THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 218, No. 14, Issue of July 25, pp. 5130-5136, 1973

Printed in U.S.A.

Function of Phosphorylated Ribosomes

THE ACTIVITY OF RIBOSOMAL SUBUNITS PHOSPHORYLATED IN VITRO BY PROTEIN I<INASE*

(Received for publication, November 10, 1972)

CHARLES EIL~ AND IRA G. WOOL

From the Department of Biochemistry, University of Chicago, Chicago, Illinois 60657

SUMMARY

The proteins of rat liver ribosomal subunits were phos- phorylated by cyclic adenosine 3’ : S’-monophosphate-acti- vated protein kinases and the activity of the particles as- sessed. While we did not examine all of the possibilities, we did not find a ribosome function that was appreciably and consistently altered by phosphorylation. The synthesis of polyphenylalanine at high concentrations of magnesium (12.5 mM), which was dependent on elongation factors EF-1 and EF-2, was not changed if the ribosomes were phosphorylated. The synthesis of polyphenylalanine at low concentrations of magnesium (6 mM), which required the initiation factors EIF-1 and EIF-2 as well as elongation factors, was increased (18%) if the concentrations of phosphorylated ribosomes were limiting but not if initiation factors were limiting. The phosphorylation of the 40 S subunit increased only slightly (13%) the EIF-l-catalyzed binding of Phe-tRNA to the particle. Finally, there was no appreciable difference in the ability of phosphorylated and nonphosphorylated ribosomes to translate encephalomyocarditis virus RNA (which required all three initiation factors).

Eukaryotic ribosomal proteins are phosphorylated in viva and in vitro by protein kinases bound to the particle or free in the cytoplasm (1-12). The implication is that the function of ribo- somes might be regulated by reversible phosphorylation of their proteins. The evidence for that impression, however, is still in the main circumstantial.

Two rat liver cytosol protein kinases catalyze phosphorylation of a number of proteins of the 40 S and of the 60 S ribosomal subunits (5, 12). In an accompanying paper we characterized the reaction catalyzed by the enzymes and determined the struc- ture of the product (12). We now report on the function of phosphorylated and nonphosphorylated ribosomes.

EXPERIMESTAL PROCEDURES

Jlaterials-Tris(hydroxymethyl)aminomethane (Sigma 7-9) and cyclic AMP’ were purchased from Sigma; puromycin dihy-

* The expenses of the research were met by grants from the John A. Hartford Foundation and the National Institutes of Health (AM-04842).

1 Medical Scientist Trainee supported by National Institute of General Medical Science Grant GiV-01939.

1 The abbreviations used are: [3H]Phe-tRNA, unfractionated

drochloride from Nutritional Biochemicals Corp. [32P]Ortho- phosphoric acid (carrier-free, in 0.02 N HCl) was obtained from Tracerlab and used to prepare [T-~~P]ATP (13). L-[3H]Phenyl- alanine (4.5 to 6.2 Ci per mmole) was purchased from New England Nuclear Corp. and used with 19 nonradioactive amino acids to aminoacylate unfractionated Escherichia coli B tRNA (14) or rat liver tRNA (see below).

1Mediu-The media most frequently used are listed here and referred to in the text by a letter. Medium A: 50 mM Tris-HCl, pH 7.7; 80 mM KCl; 12.5 mu MgCls; 10 mM MSH. Medium B: 50 mM Tris-HCl, pH 7.6; 880 mM KCI; 12.5 mM Mg&; 10 mM MSH. Medium C: 20 rnh1 Tris-HCl, pH 7.5; 125 mM KCl; 5 mM magnesium acetate; 6 mM MSH. Medium D: 10 mM Tris- HCl, pH 7.5; 10 mM KCl, 1.5 mM MgClz. Medium E: 50 mM Tris-HCI, pH 7.7; 10 mM KCl; 5 mM MgC12; 10 mM MSH; 5% glycerol. The pH of the buffers and media was determined at room temperature.

Preparation of Ribosomal Particles--Liver ribosomes were isolated from male Sprague-Dawley rats that weighed 100 to 120 g (15, 16). Ribosomal subunits were prepared by suspending ribosomes in Medium B and incubating for 15 min at 37” with 0.1 mM puromycin; the subunits were separated on linear sucrose gradients (15-17). Fractions from the gradients were dialyzed overnight against Medium A and the subunits were precipitated with 0.2 volume of ethanol (18). The concentration of ribo- somes and ribosomal subunits was calculated from the absorp- tion at 260 nm (19) ; 1 A260 unit was taken to be the equivalent of 45 pg of rRNA.

Preparation of Protein Kinase-The preparation of protein kinases I and II by chromatography on DEAE-cellulose (DEAE- protein kinase) and the further purification of protein kinase I by chromatography on hydroxyapatite (HA-protein kinase) has been described before (5, 12).

Preparation of Rat Liver and Ascites Cell Cytosol (G-25 Fraction) -The 100,000 X g postmicrosomal supernatant from rat liver (15) was passed through a column of Sephadex G-25 which had been equilibrated with 50 mM Tris-HCl, pH 7.7, and 10 mM

MSH. The void volume eluate is referred to as G-25 fraction. The 150,000 x g postmicrosomal supernatant (20) from ascites cells grown in HA/ICR Swiss mice (males, 20 to 25 g) was

Escherichia co& B tRNA or rat liver tRNA (as specified) acylated with radioactive phenylalanine and 19 nonradioactive amino acids; MSH, p-mercaptoethanol; EF-1 and EF-2, elongation fac- tors 1 and 2, formerly called aminoacyltransferase I and II; EIF-1, EIF-2, and EIF-3, eukaryotic initiation factors; cyclic AMP, cyclic adenosine 3’:5’-monophosphate; EMCV, encephalomyo- carditis virus.

