THE J O U ~ A I . OF B I O ~ I C A L CHEMISTRS 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 14, Issue of April 8, pp. 10529-10537, 1994 Printed in U.S.A.

Identification of Phosphorylation Sites in Rat Liver CTP: Phosphocholine Cytidylyltransferase*

(Received for publication, November 12, 1993, and in revised form, January 31, 1994)

James I. S. MacDonald and Claudia Kent$

Ann Arbor, Michigan 48109-0606 From the Department of Biological Chemistry, The University of Michigan Medical School,

CTPvhosphocholine cytidylyltransferase (CT) is an important regulatory enzyme in phosphatidylcholine biosynthesis. The enzyme exists as a soluble, inactive form that is highly phosphorylated; activation of the en- zyme is accompanied by dephosphorylation and trans- location to the membrane. We have used a recombinant baculovirus clone to obtain CT labeled in vivo with s2P04. The tryptic phosphopeptide pattern of the bacu- lovirus-expressed CT was the same as for CT expressed in mammalian cells, indicating that insect cells modify the same phosphorylation sites as do mammalian cells. S2P04-labeled, baculovirus-expressed CT was digested with trypsin and the peptides purified by reversed- phase high performance liquid c~omato~aphy . Phos- phoamino acid analysis of the complete protein as well as individual peptides revealed that only serine resi- dues were phospho~lated. Sequence analysis of puri- fied radioactive peptides revealed that phospho~lation of CT was confined to the carboxyl-te~inal region and that all or nearly all Ser residues from S e P to the car- boxyl terminus were labeled. S e P , SeP*, SeP", SerS=, SeP', SeF, and SeP7all reside in potential sites for proline-directed kinases. l k o other phosphorylated ser- ine residues, SerSIK and S e e , are found within protein kinase C consensus phosphorylation sites. Se*21, S e F , S e e , SeP5, Ser-, S e P , SeF2, and Se1-962 were also found to be phosphorylated. SerineM2 resides within a putative casein kinase I1 phosphorylation site, and there are five potential sites for phosphorylation by gly- cogen synthase kinase 3. Identification of these sites will allow investigations that focus on the establishment of the physiological function of phosphorylation at each site.

The biosynthesis of phosphatidylcholine, the major mem- brane lipid in eukaryotes, is regulated in part by C T P phosphocholine cytidylyltransferase (EC 2.7.7.15 (CT))' (1). Ac- tivation of CT can be achieved in vivo by reversible translocation from the soluble to the particulate fraction (1) or by the sequestration of lipid, presumably from some internal membrane, and the formation of a lipoprotein complex (2, 3). Translocation of CT from the soluble to particulate fraction may be effected by treatment of cells with phospholipase C (1,

cer Society. The costs of publication of this article were defrayed in part * This work was supported by Grant BE126 from the American Can-

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.

ferase; BBCT, recombinant baculovirus clone encoding CT CHO, Chi- ' The abbreviations used are: CT, CTP:phosphocholine cytidylyltrans-

nese hamster ovary; HPLC, high performance liquid chromatography; MAP kinase, mitogen activated protein kinase; PTH, phenylthiohydan- toin.

4,5), certain unsaturated fatty acids such as oleate (1,6), or by choline deficiency or supplementation with choline analogs (4, 7, 8).

Recent evidence has shown that CT is phosphorylated a t multiple sites in vivo and that phosphorylation plays an im- portant role in the regulation of CT activity (5, 6, 9). In both HeLa and Chinese hamster ovary (CHO) cells the soluble form of CT is phosphorylated whereas the particulate form is largely dephosphorylated (5, 6). Moreover, phosphorylation and de- phospho~lation of CT in both CHO and HeLa cells can be correlated with CT activity and localization (5,6). In phospho- lipase C-treated CHO cells, both translocation of CT and stimu- lation of phosphatidylcholine synthesis are inhibited by oka- daic acid, a potent inhibitor of types I and 2A protein phospha~ses , indicating that dephosphorylation is required for these processes (5) . Okadaie acid treatment of hepatocytes reduces the amount of membrane-associated CT, providing fur- ther evidence that dephosphorylation is important for mem- brane association (10). In HeLa cells the removal of oleate causes a rapid relocation of CT from the particulate to the soluble fraction (6). Increased phosphorylation of CT occurs concomitant with solubilization; however, extensive phospho- rylation occurs more slowly after removal from the membrane (6). Thus, it appears that CT is released from the membrane, possibly by phosphorylation at a single key site, followed by sequential phosphorylation at other sites.

