THE JOURNAL OF BIOLOGICAL CHEMISTRY ( ~ 1 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 19, Issue of July 5, pp. 12574-12580, 1991 Printed in U.S. A.

Phosphorylation of Elongation Factor 1 (EF-1) and Valyl-tRNA Synthetase by Protein Kinase C and Stimulation of EF-1 Activity*

(Received for publication, March 11, 1991)

Richard C. Venema, Holme I. Peters, and Jolinda A. TraughS From the Department of Biochemistry, University of California at Riverside, Riverside, California 92521

A high M, complex isolated from rabbit reticulocytes contains valyl-tRNA synthetase and the four subunits of elongation factor 1 (EF-1). Previously, valyl-tRNA synthetase and the a, 8, and 6 subunits of EF-1 were shown to be phosphorylated in reticulocytes in re- sponse to phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the complex was accompanied by an increase in both valyl-tRNA synthetase and EF-1 activity (Venema, R. C., Peters, H. I., and Traugh, J. A. (1991) J. Biol. Chem., 266, 11993-11998). To in- vestigate phosphorylation of the valyl-tRNA synthe- tase .EF-l complex in v i tro by protein kinase C, the complex has been purified to apparent homogeneity from rabbit reticulocytes by gel filtration on Bio-Gel A-5m, affinity chromatography on tRNA-Sepharose, and fast protein liquid chromatography on Mono Q. Valyl-tRNA synthetase and the 8 and 6 subunits of EF- 1 in the complex are highly phosphorylated by protein kinase C (0.5-0.9 mol of phosphate/mol of subunit), while EF-la is phosphorylated to a lesser extent (0.2 mol/mol). However, the isolated EF-la subunit is highly phosphorylated (2.0 mol/mol). Phosphopeptide mapping of EF- l a shows that the same sites are modi- fied by protein kinase C in v i tro and in PMA-treated cells. Phosphorylation of the valyl-tRNA synthetase. EF- 1 complex results in a %fold increase in activity of EF- 1 as measured by poly(U)-directed polyphenylalan- ine synthesis; no effect of phosphorylation is detected with valyl-tRNA synthetase and isolated EF-la. Thus, phosphorylation and activation of EF-1 by protein ki- nase C, which has been shown to occur in vitro as well as in reticulocytes, may have a role in PMA stimulation of translational rates.

Valyl-tRNA synthetase from mammalian tissues is isolated exclusively as part of a high M , complex with the four subunits of elongation factor 1 (EF-l).’ The valyl-tRNA synthetase. EF-1 complex has been purified from rabbit liver (1-4) and rabbit reticulocytes (5) and contains all of the reticulocyte valyl-tRNA synthetase activity and up to half of the total EF- 1 activity (5). Three distinct catalytic activities, each of which is potentially rate-limiting in the elongation phase of protein biosynthesis, are contained within the five polypeptides of the complex. Valyl-tRNA synthetase ( M , - 130,000) catalyzes

* These studies were supported by United States Public Health Service Grant GM21424. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. ’ The abbreviations used are: EF, elongation factor; eIF, eukaryotic

initiation factor; PMA, phorbol If-myristate 13-acetate; FPLC, fast protein liquid chromatography.

aminoacylation of tRNA””’. EF- l a (Mr - 50,000) binds to and delivers aminoacyl-tRNA to the A site on the ribosome (6-8). As with other guanine nucleotide-binding proteins, the reac- tion catalyzed by EF- l a depends on hydrolysis of tightly bound GTP. The inactive, GDP-bound form of the factor must be recycled to the active, GTP-bound form before an- other round of elongation can occur. The @ (M, - 30,000) and y (M, - 52,000) subunits of EF-1 (6-8), and possibly the 6 (M, - 40,000) subunit (9), catalyze the exchange of GDP bound to EF-la with GTP.

In a previous report (5), we showed that valyl-tRNA syn- thetase and the a, p, and 6 subunits of EF-1 are phosphoryl- ated in reticulocytes incubated with ‘“Pi. Phosphorylation of each of the four proteins is increased 2-4-fold in response to phorbol 12-myristate 13-acetate (PMA). Phosphorylation of valyl-tRNA synthetase in response to PMA is reproducibly accompanied by a 1.7-fold increase in aminoacylation activity, whereas phosphorylation of EF-1 is associated with a 2.0-2.2- fold increase in activity, as measured by poly(U)-directed polyphenylalanine synthesis. Phorbol esters have also been shown to stimulate phosphorylation of glutamyl-tRNA syn- thetase in another high M , synthetase complex, the synthetase core complex, which contains aminoacyl-tRNA synthetase activities specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline (10). Phosphorylation of glutamyl-tRNA synthetase is asso- ciated with a 40% reduction in aminoacylation activity.