5130

by guest on February 25, 2020http://w

ww

.jbc.org/D

ownloaded from

5131

passed through a column of Scphadcx G-25 which had been equilibrated with Medium C.

Preparation and Aminoacylation oj Rat Liver tRNA--Rat liver tRNA was extracted by a modification of the procedure of Yang and Novelli (21) suggested to us by J)r. J. J. Castles. Bentonite (1 mg per ml) was added to the 100,000 X g postmicrosomal supernatant (15), and RNA was extracted with an equal volume of water-saturated phenol. The concentrat,ion of potassium a&ate (pH 5.25) in the aqueous cstract was adjusted to 0.2 M

and the RNA was precipitated by the addition of 2 volumes of ethanol. The precipitate was collected by centrifugation and extracted twice with 1 M NaCl in the presence of 1 mg per ml of bentonite. The extracted material was again adjusted to 0.2 M

potassium acetate, pH 5.25; the tRNA was precipitated with 2 volumes of ethanol, collected, and dissolved in 100 mM Tris-HCl (~1~ 9.0)-10 mM MgC&l mM EDTA. The tRNA was deacylated by incubation at 37” for 20 min. The solution was adjusted to 0.2 M potassium acetate, p1-I 5.25, and the tRNA was precipi- tated by the addition of 2 volumes of ethanol. The precipitate was collected by centrifugation, dissolved in approximately 5 ml of water, and dialyzed overnight against water.

The rat liver tRNA was aminoacylated with [3H]phenylalanine ad 19 other nonradioactive amino acids in a manner similar to that used for E. coli tRNh (22) cxctpt that a crude preparation of rat liver aminoacyl-tRNh sy~~thctases was used. The syn- thetases were prepared from rat liver postmicrosomal superna- tant by fractionation with ammonium sulfate: those proteins precipitating between 30 and 70% saturation were collected by centrifugation, dissolved in buffer (50 mM ‘Iris-HCl (pH 7.5), 10 rnM KCI, 5 mM MgC12, 20 mM MSII, 10 7. glycerol), and dialyzed overnight against the same buffer. The preparation of amino- acyl-tRNA was carried out in 20 ml of reaction mixture contain- ing: 100 mM Tris-HCl, pII 7.5; 10 lllM KCl, 5 mM MgC12; 2 mM

ATP; 0.1 InM GTP; 10 InM MSH; 0.01 mM each of 19 nonradio- active amino acids; 0.005 IrlM [311]pl~cnylalanine; 30 mg of tRNA; and enzyme (the optimum arnount was determined in a trial reac- tion). After incubation for 20 min at 37”, 2.2 ml of 2 M potas- sium acetate, pH 5.25, was added followed by 18 ml of phenol (saturated with 0.2 M potassium acetate, pl-I 5.25). The amino- acyl-tRNA was isolated as von Ehrcnstein and Lipmann had (23) ; its specific activity was approximately 1000 cpm per pg of tRNA.

Preparation of Salt-Wash Factors-Salt-wash factors (initiation factor preparation) were obtained from rat liver microsomes as described by Leader et al. (24). Ascites initiation factors were prcparcd as follows. l’ackcd ascitcs cells (20) were suspended in 1 to 1.5 volumes of Medium 1) and homogenized. The ionic strength of the homogenate was adjusted with 10 X Medium D (i.e. 10 times the usual concentration of ions and Tris-HCI) so that the final composition of the homogenate was 2 X in Me- dium 1). The homogenate was centrifuged at 30,000 x g for 10 min and the pellet discarded. The supernatant was centrifuged again for 10 min at 30,000 X g. The second supernatant was centrifuged for 3 hours at 45,000 rpm in a Spinco Ti50 rotor. The supernatant was set aside for preparation of aminoacyl- tRNA synthetases (see “l’rcparation of Aminoacyl-tRNA Syn- thetases from Ascites Cells”) and the ribosome pellet was resus- pended in 2 x Medium I) by gentle homogenization. This suspension was layered over 5 ml of 1 1\1 sucrose (in 2 x Medium D) and centrifuged for 4.5 hours at 45,000 rpm in a Spinco Ti50 rotor. The initiation factors were extracted from the clear ribo- somal pellet with 20 mM ‘I’&HCl (~1-1 7.5), 500 mM KCl, 5 mM MgC&, and 10 mar MSH. The misturc was kept at 0” for 1 hour

with occasional swirling. The ribosomes were sedimented by centrifugation at 60,000 rpm in a Spinco SW 65 rotor for 4 hours at 9”. The upper four-fifths of the supernatant was dialyzed against Medium C for 3 hours. The precipitate in the solution was removed by centrifugation at 10,000 X g for 10 min. The clear supernatant which contains initiation factors is referred to as “ascites salt-wash”; it was stored at -70” or in liquid nitrogen.

Preparation and Assay of Elongation Factors-Liver elonga- tion factors, EF-1 and EF-2, were prepared by hydroxyapatite chromatography and separated on a column of Sephadex G-200 as described by Schneir and Moldave (25).

Preparation of Initiation Factor EIF-l--The initiation factor which promotes binding of aminoacyl-tRNA to 40 S subunits (EIF-1) was purified from the 100,000 X g cytosol fraction of rat liver by chromatography on hydroxyapatite and filtration through Sephadex G-200 (26).