Of central importance in understanding how CT is regulated is identi~cation of the sites at which phospho~lation occurs. Until recently this has proved a daunting task given the low levels of enzyme inherent in cells (12, 13). The cloning of the cDNA for rat liver CT (14) has allowed for the development of baculovirus expression systems for CT (15, 16). Herein we re- port that the baculovirus-expressed CT is phosphorylated in the same pattern seen in mammalian systems, and we have identified the phosphorylation sites in rat liver CT expressed in the baculovirus system.

EXPERIMENTAL PROCEDURES Materials-Sequencing grade trypsin was from Promega and carrier-

free [32Plorthophosphate (400-800 mCi/ml) was from ICN. Grace's in- sect cell culture medium, yeastolate solution, lactalbumin hydrolysate solution, and fetal bovine serum were obtained from Life Technologies, Inc. HPLC grade solvents were from Fisher and EM Science, and mi- crocrystalline cellulose thin layer chromatography plates were from Kodak. Octyl glucoside was from Calbiochem. All other chemicals and reagents were from Sigma or Boehringer Mannheim.

Maintenance of Cell Lines-Dichoplusia ni (-2 ni) cells, obtained from Invitrogen, were used as the host for baculovirus expression and were maintained in both suspension and monolayers at 27 "C in Grace's medium supplemented with yeastolate, lactalbumin hydrolysate, and 10% fetal bovine serum 115). The recombinant baculovirus clone ex- pressing rat liver CT (BBCT) has been described elsewhere (15).

Cell Labeling--2 ni cells were plated in 60-mm culture dishes at a density of 2.5 x lo6 cellddish and were infected with BBCT at a mul- tiplicity of infection between 5 and 10 as previously described (15). After

10529

Cytidylyltransferase Phosphorylation 10530

A

t I

1 - 1 e l e c t r o p h o r e s i s -

1, 13

a 12 11

a9 d

13

I- I e l e c t r o p h o r e s i s ~

C

36 h of infection, the cells were gently washed five times with phos- phate-free insect medium before being incubated for an additional 12 h in the same medium containing 5 mCi of carrier-free "'P0,and 5% fetal bovine serum. The cells were then washed three times with phosphate- buffered saline and scraped into 1 ml of lysis buffer (50 mM ?tis-CI, 50 mhr NaCI, 100 mM NaF, 1 mM Na,VO,, 2 mM dithiothreitol, 1 mM EDTA, 1 mM 4-(2-aminoethyl)-benzenesulfonylflouride, 1 mM benzamidine, 20 pg/ml leupeptin, 20 pg/ml antipain, 10 pg/ml pepstatin, and 10 pg/ml

T ni cells in suspension were infected with BBCT as previously * described (15), sedimented by centrifugation, washed with phosphate- buffered saline, resuspended in lysis buffer, and pooled with the labeled

11 7 12 4 ag 5

2 chymostatin, pH 7.5). * 8 - e 1 Z ni cells.

' 6

1 + 1

7

5 c e2 * C

6

l + I

1 - 1 e l e c t r o p h o r e s i s -

Wild type CHO K1 cells and the mutant CHO strain 58 (17) stably expressing rat liver CT (18) were also labeled with "'PO,. Approximately 2.5 x IO5 cells growing in Eagle's minimum essential medium supple- mented with 10% fetal bovine serum were plated onto 60-mm culture dishes and allowed to attach by incubating for 8 h a t 37 "C. The cells were then washed four times with 4 ml each of phosphate-free Eagle's minimun essential medium containing 5% fetal bovine serum before being incubated overnight at 37 "C in 3 ml of the same medium con- taining 5 mCi of 90,.