Phorbol esters substitute for the second messenger, diacyl- glycerol, in activating one or more isozymes of the Ca2+/ phospholipid-dependent protein kinase, protein kinase C (11). Activation of protein kinase C by phorbol esters mimics many of the effects of growth factors on intact cells through phos- phorylation of substrates involved in regulation of cell growth and differentiation. Our studies on PMA-stimulated phos- phorylation i n vivo have therefore been extended to an ex- amination of phosphorylation of the valyl-tRNA synthetase. EF-1 complex by protein kinase C i n vitro. The complex has been purified from rabbit reticulocytes and used as substrate for purified protein kinase C . Valyl-tRNA synthetase and the a, p, and 6 subunits of EF-1 in the complex, which were phosphorylated in reticulocytes in response to PMA, were also phosphorylated by protein kinase C i n vitro. The effects of phosphorylation on activity of the valyl-tRNA synthetase. EF-1 complex were also examined. Although no effect on aminoacylation activity of valyl-tRNA synthetase was de- tected, EF-1 activity, as measured by poly(U)-directed poly- phenylalanine synthesis, was stimulated by up to 3-fold.

EXPERIMENTAL PROCEDURES

Materials-’H-Labeled-L-valine, L-phenylalanine, and L-lysine were purchased from ICN. [y:’2P]ATP was obtained from Amersham Corp. Brewers’ yeast tRNA was from Boehringer Mannheim, and Bio-Gel A-5m (200-400 mesh) was purchased from Bio-Rad. Ampho-

12574

Phosphorylation and Activation of EF-1 12575

lytes for isoelectric focusing and nonequilibrium pH gradient electro- phoresis were from Pharmacia LKB Biotechnology Inc. EF-1 from rabbit reticulocytes, used to identify the subunits of EF-1 on two- dimensional gels, and polyclonal antibodies to rabbit EF-1 (raised in goat) were generously provided by Dr. William C. Merrick (Case Western Reserve School of Medicine, Cleveland, OH). EF-1 was purified from rabbit reticulocytes in our laboratory by gel filtration on Bio-Gel A-5m, affinity chromatography on tRNA-Sepharose, and FPLC on a Mono Q HR 5/5 column (12).* Protein kinase C was purified from beef brain by the method of Walton et al. (13) followed by FPLC on Mono Q as described previously (10).

Purification of the Valyl-tRNA Synthetase. EF-1 Complex-The valyl-tRNA synthetase.EF-1 complex was purified from rabbit retic- ulocytes by gel filtration on Bio-Gel A-5m and affinity chromatogra- phy on tRNA-Sepharose as described previously (5), except that the phosphatase inhibitor, 8-glycerophosphate, was omitted from the buffers. Further purification was achieved by FPLC on a Mono Q HR 5/5 column equilibrated in Buffer A (25 mM Tris-HC1, pH 7.5 a t 4 "C, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 10% (v/v) glycerol, 0.02% NaN:I, and 0.5 mM phenylmethylsulfonyl fluoride). Fractions from tRNA-Sepharose chromatography containing the valyl-tRNA synthetase. EF-1 complex were pooled, concentrated to 0.5-1.0 ml by ammonium sulfate precipitation (70% of saturation), and dialyzed overnight against Buffer A. Dialyzed protein was applied to the Mono Q column and, following washing of the column with 10 ml of Buffer A, was eluted with a 40-ml linear gradient of NaCl(0-1.0 M ) in Buffer A. One-ml fractions were collected and assayed for valyl-tRNA syn- thetase and EF-1 activity. The identity and purity of the valyl-tRNA synthetase. EF-1 complex and the dissociated a subunit of EF-1 were determined by polyacrylamide gel electrophoresis.

Assays for Aminoacyl-tRNA Synthetases and EF-1-Valyl- and lysyl-tRNA synthetase activity were determined by measuring the aminoacylation of unfractionated brewers' yeast tRNA, during puri- fication, or rabbit reticulocyte tRNA with 3H-labeled amino acids, as described previously (5). One unit of valyl-tRNA synthetase activity refers to the formation of 1 nmol of valyl-tRNA/min a t 25 "C.

EF-1 activity was assayed by measuring the rate of polyuridine- dependent ["H]polyphenylalai~ine synthesis as previously described (5). One unit of EF-1 activity is defined as the formation of 1 nmol of polyphenylalanine/min a t 37 "C.

Phosphorylation of the Valyl-tRNA Synthetase. EF-1 Complex by Purified Protein Kinase C-Highly purified valyl-tRNA synthetase. EF-1 complex (5 pg), EF-1 (5 pg), and EF-1 a (2 pg) were phosphoryl- ated in uitro by 20 units of protein kinase C and analyzed by polyacrylamide gel electrophoresis and autoradiography as described previously (14). One unit of protein kinase C activity was identified as the amount of enzyme that catalyzes the incorporation of 1 pmol of inorganic phosphate from [y-"P]ATP/min into histone 1 a t 30 "C.