Preparation of Aminoacyl-tRNA Synthetases from Ascites Cell- The supernatant obtained by centrifugation of an ascites cell homogenate at 45,000 rpm (cf. “Preparation of Salt-Wash Fac- tors”) was treated with ammonium sulfate and the proteins pre- cipitating between 60 and 75% saturation were collected by cen- trifugation (at 10,000 x g for 15 min), suspended in a small volume of Medium C and dialyzed overnight against the same buffer. The insoluble material was removed by centrifugation (at 10,000 X g for 15 min). The supernatant contained the aminoacyl-tRNA synthetases.

Preparation of EMCV RNA-Encephalomyocarditis virus of the K2 strain was grown on Krebs II ascites tumor cells (27) ; the virus was purified (28) and the RNA extracted (29).

Measurement of Protein synthesis-For the measurement of the synthesis of polyphenylalanine, 10 pg (rRNA) of ribosomal subunits (the ratio (micrograms of rRNA) of 60 to 40 S subunits was 2.5:1) were incubated for 10 min at 37” in 0.1 ml of buffer (20 mM Tris-HCl (pH 7.7), 125 mM KCl, MgClz (as indicated), 10 mM MSH) which contained: 1 mM ATP, 0.1 mM GTP, 180 pg of creatine phosphate, 20 pg of creatine phosphate kinase, 80 pg of [%]Phe-tRNA (E. coli) or 60 pg of [3H]Phe-tRNA (rat liver), 12 pg of poly(U), and protein factors (the amounts are given in the tables and legends).

For assay of the translation of EMCV RNA, 10 pg of ribo- somal subunits were incubated at 37” in 0.05 ml of Medium C which contained: 1 mM ATP, 0.1 mM GTP, 0.05 mM each of 19 unlabeled amino acids, 0.003 mM [3H]phenylalanine, 90 pg of creatine phosphate, 10 pg of creatine phosphate kinase, 4,pg of EMCV RNA, 8 fig of deacylated rat liver tRNA, 150 pg of the ascites aminoacyl-tRNA synthetascs, and ascites salt-wash pro- tein (the amount is given in the tables and legends). Protein synthesis was terminated by addition of 1 ml of 10% trichloro- acetic acid, and the samples were heated at 90-95” for 15 min. The precipitate was collected on Reeve Angel or Whatman GF/C glass fiber filters (depending on whether the product was polyphenylalanine or EMCV protein) and washed with 30 ml of 5% trichloroacetic acid containing 10 pg per ml of phenylalanine. The filter discs were dried and placed into glass vials containing scintillation fluid (30). Radioactivity was determined in a Packard Tri-Carb spectrometer; the efficiency of the determina- tion of the radioactivity was 16%.

Assay of Ribosomal Phosphoprotein Phosphatase-Ribosomal phosphoprotein phosphatase activity was determined by incu- bating a mixture of 7.5 to 10 pg (rRNA) of 32P-labeled 60 and 40 S subunits (in a ratio of 2.5:l) in 0.05 or 0.1 ml of the reaction mix- ture used to measure protein synthesis, except that the amino- acyl-tRNA or amino acids were not radioactive. The reaction

by guest on February 25, 2020http://w

ww

.jbc.org/D

ownloaded from

5132

was started by adding ribosomes. Incubation was at 37” for the times specified in the tables. The reaction was stopped by adding 1 ml of 10% trichloroacetic acid and the mixture was heated at 90-95” for 15 min. The precipitate was collected on glass fiber discs and the radioactivity was measured as described before (12).

Assay of Function of Phosphorylated Ribosomes--The 40 or 60 S ribosomal subunits (the amounts are specified in the tables or the legends to the figures) were incubated for 25 min at 37” in 0.1 ml of Medium E containing: 0.1 mM ATP, 10e5 M cyclic AMP, and protein kinase I. The reaction was stopped by cooling on ice and aliquots of the subunits were used to assay their function.

Assay if Binding of Phe-tRNA to 40 S Ribosomal Subunits- The extent to which 40 S ribosomal subunits would bind Phe- tRNA in the reaction catalyzed by EIF-1 was assayed as de- scribed by Leader et al. (31).

Concentration of Protein-The concentration of protein in sam- ples was generally determined by the method of Lowry et al. (32) with bovine serum albumin as a standard. In some cases the concentration was estimated from the absorbance at 280 and 260 nm (33).

RESULTS

Function of Phosphorylated Ribosomes-Before we could under- take to determine the effect of phosphorylation on the function of ribosomes, it was necessary to modify the conditions of the reac- tion in which the particles were phosphorylated so that they would retain activity. Our aim was to achieve the maximum phosphorylation of the ribosomes that was consistent with preser- vation of the structure and function of the particles. We knew that the concentration of potassium (75 to 125 mlw) usually used in assays of ribosome function would inhibit protein kinase (12). However, if ribosomal subunits were phosphorylated in 10 m&f potassium, the reaction was not appreciably depressed, and the subsequent capacity of the ribosomes to synthesize polyphenyl- alanine (during which assay the potassium concentration was 125 mnf) was hardly decreased. There was no synthesis of poly- phenylalanine if potassium was omitted from the preincubation medium. Similar observations on the effect of potassium had been made by Niislund and Hultin (34).

We had to take notice of an additional possible complication, the contamination of the several protein fractions used to assay ribosome activity with a phosphoprotein phosphatase. Cer- tainly, if the enzyme were present there would be no reliable way of comparing the activity of phosphorylated and nonphos- phorylated ribosomes. For that reason we determined the ribosomal phosphoprotein phosphatase activity of the several protein fractions we intended to use in the assay of ribosome activity. The cytosol (G-25 fraction) from Krebs ascites cells, which contains the three initiation and the two translation fac- tors (24) had appreciable phosphatase activity, it catalyzed hy- drolysis of 69% of the radioactive phosphate bound to the ribo- some. Rat liver supernatant, which had EF-1 and the two elongation factors (24)) was almost as active; it removed 56% of the phosphate. However, the following factor preparations had only minimal phosphatase activity: purified EF-1 and EF-2; rat liver microsomal salt-wash (which contains EIF-1 and EIF-2 activity (24)). Those preparations then were suitable for a com- parison of the activity of phosphorylated and nonphosphorylated ribosomes.