Illuo-dimensional Phosphopeptide Mapping-CHO- and BBCT-in- fected insect cells labeled with "'PO, as described above were washed three times with 4 ml each of phosphate-buffered saline. The cells were then solubilized in 40 mM sodium phosphate containing 400 mM NaCI, 100 mw NaF, 10 mv EDTA, 1 mM benzamidine, 0.2 mv dithiothreitol, and 1% (v/v) Nonidet P-40 and protease inhibitors as above. CT was immunoprecipitated, subjected to SDS-polyacrylamide gel electrophore- sis, and transferred to Immobilon-P polyvinylidene difluoride as de- scribed previously (6). The radiolabeled CT band was then excised and transferred to a microcentrifuge tube, washed several times with H,O and then several times with 0.5% polyvinylpy~~olidone-360 in 100 mM acetic acid before incubation a t 37 "C in the same solution. After 30 min the polyvinylpyrrolidone-acetic acid was removed, and the membrane was washed 10 times with H,O and then several times with 50 mM NH,HCO, containing 5% (v/v) acetonitrile. The membrane was then incubated for 12 h a t 37 "C with 40 pg/ml 1-tosylamide-2-phenylethyl chloromethyl ketone-trypsin prepared fresh in 50 mM NH,HCO, con- taining 5% (v/v) acetonitrile. An additional 40 pg of trypsin was added after 12 h, and the digestion was allowed to continue for another 12 h. The digest was evaporated to dryness, dissolved in 400 pl of H,O, and evaporated to dryness again. The dried sample was dissolved in about 20 pl of H,O and an aliquot spotted 3 cm from the right and 5 cm from the bottom of a cellulose thin layer plate (20 x 20 cm). Phosphopeptides were separated in the first dimension by electrophoresis a t 1000 V for 30 min, followed by thin layer chromatography using H,O/n-butanol/ acetic acid/pyridine (30:20:6:24) as the mobile phase. Phosphopeptides were visualized by autoradiography.

Purification of Recomhinant Cytidylyltransferase-Cytidylyl- transferase labeled with '"P was purified through the DEAE-Sepharose step as previously described (15) except that all the buffers contained 50 mM NaF and 0.1 mM Na,VO,, and the NaCl concentrations of the elution buffers were adjusted to 100,250, and 450 mM, respectively (15). Frac- tions from the DEAE-Sepharose column containing CT were pooled and guanidine HCI added to 6.0 M. The denatured cytidylyltransferase was dialyzed against 4 liters of 10 mM Tris-CI, pH 7.5, containing 20 mM NaF and 0.1 mM Na,VO,for 24 h with one change a t 12 h. Following dialysis the "P-labeled enzyme preparation was loaded onto a Bio-Gel P-6DG column which had been equilibrated with H,O, and CT was eluted from the column using H,O as the eluant. The enzyme was lyophilized and dissolved in 50 m~ NH,HCO, containing 1.0 M urea and 5% (v/v) ace- tonitrile for tryptic digestion.

Purification of 7iyptic Peptides-CT was digested for 48 h a t 37 "C with sequencing grade trypsin added sequentially over time to give a final CT to trypsin ratio of 50:l. The digest was evaporated to dryness in a Speed-Vac centrifuge (Savant Instmments, Farmingdale, N Y ) , dis-

acrylamide gel electrophoresis, and blotted. The enzyme was localized on the blot by autoradiography, excised, and digested with trypsin as described under "Experimental Procedures." Two-dimensional electrophoresiskhromatomaphy was performed as described under "Ex-

[ + 1 Derimental Procedures." &elC re&esents a mix of eaual amounts of r ~ " ~ ~

radioactivity from A and B. The numerical designation of each spot is FIG. 1. Phosphopeptide mapping of '9-labeled cytidylyltrans- arbitrary and is as described by Sweitzer (18) following the general

ferase. Recombinant cytidylyltransferase expressed in BBCT infected pattern described by Watkins and Kent (5). Gaps in the numerical insect cells ( A ) and rat liver cytidylyltransferase expressed in CHO sequence reflect discrepancies between the maps shown here and by strain 58 cells (B) was immunoprecipitated, fractionated by SDS-poly- Sweitzer (18) and those of Watkins and Kent (5).