To assess the effects of phosphorylation on EF-1 and valyl-tRNA synthetase activity, the complex was phosphorylated with 150 units of protein kinase for 30 min at 30 "C with radioactive or nonradioac- tive ATP. Nonradiolabeled samples were analyzed for EF-1 and valyl- tRNA synthetase activity as described above, and radiolabeled sam- ples were analyzed for phosphate incorporation following gel electro- phoresis.

Labeling of EF-la with "PC in PMA-treated Rabbit Reticulocytes- Rabbit reticulocytes were labeled with '2Pi for 2 h followed by an additional 30-min incubation in the presence of 1 b~ PMA as de- scribed previously (10). The valyl-tRNA synthetase. EF- l complex was purified by gel filtration and tRNA-Sepharose chromatography; all buffers contained 50 mM 8-glycerophosphate as a phosphatase inhibitor.

Gel Electrophoresis and Autoradiography-One-dimensional poly- acrylamide gel electrophoresis in sodium dodecyl sulfate was per- formed according to Laemmli (15) with either 7.5 or 10% gels. Two- dimensional isoelectric focusing/polyacrylamide gel electrophoresis was carried out according to the procedure of O'Farrell (16) and two- dimensional nonequilibrium pH gradient/polyacrylamide gel electro- phoresis was performed according to O'Farrell et al. (17). Gels were stained with Coomassie Brilliant Blue R, destained, and dried. Au- toradiography was performed by exposing medical x-ray film to gels in cassettes lined with intensifying screens (Kodak X-Omatic). Stoi- chiometry of phosphorylation of individual proteins was quantified by liquid scintillation counting of the excised protein bands.

Two-dimensional Phosphopeptide Mapping of EF-1 cu-Two-dimen- sional phosphopeptide mapping was carried out as described previ-

' R. C. Venema and J. A. Traugh, manuscript in preparation.

ously by Tuazon et al. (14). Electrophoresis in the first dimension was carried out for 1 h a t 600 V in pyridine:acetic acidwater (10:0.4:90), pH 6.5. Ascending chromatography in the second dimen- sion was carried out for 3.5 h in butano1:acetic acidwater (3:l:l). Tryptic phosphopeptides were detected by autoradiography.

Protein Determination-Protein concentrations were estimated by the method of Bradford (18) with 7-globulin as a standard.

RESULTS

The Valyl-tRNA Synthetase. EF-1 Complex-To analyze the valyl-tRNA synthetase complex and characterize the var- ious M, forms of EF-1, postribosomal supernatant from rabbit reticulocytes was subjected to gel filtration on Bio-Gel A-5m. Fractions were analyzed for EF-1, valyl-tRNA synthetase, and lysyl-tRNA synthetase activity. The latter activity was used to identify the synthetase core complex. All of the lysyl- tRNA synthetase eluted in a single peak of M , - 1.0 x lo6 (Fig. 1). All valyl-tRNA synthetase activity eluted in a distinct but overlapping peak of -0.8 X lo6. A significant portion of EF-1 activity (20%) eluted as a high M , peak, which coincided exactly with the valyl-tRNA synthetase activity (Fig. 1). Several lower M, forms of EF-1 were also observed upon gel filtration, with the lowest M , form having the M , expected for the free EF-la subunit (50,000).

To separate the two synthetase complexes, high M, frac- tions containing the valyl-tRNA synthetase. EF-1 complex and the synthetase core complex were chromatographed on tRNA-Sepharose (Fig. 2). The valyl-tRNA synthetase. EF-1 complex eluted between 75 and 120 mM NaC1, and the syn- thetase core complex eluted between 120 and 220 mM NaC1. All of the EF-1 activity applied to the column coeluted with valyl-tRNA synthetase; no activity was detected in the ma- terial that did not adhere to the column. Thus, all of the high M , form of EF-1 obtained by gel filtration was complexed with valyl-tRNA synthetase and did not represent a high M , self-aggregate of EF-1. When pooled fractions from tRNA- Sepharose corresponding to the valyl-tRNA synthetase. EF-1 complex and the synthetase core complex were analyzed on polyacrylamide gels, both high M, complexes were found to be highly purified (>go% pure) (Fig. 3, lunes 3 and 4 ) . The core complex contained nine major polypeptide bands, which correspond to eight identified tRNA synthetase activities (19),

150

LysRS ValRS EF-1

0 20 4 0 60 80 100

FRACTION NUMBER

FIG. 1. Separation of the valyl-tRNA synthetase. EF- 1 com- plex from low M, forms of EF-1 by gel filtration on Bio-Gel A-5m. Postribosomal supernatant (25 ml) from rabbit reticulocytes was subjected to gel filtration on a Bio-Gel A-5m column. Aliquots (60 pl) were assayed for valyl-tRNA synthetase (0, ValRS), lysyl- tRNA synthetase (A, LysRS), and EF-1 (0) activity. The column was calibrated with blue dextran (BDx, M, 2 X lofi), thyroglobulin (ThG, M , 670,000), and hemoglobin (Hb, M, 64,000). Pooled fractions are indicated by the bar.