We also considered the possibility that the protein fractions used to test ribosome function might be contaminated with pro- tein kinases, and that phosphorylation of control ribosomes

might occur in the relatively high concentrations of ATP (1 rnbf) necessary to assay ribosome activity. B priori that seemed un- likely since the concentrations of potassium (75 to 125 mM) and of magnesium (5 to 12.5 InM) present during the assays of func- tion inhibit protein kinase (12). Indeed, when we tested the possibility we found no phosphorylation of ribosomal subunits, if the concentration of potassium was 125 mM and of magnesium 5 mM (results not shown). Since no phosphorylation of ribosomes occurred with saturating amounts of protein kinase in the pres- ence of cyclic AMP, it is hardly likely to have happened in the absence of cyclic AMP and with the amounts of protein kinase that might contaminate the enzyme fractions used to test ri- bosome function.

Polyphenylalanine Synthesis-We began our analysis of the function of phosphorylated ribosomes by testing their ability to catalyze the synthesis of polyphenylalanine. The test was car- ried out in two ways: at high and low concentrations of mag- nesium. The synthesis of polyphenylalanine at high concen- trations of magnesium (12.5 mM) requires only elongation factors (EF-1 and EF-2), whereas synthesis at low concentrations (6 mM) of the cation requires two of the initiation factors (EIF-1 and EIF-2) as well (35).

Ribosomal subunits were preincubated separately with or without protein kinase I and ATP (Table I) ; preincubation was to effect phosphorylation of the ribosomes and to provide proper controls. The ribosomal subunits were then combined in the appropriate ratio and their ability to catalyze polyphenylalanine synthesis assessed. Preincubation of ribosomes did not in itself alter their activity. But more importantly, there was no differ-

TABLE I

Synthesis of polyphenylalanine by phosphorylated and nonphosphorylated vibosomes

Ribosomal subunits, 28.4 pg (rRNA) of 40 S or 72 pg (rRNA) of 60 S, were preincubated for 25 min at 37” in 0.1 ml of Medium Ii: containing 0.1 mM ATP, 10M5 M cyclic AMP, and 25 pg of HA-pro- tein kinase I. In some experiments protein kinase or ATP or both were omitted from the preincubat,ion. The reaction was stopped by cooling on ice and 2.8 pg (rRNA) of 40 S and 7.2 rg of 60 S subunits were combined and incubated for 10 min at 37” in 0.1 ml of buffer (20 mM Tris-HCl (pH 7.7), 125 mM KCI, 10 mM MSH, and either 6 or 12.5 mM MgCl,) which contained 1 IIIM ATP, 0.1 m&x GTP, 180 pg of creatine phosphate, 20 pg of creatine phos- phokinase, 80 rg of Escherichia coli [sH]Phe-tRNA, and 12 pg of poly(U). When the assay was at 12.5 mM MgC12, 15 fig of EF-1, and 80 pg of EF-2 were added; when the assay was at 6 mM, 285 pg of liver ribosomal salt-wash were added as well.

Subunit

Control. 40s. 60 S. 40 8. 60s.. : 40s.. 60s. 40s.. 60 S. 408.. : 60 S..

Preincubation

Protein kinase ATE

No preincubation - -

+

+ + + +

- - - - - + + - + +

Phenylalanine incorporated

6m1d 12.5 m&l MgClz M&12

pmo1es

9.67 13.29

9.08 15.28

10.24 15.94

11.58 14.77

11.94 14.39

12.06 14.85

by guest on February 25, 2020http://w

ww

.jbc.org/D

ownloaded from

a)

0

r

2.5

b)

/

I / I

1 10 20 30

EF-1 (,ug) EF-2 (pg)

FIG. 1. Synthesis of polyphenylalanine by phosphorylated and

0 ~ 7 ---r--

25 50 77

nonphosphorylated ribosomes measured as a function of the con centration of elongation factors. Ribosomal subunits were pre- incubated just as described in Table I with (0) or without (0) 0.1 mM ATP. The capacity of the subunits to synthesize poly- phenylalanine was then determined at a concentration of mag- nesium of 12.5 InM using rat liver [3H]Phe-tRNA. In a 90 pg of EF-2 were present; in b 15 pg of EF-1 were present.

ence in the capacity of phosphorylated and nonphosphorylated ribosomes to participate in the elongation reactions of poly- phcnylalaninc synthesis, assayed at 12.5 mM MgClz with EF-1 and EF-2 (Table I). There was however, a small (18%) in- crease in the initiation of the translation of poly(U) by phos- phorylated ribosomes, the reaction carried out at 6 mM MgC12 required EIF-1 and EIF-2 (liver ribosomal salt-wash) in addi- tion to EF-1 and EF-2 (Table I).

The experiments just described were carried out in circum- stances where the concentration of ribosomes restricted the syn- thesis of polyphenylalanine. It seemed possible that phos- phorylation of ribosomal subunits might change their ability to utilize limiting amounts of the various factors. To test the pos- sibility we measured polyphenylalanine synthesis at high con- centrations of magnesium (12.5 mM) as a function of the amount of EF-1 (Fig. la) or EF-2 (Fig. lb) ; and at low concentrations of magnesium (6 Inn/r) as a function of the amount of liver ribo- somal salt-wash fraction, which contains EIF-1 and EIF-2 (Fig. 2). There was no difference in the ability of phosphoryl- ated and nonphosphorylated ribosomes to synthesize polyphenyl- alanine even when the amount of EF-1 (Fig. la) or of EF-2 (Fig. lb) was limiting. Nor was there a difference when we tested the initiation of the translation of poly(U) as a function of the amount of initiation factor preparation (Fig. 2). In- deed, this latter experiment did not substantiate the small dif- ference found when the amount of ribosomes restricted synthesis of polyphenylalanine (cf. the experiment in Table I).