10531

-T~ 1 I

I - I - 60

I 1 I I I 1 I 1 20 40 BO 80 100 120 140 160

R E T E N T I O N T I M E lmlnl he. 2. Separation of tryptic peptides of recombinant cytidylyltransferase expressed in BBCT-infected insect cells. Recombinant

cytidylyltransfera~ was labeled with 32P0, and partially purified as described under "Experimental Procedures." The partially purified enzyme was digested with trypsin and peptides separated by reverse-phase HPLC using a linear gradient of 6 5 0 % buffer B (75% CH,CN, 0.1% trifluoroacetic acid) over 160 min followed by a linear gradient of 50-100% buffer B over 10 min.

FIG. 3. Radioactive analysis of pep tides isolated following tryptic diges- tion of t2P-labeled cytidylyltrans- ferase. Recombinant cytidylyltransferase was labeled with %POg as described under "Experimental Procedures," digested with trypsin, and peptides fractionated as de- scribed in Fig. 2. kactions of 0.5 ml were collected and monitored for Cerenkov ra-

into the regions shown based on the dis- diation. The chromatogram was divided

tribution of radioactivity. Region TV was further divided into two peaks designated i and ii. No further radioactive material was eluted from the column following the initial 0-50% buffer B gradient.

35

30

c

* 2 0

I a v 15

10

5

0

RETENTION TIME tminf

solved in H,O, and the resulting peptides were separated by reversed- phase HPLC on a C-18 (Vydac) column using a gradient of &50% buffer B (75% acetonitrile, 0.1% trifluoroacetic acid) over 165 min. Radioactive peaks were identified by Cerenkov radiation and further purified by HF'LC on a C-18 column using a gradient of acetonitrile, 0.1% triflu- oroacetic acid adjusted to expand the gradient used for the original total peptide separation. Secondary gradients were extended over 165 min unless indicated otherwise in the figure legends. In some instances radioactive peptides were purified by two-dimensional electrophoresis/ thin layer chromatography. The labeled peptides were localized by ex- posure of the thin layer plate to x-ray Elm, scraped into a microcentri- fuge tube, and eluted from the cellulose with HPLC grade H,O. Peptides were then evaporated to dryness as described above. Purified peptides were sequenced using an Applied Biosystems (Foster City, CAI AB470 gas-phase sequencer by the Biomedical Research Core Facility, Univer- sity of Michgan.

P h o s ~ h ~ m i ~ o Acid A ~ l y s i ~ - P h o s p h o a ~ n o acid analysis was per- formed as previously described (9). Aliquots of 32P-labeled CT were hydrolyzed in 6 N HCl a t 100 "C for 30, 60, and 120 min. The digests were evaporated to dryness, dissolved in H,O, spotted onto a cellulose thin layer plate, and separated by electrophoresis at 1000 V for 45 min in a buffer consisting of 10% (v/v) acetic acid and 1% (v/v) pyridine. Phosphoserine, phosphot~onine, and phosphot~osine were included as standards. The plate was sprayed with 0.5% ninhydrin in acetone; phosphoamino acids were located by autoradiography and identified by comparison with phosphoamino acid standards.

Miscellaneous Procedures-Protein was determined by the procedure of Bradford (19) using the reagent supplied by Eo-Rad, with bovine

serum albumin as the standard. Po~yac~lamide gel electrophoresis was performed as described by Laemmli (20). Cyanogen bromide cleavage of CT was performed as described (21). Briefly, CT was incubated at room temperature for 5 h in 100 mg/ml cyanogen bromide in 70% formic acid. At the end of the incubation time, the mixture was diluted 10-fold with H,O, lyophilized, and digested with trypsin as described above. Carbo- hydrate analysis of CT was performed using the Glycotrack Carbohy- drate Analysis kit (Oxford Glycosystemsf following the instructions sup- plied therein. Ovalbumin supplied with the kit was used as the standard.