12576 Phosphorylation and Activation of EF-1

0 1 0 2 0 3 0 4 0

FRACTION NUMBER

FIG. 2. Separation of the valyl-tRNA synthetase .EF- 1 com- plex from the synthetase core complex by tRNA-Sepharose chromatography. High M, fractions from gel filtration on Bio-Gel A-5m were pooled and applied to a tRNA-Sepharose column. Frac- tions were assayed for valyl-tRNA synthetase (0), lysyl-tRNA syn- thetase (A), and EF-1 (0) activity. Pooled fractions for each of the high M, synthetase complexes are indicated by the bars. Abbreviations are as for Fig. 1.

- " V a l R S

E F - l Y I j53 - EF- Ip

1 2 3 4 5 6

FIG. 3. Analysis of the purity of the synthetase complexes by polyacrylamide gel electrophoresis. Proteins (5-10 pg) from each chromatographic step were subjected to polyacrylamide gel electrophoresis followed by staining with Coomassie Blue. Lane 1 , postribosomal supernatant; lane 2, valyl-tRNA synthetase ( ValRS) pooled from Bio-Gel A-5m; lane 3, valyl-tRNA synthetase-EF-1 complex from tRNA-Sepharose; lane 4, synthetase core complex from tRNA-Sepharose, lane 5, EF- la from FPLC on Mono Q; lane 6, valyl- tRNA synthetase.EF-1 complex from FPLC on Mono Q.

and a 37-kDa protein, previously identified as an inactive form of casein kinase I (20). The purified valyl-tRNA synthe- tase EF-1 complex contained five major polypeptides identi- fied as valyl-tRNA synthetase (130 kDa) and the four subunits of EF-1 (52,50,40, and 30 kDa) (1-5).

At this stage of purification the valyl-tRNA synthetase. EF-1 complex still contained minor protein contaminants, including a small amount of protein kinase activity. There- fore, the complex was subjected to FPLC on a Mono Q column. With this chromatographic step, two different forms of EF-1 activity were obtained. A small but broad peak of activity eluted between 150 and 400 mM NaCl and a second, larger peak of EF-1 activity eluted a t about 600 mM NaC1, exactly coincident with the single peak of valyl-tRNA synthetase activity (Fig. 4, panel A ) . Analysis of column fractions by one- dimensional (Fig. 4, panel B ) and two-dimensional gel elec- trophoresis (data not shown) revealed that the first EF-1 activity peak represented the dissociated a subunit of EF-1. Between 60 and 90% of the EF-la subunit was dissociated during this step. Valyl-tRNA synthetase, the p, y, and 6 subunits of EF-1, and 10-40% of EF-la eluted with the second, larger EF-1 activity peak (Fig. 4, panel B) . No valyl- tRNA synthetase or EF-1 activity was detected in the material

A 3 4 ValRS-EF-1 -

0 1 0 20 30 4 0

FRACTION NUMBER

- V o l R S

/ E F - l Y

LEF- la - E F - 16 - E F - IP

FIG. 4. Purification of the valyl-tRNA synthetase.EF-1 complex and EF-la by FPLC on a Mono Q HR 5/5 column. The valyl-tRNA synthetase. EF-1 complex from chromatography on tRNA-Sepharose was concentrated by ammonium sulfate precipita- tion, dialyzed, and applied to a Mono Q HR 5/5 column. Panel A, aliquots (60 pl) of fractions were assayed for valyl-tRNA synthetase ( VulRS) and EF-1 activity; panel H , analysis of aliquots (30 pl) of fractions by polyacrylamide gel electrophoresis and staining with Coomassie Blue.

that did not adhere to the column. The purification of the valyl-tRNA synthetase. EF-1 com-

plex is summarized in Table I. Valyl-tRNA synthetase in the heterotypic complex was purified 850-fold with an overall yield of 49% and a specific activity of 144 nmol/min/mg at 25 "C. EF-1 was purified 175-fold with a 10% yield. The lower yield of EF-1 was due to separation of the low M , forms of EF-1 upon gel filtration. The complex, highly purified after two chromatographic steps, was homogeneous after the third step as judged by failure to detect any other protein bands either by Coomassie staining (Fig. 3, lane 6 ) or by silver staining (data not shown). In addition, only a single protein of -50 kDa was detected by Coomassie (Fig. 3, lane 5) and silver staining (data not shown) in fractions containing EF- la. Following tRNA-Sepharose chromatography, the relative molar ratios of protein components in the complex were estimated to be 1:1:1:1:1 by densitometric scanning of Coo- massie-stained gels. Following FPLC on Mono Q, the molar ratio of E F - l a in the complex was typically reduced to 0.1- 0.4.