Binding of Phe-tRNA to 40 S Ribosomal Subunits-The initia- tion factor EIF-1 (formerly called 40 S binding factor (31)) catalyzes the template specific binding of Phe-tRNA to 40 S ribosomal subunits (26, 31). Phosphorylated 40 S subunits bound slightly more (13 %) Phe-tRNA than did nonphosphoryl- ated particles (Table II). The small difference was dependent on the presence of cyclic AMP, as well as protein kinase and ATP during preincubation. In the assay, the amount of 40 S subunits was limiting, whereas the amount of EIF-1 was satu- rating. When binding was measured as a function of the amount of EIF-1, a small difference was seen again with all amounts of EIF-1 tested.

Translation of EMCV RNA-The most economical way to regulate protein synthesis would seem to be the control of the initiation of peptide chains, and the easiest way to accomplish that might be to condition the binding of mRNA to the ribo-

5133

r

80 160 240

SALT WASH PROTEIN (pg)

FIG. 2. Synthesis of polyphenylalanine by phosphorylated and nonphosphorylated ribosomes measured as a function of the con- centration of ribosomal salt-wash factors. Ribosomal subunits were preincubated just as described in Table I with ( l ) or with- out (0) 0.1 mM ATP. The capacity of the subunits to synthesize polyphenylalanine was then determined at a magnesium concen- tration of 6 mM using rat liver [3H]Phe-tRNA; 15 pg of EF-1 and 80 fig of EF-2 were added.

TABLE II

Rinding of Phe-tRNA to phosphorylated and non-

phosphorylated 40 S ribosomal subunits

The 40 S ribosomal subunits, 13.2pg (rRNA), were preincubated just as described in Table I. The reaction was stopped by cooling and the binding of Escherichia coli [$H]Phe-tRNA to 1.32rg of the preincubated 40 S subunits was measured at 5 mM MgClz in the presence of 72 pg of EIF-1.

Preincubation

Phe-tRNA bound0

Protein kinase AI-P

I I

cyclic AMP

fmole

Control, no preincubation 0.378 - - - 0.382 + - + 0.372 + + - 0.386 + + + 0.428

a The binding in the absence of EIF-1 has been subtracted; no binding occurred in the absence of poly(U).

some. Three or more protein factors are required for the initia- tion of a peptide chain (36). Of those factors, it would seem the ability of phosphorylated ribosomes to use two, EIF-1 and EIF-2, is not appreciably different than that of control nonphosphoryl- ated ribosomes. It remained to test the ability of phosphoryl- ated ribosomes to use EIF-3, the factor that probably catalyzes binding of mRNA to the 40 S subunit. The test we chose for that purpose was the translation of EMCV RNA. In the proper conditions eukaryotic ribosomes will translate EMCV RNA with fidelity, for virus-specific peptides are synthesized in vitro

(20). However, we encountered a serious complication. There is

no appreciable translation of EMCV RNA if aminoacyl-tRNA is used as substrate instead of amino acids.2 That means that one must use enzyme preparations that contain aminoacyl- tRNA synthetases and elongation and initiation factors to test translation of EMCV RNA by phosphorylated ribosomes. All of those enzymes are present in ascites cell cytosol (G-25 frac-

2 C. Eil and I. G. Wool, unpublished observation.

by guest on February 25, 2020http://w

ww

.jbc.org/D

ownloaded from

5134

tion), but unfortunately it also contained appreciable amounts of ribosomal phosphoprotein phosphatase and for that reason was not satisfactory for our purposes. Initiation factors, elon- gation factors, and aminoacyl-tRNA synthetases, having re- duced phosphoprotein phosphatase activity, could be prepared from ascites cells if they were homogenized in a medium of low ionic strength. When that procedure was followed the initiation factors remained bound to the ribosomes,3 while the bulk of the phosphatase activity was in the supernatant (results not shown). Aminoacyl-tRNA synthetases and elongation factors could then be prepared from the supernatant by ammonium sulfate frac- tionation. The preparation had a tolerable amount of phospha- tase activity. Initiation factors (36) were extracted from ascites ribosomes with 0.5 M KC1 (ribosomal salt-wash preparation). The salt-wash preparation had phosphatase activity also. In control assays the phosphoprotein phosphatase in the amino- acyl-tRNA synthetase and elongation factor preparation, and in the salt-wash removed 25 to 30% of the radioactive phosphate in the ribosomal subunits. Our efforts to reduce further the amount of phosphoprotein phosphatase resulted in a loss of ability of the preparations to catalyze translation of EMCV RNA.

The assay of the translation of EMCV RNA was for two time periods, 20 or 45 min: at 20 min the amount of phosphate re- leased from ribosomal protein (25%) was somewhat less than at 45 min (30%), but so too was the amount of protein that was synthesized. The reaction was carried out with limiting (5 pg) or saturating amounts (15 pg) of initiation factors (ribosomal salt-wash). In none of these circumstances (either after 20 or 45 min or with limiting or saturating amounts of initiation fac- tors) was there a consistent or appreciable difference in the ability of phosphorylated and nonphosphorylated ribosomes to trans- late EMCV RNA (Table III).