RESULTS

Insect cells infected with BBCT express large amounts of highly phosphorylated rat liver CT. In order to verify that the phosphorylation pattern of recombinant CT expressed in insect cells is the same as that observed in mammalian systems, we compared the 32P-labeled phosphopeptide pattern from BBCT- infected insect cells with that from CHO strain 58 cells stably expressing rat liver CT. Strain 58 is a CHO cell mutant in which the endogenous CT is temperature sensitive (17) and is present in considerably lower amounts than that found in wild type CHO cells (18, 22). CT was immunoprecipitated from the 32P-labeled cells, digested with trypsin, and the tryptic phos- phopeptides mapped by two-dimensional electrophoresis/ chromatography (Fig. 1). For the most part, the tryptic phos-

10532 Cytidylyltransferase Phosphorylation

0.4- A - - 30 E

v) 0.3- A C -

c N . - - .

.”

u‘: v 0 0 . 2 -

2 %

I

- 2 0 m

2 - K W U

K

In =x

0.1 - -10 5 m

m H

- 0

FIG. 4. Further purification of pep- tides in region 11. The fractions com- I I

prising region I1 (Fig. 3) were pooled, con- centrated, and further fractionated by HPLC as described under “Experimental Procedures.” Fractions were collected and monitored for radioactivity ( B ) . Several radioactive peaks were identified and were designated A-E. Only that portion of the gradient in which peptides eluted is shown.

t I :

5 0 1 0 0

R E T E N T I O N T I M E lmlnl

2 0 - A /

,- 30 / ,

/

1 5 - , B

/

/ / - 2 0 ,

/ / ,

1 0 - , C’

- 10

5 - ,

, , , 0 - . o ’ I I I I

2 0 40 60 80 100 120 140

R E T E N T I O N T I M E lmlnl

phopeptide maps of the recombinant baculovirus-expressed enzyme and the rat liver enzyme expressed in strain 58 cells were identical, although several anomalies were apparent. The most notable differences in the phosphorylation patterns were that spot number 4 was more highly labeled in the CHO cells while spot number 1 was more highly labeled in the insect cells. In addition, a phosphopeptide appeared between phosphopep- tides 6 and 8 in Fig. 1B which is not evident in Fig. lA. How- ever, when equal amounts of radioactivity in the two digests were mixed and the phosphopeptides separated, the phos- phopeptide observed between peptides 6 and 8 in Fig. lB dis- appeared (Fig. 1C) suggesting that its original appearance was artifactual. We also ran tryptic digests of CT immunoprecipi- tated from wild type CHO-K1 cells and observed a pattern qualitatively identical to that observed for the baculovirus- expressed rat liver enzyme (not shown). Previous work in our laboratory had shown that CT in HeLa cells is phosphorylated only on serine residues (9). In agreement with the previous results, phosphoamino acid analysis of 32P04-labeled CT from infected insect cells revealed only phosphoserine (not shown).

In order to identify the phosphorylation sites in CT, we de- cided to purify the labeled enzyme up through the DEAE- Sepharose step, completely digest the enzyme with trypsin, and characterize all 32P-containing peptides. We chose to use the DEAE fraction for these analyses because the next and final step in the purification, hydroxylapatite chromatography, re- sults in a considerable loss of enzyme (15). Although the DEAE- Sepharose step does not result in a homogeneous preparation of cytidylyltransferase, CT is by far the major protein after this purification step (15). Typically, about 10 mg of CT was ob-

tained from 250 ml of infected cells. Baculovirus-expressed cytidylyltransferase was digested with trypsin, and the tryptic fragments were separated by reverse-phase HPLC with a lin- ear gradient of 0-37.5% acetonitrile over 165 min (Fig. 2). The chromatogram was divided into regions corresponding to peaks of radioactivity (Fig. 3), and these were further separated by HPLC with a more shallow gradient. When the fractions in region I (Fig. 3) were pooled and subjected to secondary anal- ysis on HPLC with a linear gradient of 0-20% acetonitrile, no distinct radioactive peaks were observed, and no further anal- ysis on this region was carried out.