To confirm the identity of the protein components of the valyl-tRNA synthetase. EF-1 complex, purified preparations were analyzed in two different two-dimensional gel systems (Fig. 5). Both systems were utilized, because valyl-tRNA synthetase did not focus in the first dimension during non- equilibrium pH gradient electrophoresis (Fig. 5, panel A ) , as reported previously (2, 5), and EF-la did not focus when conventional isoelectric focusing was employed (Fig. 5, panel B ) , since it is outside the PI range of the gel.

The components of the complex were identified by molec- ular mass and isoelectric point as valyl-tRNA synthetase (130 kDa, PI 6.01, EF-la (50 kDa, PI 9.0), EF-1/3 (30 kDa, PI 5.01, EF-1y (52 kDa, PI 6.0), and EF-16 (40 kDa, PI 5.5). The four

Phosphorylation and Activation of EF-1 12577

TABLE I Purification of ualyl-tRNA synthetase.EF-1 complex from rabbit reticulocytes

EF-1 activity was measured by poly(U)-directed [1H]polyphenylalanine synthesis and valyl-tRNA synthetase activity was measured by aminoacylation of unfractionated tRNA with ["Hlvaline. The specific activity of EF-1 is expressed as nmol/min/mg of total protein or nmol/min/mg of EF-la (in parentheses). The specific activity of valyl-tRNA synthetase is expressed as nmol/min/mg of total protein.

Valyl-tRNA synthetase Elongation factor 1

mg units units/ mg

% -fold % -fold units units/ mg

Postribosomal supernatant 3411 580 0.2 100 1 15.7 0.005 100 1 Bio-Gel A-5m 124 384 3.1 66 18 2.3 0.019 15 4 tRNA-Sepharose 4 308 77 53 520 1.9 0.475 12 114

Mono Q 2 288 144 49 850 1.6 0.810 10 175 (3.2)

(12.2)

A NEPHGE +

"

Acidic Basic

- 31,000

B IEF __*

- 116.000

"97.400

r "68.000

- .- I I I

4 3 61 6.5

PH

FIG. 5. Identification of polypeptide components of the va- lyl-tRNA synthetase.EF-1 complex by two-dimensional gel electrophoresis. Valyl-tRNA synthetase.EF-1 complex was puri- fied by gel filtration and tRNA-Sepharose chromatography. Panel A , the complex (30 pg) was analyzed by nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension and sodium dode- cy1 sulfate-polyacrylamide gel electrophoresis (SDS) in the second dimension. Molecular weight standards run in the second dimension were bovine serum albumin (68,000), creatine kinase (40,000), and carbonic anhydrase (31,000). Protein was visualized with Coomassie Blue. Panel R, the complex (25 pg) was analyzed by isoelectric focusing ( IEF) in the first dimension and sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) in the second di- mension and stained with Coomassie Blue. Molecular weight stand- ards run in the second dimension were myosin heavy chain (205,000), &galactosidase (116,000), phosphorylase b (97,4001, bovine serum albumin (68,000). and creatine kinase (40,000).

subunits of EF-1 in the purified complex comigrated exactly (data not shown) with the subunits of EF-1 purified by Merrick and co-workers (21), although the nomenclature used in this study and described previously (5) is different from that employed by Merrick et al. (21). Furthermore, proteins identified as EF-1 reacted with anti-EF-1 antibodies in en- zyme-linked immunoabsorbent assays and in immunoblotting experiments (data not shown).

Phosphorylation of the Valyl-tRNA Synthetase. EF-1 Com- plex by Protein Kinase C-When highly purified valyl-tRNA

RADIOGRAM AUTO-

I 2 3 4

FIG. 6. Phosphorylation of the valyl-tRNA synthetase.EF- 1 complex by protein kinase C. Valyl-tRNA synthetase ( ValRS). EF-1 complex (5 pg) purified to homogeneity was incubated with 20 units of protein kinase C and [y-:'2P]ATP. Lane 1, protein kinase C alone with Ca'+, phospholipids, and diolein; lane 2, the valyl-tRNA synthetase .EF-1 complex alone with Ca2+, phospholipids, and diolein; lane 3, protein kinase C plus the valyl-tRNA synthetase.EF-1 com- plex; lane 4, protein kinase C plus the valyl-tRNA synthetase-EF-1 complex with Cay+, phospholipids, and diolein.