DISCUSSION

We did not find an appreciable and consistent difference in the activity of phosphorylated and nonphosphorylated ribosomes. The ability of phosphorylated ribosomes to use the elongation factors (EF-1 and EF-2) was assessed by measuring the synthesis of polyphenylalanine at high concentrations (12.5 mM) of mag- nesium. The assay provides a gauge of the ability of ribosomes to bind aminoacyl-tRNA, to catalyze peptide bond formation, and to carry out the translocation of peptidyl-tRNA. We found no difference in the competence of phosphorylated and nonphosphorylated ribosomes for the elongation reactions in polyphenylalanine synthesis. It is important that the test was made in two ways: where ribosomes limited synthesis and where elongation factors were limiting. The latter tested the ability of phosphorylated ribosomes to use reduced amounts of factors and is probably a more critical trial of the effect of phosphoryla- tion on ribosome function.

We next examined seriatim the ability of phosphorylated ribosomes to use initiation factors. The binding of Phe-tRNA to the 40 S ribosomal subunit, a reaction that is catalyzed by EIF-1 and is a paradigm for the formation of a proper initiation complex, was increased 13a/, if the particle was phosphorylated. The ability of ribosomes to use a second initiation factor, EIF-2, can be determined by measuring the synthesis of polyphenyl- alanine at low concentrations of magnesium (6 mM). The reac- tion requires EIF-I, EF-I, and EF-2 in addition to EIF-2. The synthesis of polyphenylalanine was increased by 18% if the ribo-

3 R. S. Ranu and I. G. Wool, unpublished observation,

T:m~n III

Translation of EMCV RNA by phosphorylated and nouphosphorylaled ribosomes

Ribosomal subunits, 28.5 pg (rRNA) of 40 S or 71.5 pg (rRNA) of 60 S, were preincubated for 25 min at 37” in 0.1 ml of Medium E containing 0.1 mM ATP, low5 M cyclic AMP, and 16 fig of HA-pro- tein kinase I. In some experiments the ATP was omitted from the preincubation. The reaction was stopped by cooling on ice, and 2.85 pg of 40 S and 7.15 fig of 60 S were combined and incubated for either 20 or 45 min at 37” in 0.05 ml of buffer (20 mM Tris-HCl (pH 7.7), 125 mM KCl, 5 mM magnesium acetate, 10 mM MSH) which contained 1 mM ATP, 0.1 mM GTP, 0.05 mM of each of 19 unlabeled amino acids, 0.003 mM [“H]phenylalanine, 90 rg of creatine phosphate, 10 pg of creatine phosphokinase, 4 rg of EMCV RNA, 8 pg of deacylated rat liver tRNA, 150 rg of ammo- nium sulfate-fractionated ascites cell cytosol which contains aminoacyl-tRNA synthetases and elongation factors, and 5 or 15 pg of ascites cell ribosomal salt-wash which has initiat,ion factors.

Preincubation with ATP

-

+ - +

-

Incubation

Time

min

20 20 20 20 20 45 45 45 45 45

-

ISMCV Ribosomal RSA salt-wash

- + + + + - + + + +

fig 5 or 15

5 5

15 15

5 or 15 5 5

15 15

I’ HlPhenylalanine incorpornted

pmozes

0.199 0.155 0.198 0.600 0.552 0.235 0.497 0.461 1.194 1.303

somes were phosphorylatcd and present in amounts which limited the reaction. However, thcrc was no difference in the synthesis of polyphenylalanine if the initiation factors rather than the phos- phorylated ribosomes were limiting. The failure to find a dif- ference in the latter circumstance mitigates our confidence in the importance of the findillg.

The most critical test of the function of phosphorylated ribo- somes that we carried out was of the ability of the particles to use the third initiation factor EIF-3 to translate a natural tem- plate, EMCV RNA. The reaction, of course, also requires the other initiation and elongation factors. Unfortunately the con- ditions we used for the assay wcrc less than optimal. A com- promise had to be struck bctwcen the need to reduce ribosomal phosphoprotein phosphatase activity and the best conditions for synthesis of virus-specific peptides. Synthesis is greatest in the presence of crude ascites cell cytosol, but that preparation con- tains a good deal of ribosomal phosphoprotein phosphatasc. Fractionation of the cytosol Icduced phosphatase activity and the translation of EMCV RNA as well. The assay was finally conducted with enzyme preparations that had some phosphatase activity but supported translation of EMCV RNA. The less than satisfactory circumstancc,s of the assay aside, there was no appreciable, consistent diffcrcncc in the translation of EMCV RNA by phosphorylated and rlonphosphorylated ribosomes. As with all negative results the failure to observe a meaningful effect of phosphorylation on ribosomal function may well have been due to unsuitable experimental conditions.

This has been to our knowledge the first test of the effect of phosphorylation on the function of cukaryotic ribosomcs in vitro. There are, on the other hand, several reports correlating