The majority of the radioactivity eluted in a peak with a retention time of 62-69 min (region ZZ, Fig. 3), and when this peak was rechromatographed with a linear gradient of 0-25% acetonitrile over 165 min, several well defined peaks were re- solved (Fig. 4A). The major radioactive peaks from this column, designated A-E (Fig. 4B), were collected and sequenced. The amino acid sequences of peaks A-C were QS319PS321-

S347PAS350LS352R, respectively. The locations of the phospho- rylated serine residues within these sequences were identified by the appearance of PTH-dehydroalanine in place of PTH- serine during sequencing. Dehydroalanine arises by a-elimina- tion of phosphate from phosphoserine during automated se- quencing (23). (As a control, when phosphoserine was included as an amino acid standard prior to sequencing, it was converted to dehydroalanine.) Small amounts of dehydroalanine can also occur by a-elimination of 0-linked carbohydrates; however, when CT was analyzed for carbohydrate content by periodate oxidation followed by biotinylation of oxidized carbohydrates

S322S323pTHER, S329pS331pS333FR and TS343pS345S346-

Cytidylyltransferase Phosphorylation 10533

I " 20 40 60 80 100

RETENTION T IME lmlnl

f I I I

I I

I L

. . . /

/ /

/ /

r /

/

c

c I I I I I I I

20 40 60 80 100 120 140

2 0

10

0

RETENTION TIME lminl

FIG. 5. Further purification of peptides in region 111. The fractions comprising region I11 (Fig. 3) were pooled, concentrated, and further fractionated by HPLC (A). The conditions consisted of a linear gradient of 0-15% buffer B (75% CH3CN, 0.1% trifluoroacetic acid) over 10 min followed by a linear gradient of 15-25% buffer B over 105 min. Fractions of 0.5 ml were collected and Cerenkov radiation was determined ( B ) . The two radioactive peaks designated F and G are shown (A ).

and detection on Western blots with a biotin-specific antibody, no carbohydrate residues were found (not shown). Using dehy- droalanine as an indicator for phosphoserine, we found that all serines in peptides A-C (Fig. 4A) were phosphorylated to some extent. S e P 9 , Ser3", Ser331, and Ser343 were the most highly phosphorylated, with greater than 90% of the PTH-Ser plus PTH-dehydroalanine present as PTH dehydroalanine. For the

between 50 and 75% of the PTH-Ser + PTH-dehydroalanine was PTH-dehydroalanine.

The peak designated in Fig. 4 as D consisted of a mixture of three tryptic peptides; the first was identical in sequence to peptide A (Fig. 4), the second corresponded to the carboxyl- terminal sequence of CT, AVTCDIS362EDEED, and the third was a CT sequence which contained no phosphorylatable resi- dues. Sequence analysis of peak E (Fig. 4) gave the peptide MLQAIS315PK. Both Ser315 and Ser362 were phosphorylated over 90%, as indicated by PTH-dehydroalanine content.

When region I11 of Fig. 3 was pooled and rechromatographed in a shallower gradient, a major radioactive peak, designated peak F, was observed, the sequence of which was AVTCDIS362EDEED (Fig. 5). This peptide, therefore, eluted from the HPLC column both discreetly and as a contaminant in peak D (Fig. 4). The peptide contains a threonine residue and,

other serines, Ser321 ~ 3 2 2 ~ 3 2 3 s 3 3 3 s345 s346 s347 ~ 3 5 0 , , , , , , , , and S352,

although no phosphothreonine was detected in the total protein hydrolysate, it was conceivable that the altered chromato- graphic properties of the two fractions could have been due to the presence of phosphothreonine in one of the peaks. However, phosphoamino acid analysis of both peak D (Fig. 4) and peak F from Fig. 5 revealed only phosphoserine, so the reason for the migrational anomaly of this peptide remains unknown. A sec- ond radioactive peak, peak G, was also isolated from region I11 (Fig. 5 , A and B ); however, when analyzed by two-dimensional electrophoresis/chromatography the peak was resolved into several radioactive peptides with greater than 70% of the total radioactivity resident in a peptide with the same migrational properties as the peptide AVTCDISEDEED.