synthetase.EF-1 complex (partially depleted of EF-la) was phosphorylated by protein kinase C, valyl-tRNA synthetase and the a, p, and 6 subunits of EF-1 were selectively phos- phorylated; no phosphate was incorporated into EF-17 (Fig. 6). Phosphorylation was completely dependent on phospho- lipids, diolein, and Ca2+. Up to 0.5 mol of phosphate was incorporated/mol of valyl-tRNA synthetase, 0.9 mol/mol for EF-lP, and 0.9 mol/mol for EF-16. EF-lcu was phosphorylated to a lesser extent, with 0.2 mol of phosphate incorporated/ mol of the subunit. When the valyl-tRNA synthetase.EF-1 complex purified through the tRNA-Sepharose step (1:l ratio of EF-1 subunits) was phosphorylated by protein kinsae C, the same stoichiometry of phosphorylation was observed (data not shown). This indicates that the extent of phosphorylation of EF-la was not dependent on the stoichiometry of this subunit in the complex.

When the low M , form of EF-1 was examined as substrate for protein kinase C, the same specificity and stoichiometry of phosphorylation was observed (Fig. 7, panel A ) . The @ and 6 subunits were highly phosphorylated, and the a subunit was phosphorylated to a lesser extent. Phosphorylation was com- pletely dependent on phospholipids, diolein, and Ca". In striking contrast to the a subunit in the valyl-tRNA synthe- tase-EF-1 complex or in the low M , form of EF-1, isolated

12578 Phosphorylation and Activation of EF-1

A. B.

RADIOGRAM RADIOGRAM AUTO- AUTO- EF-'y>[=rl EF- la E F - la-[-pl

EF- 16 - " E F - I P - -- 0

1 2 I 2

FIG. 7. Phosphorylation of EF-1 and EF-la in vitro by pro- tein kinase C. Purified EF-1 and EF-la were incubated with 20 units of protein kinase C and [y""P]ATP. Panel A , EF-1 (5 pg); Panel R, EF-la (2 pg). Lane I, EF-1 or EF-la plus protein kinase C; lane 2. EF-1 or EF-la DIUS Drotein kinase C with Ca2+, phospholipids, and diolein.

. .

In v i v o In v l t r o

T - ELECTROPHORESIS __*

FIG. 8. Two-dimensional phosphopeptide maps of elonga- tion factor la. :"P-Labeled valyl-tRNA synthetase. EF-1 complex (5 pg) was purified from PMA-stimulated reticulocytes through the tRNA-Sepharose step (in vivo). The complex purified through the tRNA-Sepharose step (5 pg) was phosphorylated by incubation with 20 units of protein kinase C and [y:'"P]ATP for 15 min (in vitro). Labeled complex was analyzed by polyacrylamide gel electrophoresis, and the EF-la band was excised from the gel, trypsin-digested, and subjected to two-dimensional phosphopeptide mapping. Arrows indi- cate the origins.

EF-la was an excellent substrate for protein kinase C; up to 2.0 mol of phosphate were incorporated/mol of EF- l a (Fig. 7, panel B ) .

Two-dimensional Phosphopeptide Mapping of EF-1 a--"'P- Labeled valyl-tRNA synthetase. EF-1 complex was purified through the tRNA-Sepharose step from PMA-stimulated rab- bit reticulocytes. The nonphosphorylated complex (purified through the tRNA-Sepharose step in the absence of phospha- tase inhibitors) was phosphorylated by protein kinase C in vitro under conditions where 0.2 mol of phosphate was incor- porated/mol of EF-la subunit. Mapping of EF- la , labeled either in vivo or in vitro, generated the same two tryptic phosphopeptides (Fig. 8), suggesting that the PMA-stimulated phosphorylation of EF-la reported previously (5) occurred through direct phosphorylation by protein kinase C.

Effect of Phosphorylation on Valyl-tRNA Synthetase and EF-1 Activity-The possible role of phosphorylation in mod- ulating valyl-tRNA synthetase and EF-1 activity was also investigated. The valyl-tRNA synthetase e EF-1 complex (ex- tensively purified through FPLC on Mono Q and thus par- tially depleted of EF-la), complex highly purified through tRNA-Sepharose and containing the a subunit in 1:l ratio with the other subunits, and isolated EF- la were incubated with phospholipids, diolein, and Ca2' in the presence or absence (control) of protein kinase C. Under these conditions, approximately 0.2, 0.9, and 0.9 mol of phosphate was incor- porated per mol of a, p, and 6 subunit in the complex and 2.0 mol/mol were incorporated into the isolated EF-la subunit. Following phosphorylation, the various substrates were as-

sayed for EF-1 or valyl-tRNA synthetase activity. Care was taken to ensure that activity determinations were kinetically valid. EF-1 activity in the phosphorylated complex was in- creased 3-fold over that of the control (Table 11). However, phosphorylation of isolated EF-la, even to the extent of 2.0 mol of phosphate/mol of subunit, was not associated with a detectable change in activity. Incubation of the complex with heat-inactivated protein kinase C had no effect on activity. Phosphorylation of valyl-tRNA synthetase (0.5 mol/mol) did not result in a detectable change in aminoacylation activity.