by guest on February 25, 2020http://w

ww

.jbc.org/D

ownloaded from

~~l~os~~l~orylatioii with :lltc:ratiolls of ribosomc function in vivo. Kabat (2) found that tlra 60 S ril)osomal subunit protein Si is ollly phosphorylated in singk ribosomcs. The presumption from that observation was that l,llospllorylatioi1 of Si inacti- vated the particles ant1 prcvciitotl the monomers from partici- pating in the ribosomo c.yclc. It ~rc~ds emphasizing that it was not directly shown that ribosomcs with a phosphorylated Si protein were inactive and, in fact, the correlation was subse- quently found (10, 11) not to bc compelling. IIlat and Loeb (6) found that glucagon ildlni,list,rat,iol~ to rats led to an increase in the phosphorylation of a single liver ribosomal protein band (M). Glucagon administration is known to induct several hepatic cnzymcs among which arc tyrosillc aminotransferase (37) and serinc dchydratase (38). The t&it assumption would seem to be that l.‘hosphorylatiol1 of the x4 protein (band) in some way conditions the binding and trallslatiol~ of thr mRNA for specific tt~lzyn~s. The rescrvatioll one 11:~s n-it11 the cxperimcnts and the illtcrpretation is that the ribosomcs may not have been washed sufficiently carefully in high ionic strength solutions to rcmovc all nonribosomal contaminant,s; thus, the M-band may not b(, a ribosomal protein. Some 40% of the 3T radioact,ivity assoc%Ltcd with reticulocytc ribosomcs is present in ten or so different loosely bound phosphoprotcins that can be removed if the ribosomes are washed in high ionic strength solutions (2). Careful washing is a necessary precaution for in vivo experiments. Li and Amos (7) found that the extent of the phosphorylation of fibroblast ribosomes in vitro by a protein kiuase was conditioned by the nature of the media in which the cells were grown; ribo- somcs from cells cultured in scram wcrc a bet.ter substrate than those grown in protein-free media. However, the difference was lost when the ribosomrs wcrc purified by treatment with dcoxy- cholatc and 0.4 M KCl. Thus it is unlikely that a functionally sigllificaant change in the ribosomc was to be attributed to a change> in phosphorylation of a ribosomal protein. Correze et al. (9) found that thyroidcctomy of rats reduced the phosphorus contrnt by 357; alit1 tlccrcascd t,hc: phosphorylation of liver ribosomes on administration of [ “‘l’]ortllol)hosphate. Thyroid hormollc increases the synthesis of protein in the liver (39); the suggestion is made (9) that the il1cr(‘asc could flow from phos- phorylntion of ribosomcs. Once again the ribosomes were not shown to be free of l)hosl)liol”‘otc~ill contaminants before the l)utatiT-c ribosomal protcitls wcrc cstractcd, nor were the radio- active 1)hosphoproteilis shown to migrate with ribosomal proteins on c,lcctrophoresis. Thcrc arc thou a luunbcr of reports de- scribing experiments, more or less rigorously controlled, in which the phosphorylation of ribosomal l)rotoins is correlated with a llutatirc change in ribosomc function. Correlation is not syn- onymous with causatioll, and in no itlstallcc has the function of the ribosome actually been shop to bc altered, no less that a change in ribosome function was tluc to phosphorylation of a protein.

OIIO might push the analysis 0110 stq) further and ask whether the evidence is strong that l~llosl~l~o~~ylatio~~ of ribosomal proteins actually occurs in eukaryotic cells. I f one uses satisfaction of tha tllree criteria previously givcll (12) as the standard, one must conclude t.hat it is almost ccrtaiu that phosphorylation of reticulocyte ribosomal proteins OU:UIX in vivo (2, 10) ; likely, but less certain, that rat liver ribosomal proteins are phosphorylated in vivo (I, 6, 9). The uuccrtainty in the latter experiments de- rives from a failure to establish d(,finitcly that all contaminating phosphoproteins were rcmovcd bcforc the ribosomal proteins were cstracted. The same rcscrvatioll pertains to the experi- mctrts with reticulocyte ribosomrs (2, LO) which were washed

5135

with buffers containing 0.25 31 KC1 and 10 mhl hfg$12. The concentration of KC1 may not hare been sufficiently high to rc- move nollribosomal proteins sucl~ as initiation factors. It is perhaps apropos that bacterial ribosomal proteins are not phos- phorylated. Escherichia coli ribosomes and isolated proteins can be phosphorylatcd in vitro with rabbit muscle protein kinase (40) ; howcvcr, Gordon (41) failed to find incorporation of 321’ from orthophosphatc into ribosomal proteins during growth of the bacteria.

I f we assume for the moment that eukaryotic ribosomes are phosphorylated in vivo and that phosphorylation alters their activity, how then do WC rationalize our failure to detect the change in vitro. It is possible that liver ribosomes are nearly maximally phosphorglated when they are isolated, and that the phosphorylation that occurs in vitro is, thcreforc, only a small fraction of that which can occur. WC have not attempted to remove phosphate from ribosomes with phosphatase and to study the function of those part.iclcs. It is conceivable that the protein kinases we used arc not the physiologically relevant ribosomal protein kinas-s and thus the proper serine and threo- nine residues were not phosphorylated in our experiments. Moreover, it may be necessary to phosphorylate and dcphos- phorylate ribosomal proteins cyclically to alter function, and that may require the coupling of ribosomal phosphoprotein phospha- tase to the kinase; one or both enzymes may bc lacking from our in vitro assay.

It is a distinct possibility that me have failed to detect an effect of 1)hosphorylation on ribosome function because WC have not tested the proper function. Among the many functions we have not analyzed arc : the translation of specific cellular mRNAs; the binding of ribosomes to the endoplasmic reticulum; and the tcr- mination of protein synthesis. The suggestion that phospho- rylation of ribosomal proteins might affect those functions is not frivolous. Finally, our assays may not have been carried out in the proper way or may have lacked some essential factor.

1.

2. 3. 4.

5.

G.

7.

8.

9.

10. 11.

12. 13.

14.

I,oI~:B, J. 15., AR’I) BUT, C. (1970) Fed. Eul. Hiochem. Sot. I,elt. 10, 105-108

K.\IJ.\T, J>. (1970) IGochenzistry 9, 41W4175 K.UUT, I). (1971) Hiochemislry 10, 197-203 WALTON, C. iU., GILL, G. N., AISRGS, I. G., AR’D Ga~wx,

L. I>. (1971) 1’1.0~. Xat. Acad. Sci. U. S. A. 68,880-884 EIL, C., >LND Wool,, 1. G. (1971) Biochem. Hiophys. Res, Com-

mun. 43, 100-1009 BLOT, C., >\ND Lam, J. 15. (1971) Fed. &UT. Biochem. Sot. Lett.