Region IV (Fig. 3) contained two prominent radioactive peaks (designated i and ii). The main radioactive peak in re- gion IV, i, consisted of a mixture of two peptides, one being the previously identified phosphopeptide MLQAIS315PK and the second corresponding to the peptide EVPGPNGATEEDG- IPS3'K. No dehydroalanine peak was observed at cycle 16 of the latter sequence suggesting that S e P was not phospho- rylated. To investigate further whether or not Ser3' is phospho- rylated, peak i was digested with V8 protease. The peptide EVPGPNGATEEDIPS3'K is cleaved with V8 protease into two peptides, EVPGPNGATE and DIPS3'K. Separation of the V8

10534 Cytidylyltransferase Phosphorylation

0 .4 - A /

2- 30 - /

”- -

c /

.... /

EVPGPNGATE - - / - 2 0 - m

0.2 - R

- MLOAISPK

/ - 1 0 E c

/

- - U 3 -

/ s ~

” ” A 0 - f - 0 n

I I

100

75

(?

b, x 5 0

I n 0

2 5

0

40 80 120

R E T E N T I O N T I M E I min I

B MLQAISPK ,

/

/ , , , I

/ , A

I 1 I I I I I 2 0 40 0 0 80 100 120 140

R E T E N T I O N T I M E lminl

FIG. 6. HPLC fractionation following digestion with V8 protease of the major radioactive peak from region IV. The fractions comprising the main radioactive peak of region IV (i from Fig. 3) were pooled, concentrated, and further separated by HPLC (a linear gradient of 030% buffer B (75% CH,CN, 0.1% trifluoroacetic acid) over 160 min). A single radioactive peak was isolated, evaporated to dryness, and digested with V8 protease. Secondary HPLC fractionation of the digest was performed using conditions identical to those described above for peak i (A). Fractions of 0.5 ml were collected and Cerenkov radiation was determined ( B ) . The peptide composition of the three peaks inA and the radioactive peak ( B ) is shown. Only that portion of the gradient in which peptides eluted is shown.

digestion products on HPLC gave three peaks which, when sequenced, proved to be the two expected V8 peptides and MLQAIS315PK (Fig. 6 A ) and again, no dehydroalanine was found at Ser3’. All the radioactivity was confined to the peptide MLQAIS315PK (Fig. 6B). Sequence analysis of the first six amino acids of peak ii in region IV gave the sequence MLQAIS315, but the actual peptide was apparently incom- pletely digested, so that the complete peptide sequence was probably MLQATSPKQSPSSSPTHERSPSPSFR in which the tryptic cleavage site is KQ. Previous data showed that S319 in the sequence QS319P is highly phosphorylated (Fig. 41, and it is known that trypsin cleaves inefficiently in the sequence k g / Lys-X-pSer/pThr (24). This was confirmed by exhaustively di- gesting peak ii with trypsin, then subjecting the products to two-dimensional electrophoresidchromatography. The migra- tional properties of the products of peak ii after redigestion with trypsin were identical to those of peaks A, B, and E (Fig. 4).

Chromatography of region V (Fig. 3) resulted in one major radioactive peak (Fig. 7, A and B ) , the sequence of which proved to be WPFS339GK. We could not clearly identify a dehy- droalanine peak at cycle 4 of the sequence, although a promi- nent unknown peak did appear with a slightly retarded reten- tion time relative to dehydroalanine. The peak migrated as a single radioactive spot on two-dimensional electrophoresis/ chromatography, and no change in migration was observed af- ter redigestion of the peptide with trypsin. Thus, the data sug- gest that Ser339 is phosphorylated.

Cytidylyltransferase contains 2 serine residues at the amino terminus confined to the tryptic peptide MDAQS‘S‘AK. The

possibility existed that these serines may be phosphorylated but, because of the blocked amino terminus (25) no sequence would be obtained. Radiolabeled CT was treated with cyanogen bromide to cleave the terminal methionine residue and then digested with trypsin. The chromatographic pattern of the cy- anogen bromide-treated material was identical to that shown in Figs. 2 and 3 except that one peak in region I appeared to have a slightly retarded retention time relative to that ob- served for the non-cyanogen bromide-treated sample. This peak contained only 1-2% of the total radioactivity in the sample, and the only sequence we could obtain was FZg3GPEGALK, which contains no phosphorylatable residues. That CT was successfully cleaved with cyanogen bromide was indicated by the fact that the amino acid immediately preced- ing F293 is a methionine. We isolated each region and carried through the analysis as before, but the results with cyanogen bromide-treated CT were identical to those obtained when CT was treated with trypsin alone. Phosphopeptide maps of CT digested with trypsin following cyanogen bromide cleavage are identical to those obtained after digestion with trypsin alone (not shown). Thus, it is highly unlikely that Ser‘ and/or Ser‘ are phosphorylated.