DISCUSSION

The valyl-tRNA synthetase-EF-1 complex has been highly purified from rabbit reticulocytes by a rapid and simple pro- cedure that has several advantages over those previously published by Motorin et al. (1) and by Bec et al. (3) for the complex from rabbit liver. This method results in purification of the complex to apparent homogeneity and in high yield after three chromatographic steps (as compared with five for both of the previously published methods). Additionally, the three steps of gel filtration on Bio-Gel A-5m, affinity chro- matography on tRNA-Sepharose, and FPLC on a Mono Q HR 5/5 column allow for the simultaneous purification of the synthetase core complex (containing activities specific for Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met, and Pro) and the a subunit of EF-1, both in high purity and in high yield. The activity of both the valyl-tRNA synthetase and EF-1 during each chromatographic step has not previously been reported and led to the observation that all of the valyl-tRNA synthe- tase activity and at least 20% of the EF-1 activity in reticu- locytes is contained in the high M , complex. When phospha- tase inhibitors are included in the purification buffers, up to 50% of the cellular EF-1 activity copurifies with valyl-tRNA synthetase in a high M , complex (5).

The activity of EF-la in the complex, as measured by poly(U)-directed polyphenylalanine synthesis, is dramatically higher than that of the purified EF-la subunit. Corresponding specific activities are 12.2 and 1.2 nmol/min/mg EF- la a t

TABLE I1 Effects of phosphorylation by protein kinase C on valyl-tRNA

synthetase and EF-I activity The valyl-tRNA synthetase (ValRS) .EF-1 complex was incubated

with (+) and without (-) protein kinase C (PKC), then assayed for valyl-tRNA synthetase and EF-1 activity as described under "Exper- imental Procedures." Either 2 pg of the complex purified through Mono Q (a-reduced), 5 pg of the complex purified through tRNA- Sepharose, or 5 p g of EF-la was used as substrate for protein kinase C. The stoichiometry of phosphate incorporated into each subunit in these experiments was 0.5 mol/mol for ValRS and 0.2, 0.9, and 0.9 mol/mol, respectively, for the a, 8, and d subunits of EF-1 in the complex. Isolated EF-la was phosphorylated to the extent of 2.0 mol/ mol. Values represent the average of duplicates from a representative experiment.

Substrate Protein "H-Phe or "-Val kinase C incorporated

cpm -fold

ValRS. EF-1 - 6,000 3.3

ValRS-EF-1 (a-reduced) - 2,815 3.0

ValRS .EF-1 (a-reduced) + - 2,106 1 .o

EF-1 activity

+ 19,691

+ 8,308

heat-inactivated PKC + 2,150

+ 2,410 EF- 1 CY - 2,448 1.0

ValRS activity ValRS.EF-1 (a-reduced) - 27,211 1.0

+ 26,133

Phosphorylation and Activation of EF-1 12579

37 "C for the valyl-tRNA synthetase. EF-1 complex and EF- la , respectively. Bec et al. (3) made a similar observation, that rabbit liver E F - l a contained in the valyl-tRNA synthe- tase.EF-1 complex had a 4-fold higher specific activity than isolated EF- l a subunit from calf brain. Those authors pro- posed that the higher activity of EF-la in the complex was probably attributable to the presence of the complementary factors, EF-lPy(G), which catalyze GDP/GTP exchange on EF-la. In these studies, we show a 10-fold increase in overall EF-1 activity due to the presence of EF-1Py6, suggesting that recycling of EF- l a by EF-lPy(6) may be the rate-limiting step in the elongation cycle, as has been recently proposed by Janssen and Moller, based on kinetic studies of the factor from Artemia salina (22).