18, 124-126 LI, C.-D., axn Autos, H. (1972) Biochem. Biophys. Res. Com-

mun. 46, 1398-1407 YAM,\MUIU, I-I., INOUIS, Y., SHIMOMUIU, II., ‘\ND NISIIIZUIU,

Y. (1972) Biochem. Bioph,ys. Res. Commun. 46, 589-596 CORI~EZI~, C., PINELL, P., AND NUNEZ, J. (1972) Fed. Eur. Uio-

them. Sot. Lell. 23, 87-91. K,IB~T, I>. (1972) J. Biol. Chem. 247, 5338-5344 BITT>;, L., ~XI) KIIUT, I>. (1972) J. Bid. Chem. 247, 5345-

5350 EIL, C., .~ND Wool,, I. G. (1!)73) J. Biol. Chem. 248, 5122-5129 GLYNN, I. iV., .a~ CH~ITELL, J. I!%. (19G4) Biochem. J. 90,

147-149 WOOI,, I. G., .IND C.~v~cwr, 1’. (196B) Biochemislry 6, 1231-1242

15. ~~LIILTIN, T. I<., : IXD WOOL, I. G. (1!%9) J. 1MoZ. Viol. 43,151-161 16. MARTIN, T. 15., .IND WOOL, I. G. (19tiS) 1’1.0~. Mat. Acad. Sci.

U. S. A. 60, 569-574 17. STIRISW.~LT, W. S., Cas~r,ws, J. J., AND Wool,, I. G. (1971)

Biochemistry 10, 1594-1598 18. ~~~~~~~~~~~~ M. S. (1971) Anal. Uiochem. 41, 12G-131 lg. Wool,, I. G., .LSI) C.~IC~III, P. (19%) I’wc. Xat. Acad. Sci.

U. S. A. 66, 991-998

by guest on February 25, 2020http://w

ww

.jbc.org/D

ownloaded from

5136

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

?\lz~~~~~.:n-~, M. B., ‘END K~RXI~R, A, (1970) Eur. J. Biochem. 17, 328-338

YANG, W.-K., AND NOVELLI, G. D. (1968) Proc. Nat. Acad. Sci. U. S. A. 69, 208-215

MUENCH, It. I-T., AND BERG, P. (1966) in Procedures in Nucleic Acid Research (CANTONT, G. L., AND DAVIES, D. R., eds) p. 375, Harper and Row, Publishers, Inc., New York

VON EHRENSTEIN, G., AND LIPMANN, F. (1961) Proc. Nat. Acad. Sci. U. S. A. 47, 941

LEADER, D. P., KLEIN-BREMHAAR, H., WOOL, I. G., AND Fox, A. (19721 Biochem. Biovhus. Res. Commun. 46.215-223

SCHN&R, h., AND MOL~A;E, K. (1968) Biochim. Biophys. Acta 166, 58-67

LIUDER, I>. P., AND WOOL, I. G. (1972) Biochim. Biophys. Acta 262, 360-370

BELLETT, A. J. D., AND BURNESS, A. T. H. (1963) J. Gen. Microbial. 30, 131

CASTLES, J. J., AND WOOL, I. G. (1970) Biochemistry 9, 1909- 1961

AVIV, H., BOIME, I., AND LEDI~R, P. (1971) Proc. Nat. Acad. Sci. U. S. A. 68, 2303-2307

KI~I~R, I. M., COHEN, N., AND WORK, T. S. (1966) Biochem. J. 93, 826-835

31. LEADER, D. P., WOOL, I. G., AND CASTLES, J. J. (1970) Proc. Nat. Acad. Sci. U. S. A. 67, 523-528

32. LOWRY, 0. F ROSISBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (195;, J. Biol. Chem. 193,265275

33. WARRURG, O., -END CHRISTIAN, W. (1942) Biochem. 2.310,384 34. N&LUND, P. H., AND HULTIN, T. (1970) Biochim. Biophys.

Acta 204, 237-247 35. SHAFRITZ, D. A., AND ANDERSON, W. F. (1970) J. Biol. Chem.

246, 5553-5559 36. PRICHARD, P. M., GILBERT, S. M., SHAFRITZ, D. A., AND AN-

DERSON, W. F. (1970) Nature 227, 511-514 37. WICKS, W. D., KICNNEY, F. T., AND LEE, K. L. (1960) J. Biol.

Chem. 244, 6008-6013

41. GORDON, J. (1971) Biochem. Biophys. Res. Commun. 44, 579- 586

38. JOST, J. P., HSIL, A. W., AND RICI~ENBERG, H. V. (1969) Bio- them. Biophys. Res. Commun. 34, 748-754

39. TATA, J. R. (1964) in Action of Hormones on &Iolecular Processes (LIT~ACI~, G., AND KRITCHEVSKY, D. eds) pp. 58-131, John Wiley and Sons, New York

40. TRAUGH, J. A., AND TRAUT, R. It. (19i2) Biochemistry 11, 2503-2509

by guest on February 25, 2020http://w

ww

.jbc.org/D

ownloaded from

Charles Eil and Ira G. WoolSUBUNITS PHOSPHORYLATED IN VITRO BY PROTEIN KINASE

Function of Phosphorylated Ribosomes: THE ACTIVITY OF RIBOSOMAL

1973, 248:5130-5136.J. Biol. Chem. 

  http://www.jbc.org/content/248/14/5130Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/248/14/5130.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on February 25, 2020http://w

ww

.jbc.org/D

ownloaded from