addition, mRNA encoding growth regulatory proteins has a very short half-life; thus, even a modest reduction in translation rate such as the 20–30% decrease seen in rpS13 mutants and the 23% reduction observed with underphosphorylated S18 would result in the rapid degradation/reduction of these nascent message levels in the cell. The co-immunoprecipitation of a 20-kDa protein with CaMK II only in the presence of mitogenic stimulation has been reported earlier (32Tazi K.A. Bonnafous M. Favre G. Soula G. Le Gaillard F. Biochem. J. 1995; 307: 557-561Crossref PubMed Scopus (7) Google Scholar). However, further studies to identify the protein were not reported. There is one prior observation of differential phosphorylation of a ribosomal protein (33Fawell E.H. Boyer I.J. Brostrom M.A. Brostrom C.O. J. Biol. Chem. 1989; 264: 1650-1655Abstract Full Text PDF PubMed Google Scholar). However, this effect was independent of the cytosolic free Ca2+ pool and thus was not a result of calmodulin-dependent protein kinases; instead, it is a reflection of the sequestered pool of intracellular Ca2+. In contrast, serum treatment causes an increase in intracellular Ca2+ levels, a key factor in proliferative responses. Exploring the connection between CaMK II and S18 should provide a better understanding of the antiproliferative mechanism of heparin and may shed new light on the relationship between Ca2+-mobilizing agents and protein translation." @default.
• W2140699300 created "2016-06-24" @default.
• W2140699300 creator A5005235946 @default.
• W2140699300 creator A5034176671 @default.
• W2140699300 creator A5046742673 @default.
• W2140699300 date "2002-09-01" @default.
• W2140699300 modified "2023-09-29" @default.
• W2140699300 title "The S18 Ribosomal Protein Is a Putative Substrate for Ca2+/Calmodulin-activated Protein Kinase II" @default.
• W2140699300 cites W1483678770 @default.
• W2140699300 cites W1504410512 @default.
• W2140699300 cites W1505781390 @default.
• W2140699300 cites W1522104342 @default.
• W2140699300 cites W1560941354 @default.
• W2140699300 cites W1568808038 @default.
• W2140699300 cites W1664151389 @default.
• W2140699300 cites W1937973444 @default.
• W2140699300 cites W1966125684 @default.
• W2140699300 cites W1968054859 @default.
• W2140699300 cites W1972570307 @default.
• W2140699300 cites W1979012545 @default.
• W2140699300 cites W1988308818 @default.
• W2140699300 cites W1993733800 @default.
• W2140699300 cites W1994786303 @default.
• W2140699300 cites W1998232144 @default.
• W2140699300 cites W2000878083 @default.
• W2140699300 cites W202786147 @default.
• W2140699300 cites W2033187834 @default.
• W2140699300 cites W2034856894 @default.
• W2140699300 cites W2045259997 @default.
• W2140699300 cites W2054602879 @default.
• W2140699300 cites W2056379355 @default.
• W2140699300 cites W2063356470 @default.
• W2140699300 cites W2072696446 @default.
• W2140699300 cites W2146087296 @default.
• W2140699300 cites W2157756102 @default.
• W2140699300 cites W2158034990 @default.
• W2140699300 cites W565127267 @default.
• W2140699300 doi "https://doi.org/10.1074/jbc.c200342200" @default.
• W2140699300 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12145273" @default.
• W2140699300 hasPublicationYear "2002" @default.
• W2140699300 type Work @default.
• W2140699300 sameAs 2140699300 @default.
• W2140699300 citedByCount "10" @default.
• W2140699300 countsByYear W21406993002012 @default.
• W2140699300 countsByYear W21406993002013 @default.
• W2140699300 countsByYear W21406993002017 @default.
• W2140699300 countsByYear W21406993002021 @default.
• W2140699300 crossrefType "journal-article" @default.
• W2140699300 hasAuthorship W2140699300A5005235946 @default.
• W2140699300 hasAuthorship W2140699300A5034176671 @default.
• W2140699300 hasAuthorship W2140699300A5046742673 @default.
• W2140699300 hasBestOaLocation W21406993001 @default.
• W2140699300 hasConcept C104317684 @default.
• W2140699300 hasConcept C181199279 @default.
• W2140699300 hasConcept C184235292 @default.
• W2140699300 hasConcept C185592680 @default.
• W2140699300 hasConcept C18903297 @default.
• W2140699300 hasConcept C2777289219 @default.
• W2140699300 hasConcept C29688787 @default.
• W2140699300 hasConcept C38062823 @default.
• W2140699300 hasConcept C55493867 @default.
• W2140699300 hasConcept C67705224 @default.
• W2140699300 hasConcept C86803240 @default.
• W2140699300 hasConcept C88478588 @default.
• W2140699300 hasConcept C97029542 @default.
• W2140699300 hasConceptScore W2140699300C104317684 @default.
• W2140699300 hasConceptScore W2140699300C181199279 @default.
• W2140699300 hasConceptScore W2140699300C184235292 @default.
• W2140699300 hasConceptScore W2140699300C185592680 @default.
• W2140699300 hasConceptScore W2140699300C18903297 @default.
• W2140699300 hasConceptScore W2140699300C2777289219 @default.
• W2140699300 hasConceptScore W2140699300C29688787 @default.
• W2140699300 hasConceptScore W2140699300C38062823 @default.
• W2140699300 hasConceptScore W2140699300C55493867 @default.
• W2140699300 hasConceptScore W2140699300C67705224 @default.
• W2140699300 hasConceptScore W2140699300C86803240 @default.
• W2140699300 hasConceptScore W2140699300C88478588 @default.
• W2140699300 hasConceptScore W2140699300C97029542 @default.
• W2140699300 hasIssue "37" @default.
• W2140699300 hasLocation W21406993001 @default.
• W2140699300 hasOpenAccess W2140699300 @default.
• W2140699300 hasPrimaryLocation W21406993001 @default.
• W2140699300 hasRelatedWork W1991011805 @default.
• W2140699300 hasRelatedWork W2052920071 @default.
• W2140699300 hasRelatedWork W2056481979 @default.
• W2140699300 hasRelatedWork W2082554893 @default.
• W2140699300 hasRelatedWork W2087473022 @default.
• W2140699300 hasRelatedWork W2182569872 @default.
• W2140699300 hasRelatedWork W3034530439 @default.
• W2140699300 hasRelatedWork W3210429714 @default.
• W2140699300 hasRelatedWork W347205979 @default.
• W2140699300 hasRelatedWork W4236835567 @default.
• W2140699300 hasVolume "277" @default.
• W2140699300 isParatext "false" @default.
• W2140699300 isRetracted "false" @default.
• W2140699300 magId "2140699300" @default.
• W2140699300 workType "article" @default.