We show, for the first time, that protein kinase C phospho- rylates valyl-tRNA synthetase and the a, P, and 6 subunits of EF-1. These results complement our previous investigation (5), which showed that phosphorylation of the same four proteins is stimulated in reticulocytes in response to PMA. Though the correlation does not exclude the possibility of PMA-induced activation through another protein kinase, the strong suggestion is phosphorylation of these proteins in response to PMA occurs through direct modification by pro- tein kinase C. This conclusion has been confirmed for the EF- l a subunit by showing that two-dimensional phosphopep- tide maps are identical for the subunit modified in PMA- stimulated cells and i n vitro by protein kinase C. Thus, coordinate control of protein synthesis by protein kinase C appears to involve phosphorylation of valyl-tRNA synthetase and EF-1. Protein kinase C has previously been shown to modify the translational initiation factors, eIF-2, eIF-3, eIF- 4B, and eIF-4F (14, 23), and glutamyl-tRNA synthetase in the synthetase core complex (10).

The effects of phosphorylation of EF-1 i n vitro on activity has also been investigated and found to be similar to that observed in PMA-stimulated cells. Phosphorylation of the factor both in reticulocytes (5) and i n vitro results in a 2-3- fold increase in EF-1 activity, as measured by poly(U)-di- rected polyphenylalanine synthesis. This effect of phosphoryl- ation on EF-1 activity may occur through an enhancement of the nucleotide exchange activity of EF-lPy 6, since the activity of isolated EF- l a does not appear to be affected by phos- phorylation. This interpretation is consistent with the pref- erential phosphorylation of the P and 6 subunits in the com- plex (as opposed to the a subunit) that we reproducibly observe. Valyl-tRNA synthetase is also unaffected by phos- phorylation i n uitro, even though phosphorylation of the enzyme in reticulocytes in response to PMA is associated with a 1.7-fold increase in aminoacylation activity (5). Alteration of the activity by phosphorylation may occur through inter- action of the phosphorylated synthetase with an effector molecule that is not present in the highly purified complex.

Previously, E F - l a was shown to be phosphorylated in ri- bosome fractions from rabbit reticulocytes (24). The only modification of this subunit previously identified i n vivo was methylation of lysine residues and addition of glycerylphos- phoryl-ethanolamine to glutamic acid residues (25, 26). EF- 1P is phosphorylated i n vitro (27-30) and in vivo (30) by casein kinase 11. EF-16 can also be phosphorylated by casein kinase I1 in vitro under certain conditions (28, 30). EF- ly is phosphorylated in Xenopus oocytes and i n uitro by the cell division control kinase, ~ 3 4 " ~ " ' (31). However, in none of these cases have the functional consequences of modification on EF-1 activity been analyzed.

We have consistently observed a 10-fold greater extent of phosphorylation of isolated E F - l a by protein kinase C (2.0

mol/mol) as compared with EF- l a in the complex (0.2 mol/ mol). This may be a characteristic feature of guanine nucleo- tide-binding proteins, since the same phenomenon has been reported previously for the a subunit of transducin (32) and the a subunit of Gi (33). In both of these studies, the purified a subunit of the guanine nucleotide-binding protein was a better substrate for protein kinase C than the a subunit complexed with the complementary Py subunits. It is sug- gested that this effect reflects conformational and/or steric differences between the complexed and non-complexed a- subunits.

Several lines of evidence suggest that EF-la has an espe- cially important role in cellular function. I t is a highly abun- dant protein, comprising 5% or more of total cellular protein (34, 35), which has led to the hypothesis that it may have functions beyond that of catalyzing binding of aminoacyl- tRNA to the ribosome. Even when present at these high levels, EF- l a is one of the major proteins induced by serum or epidermal growth factor in Swiss 3T3 cells (36). The amino acid sequence of the protein is extremely highly conserved, with 81% sequence identity between yeast and human (37), suggesting stringent functional restrictions on the three-di- mensional structure of the protein. Therefore, phosphoryla- tion of either E F - l a or its complementary subunits is likely to have functional consequences. At least one of the functional consequences of phosphorylation is an enhancement in the activity of the factor, as demonstrated in this study.

Another important point can be made from the data pre- sented. Fully 100% of the high M , form of EF-1 ( M , - 0.8 x IOfi) in rabbit reticulocytes is specifically associated in a complex with valyl-tRNA synthetase. This observation has relevance in interpreting the results of several previous stud- ies, which report developmental regulation of high and low M , forms of EF-1. In Artemia, for example, development of dehydrated cysts into hatched embryos is accompanied by a decrease in the high M , form and an increase in the low M , form (38). A similar conversion of high M , form to low M , form occurs during germination of wheat seeds (39). In con- trast, development of spores of the fungus Mucor racemosus into mycelia is associated with a significant increase in the amount of the high M , form (40). It has previously been assumed that conversion between high and low M , forms represents an aggregation/disaggregation phenomenon. These studies should now be reevaluated to consider the possibility that conversion actually represents complexation with valyl- tRNA synthetase.

Acknowledgment-We wish to thank Dr. W. C. Merrick for gen- erously providing purified EF-1 to use as a standard and for providing antibody to EF-1.

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