DISCUSSION Reversible phosphorylation is a universal mechanism for the

regulation of enzymatic processes in eukaryotic cells. It has been known for some time that CT is regulated by phosphorylatioddephosphorylation (1, 5, 6, 9); however, given the difficulty in obtaining appreciable amounts of enzyme from tissues (12,13), the nature of these phosphorylation events has

Cytidylyltransferase Phosphorylation

A I 1

0.4 - - 40 - "

- 0.3 - - - - -

- / - /

- - - 3 0 - m - c - -

0 . 2 - - - c - a - - - - - - - 2 0 ," - -

c

r - Y 3

0.1 - / I - 1 0 x

m

0 1-J- I

- 0 I I I I I

2 0 4 0 8 0 8 0 100 1 2 0

R E T E N T I O N T I M E lmlnl

10535

3

m I 0 r

x 2

I V n.

1

0

, . , , , , , , /

I I

I I

I I -

2 0 40 6'

, ,

, , , - I , , , - 30

- 2 0

- 10

I I I I 80 100 120 140

0

Only that portion of the gradient in which peptides eluted is shown.

remained a mystery. The availability of baculovirus-expressed CT has afforded us the opportunity to determine the sites phos- phorylated in rat liver CT expressed in insect cells. That the phosphorylation sites in baculovirus-expressed CT are the same as those in mammalian systems is strongly suggested by the similarity of tryptic phosphopeptide maps of baculovirus- expressed CT to phosphopeptide maps of CHO CT and of rat liver CT stably expressed in CHO strain 58 cells. Major phos- phorylation sites of other mammalian proteins have been found to be similar or identical to the sites phosphorylated when those proteins are expressed in a baculovirus system (26-29).

The location of phosphorylated serines in CT was indicated by the appearance of PTH-dehydroalanine in place of PTH- serine at given cycles during sequencing. Another method for identifying the location of phosphorylated serines is to derivat- ize the dehydroalanine by addition of ethanethiol to form S- ethylcysteine (23, 30). However, the dehydroalanine formed as an intermediate in the reaction is optically inactive and, there- fore, addition of ethanethiol results in racemization. We deri- vatized the CT peptides containing multiple serine residues with ethanethiol and, when the cysteinylated peptides were injected onto the HPLC column, the appearance of 32P radioac- tivity in the first column fractions indicated that the P-elimi- nation reaction had occurred. Unfortunately, we could recover no distinct peaks from these samples. Apparently, since the C-18 column used in this study is capable of separating diaste-

reomers, the extent of racemization of the peptides was too great to allow the cysteinylated peptides to elute as a sharp peak.

Phosphorylation of CT is confined to 15-16 Ser residues near the carboxyl terminus of the enzyme (Fig. 8). From Ser315 to the carboxyl terminus, all Ser residues, with the possible exception of SeP9, are phosphorylated. Some of these residues appear to be only partially phosphorylated, judging from the percent PTH-dehydroalanine at these positions. Whether this partial phosphorylation is due to the overproduction of CT in the insect cells or whether it bears a functional resemblance to that tak- ing place in a more natural system is not known.

A number of the phosphorylated residues in CT lie within consensus sequences for well known protein kinases. Two phos- phorylated serine residues, Ser315 and Ser333, as well as possibly phosphorylated are found within sequences resembling protein kinase C phosphorylation sites, S/T-X-K/R (31). In fact the sequence, SPSPS333FR is nearly identical to the sequence SPSPSFK in nuclear lamin B, in which the Ser residue closest to the lysine is phosphorylated by protein kinase C (32). Cyti- dylyltransferase can be phosphorylated in vitro by protein ki- nase C: and phorbol ester treatment of HeLa cells influences CT activity in vivo (9). Phorbol ester treatment of HeLa cells does not, however, affect the phosphorylation state of CT (9).

* J. D. Watkins and C. Kent, unpublished observations.

10536 Cytidylyltransferase Phosphorylation

* * *** * * * 3'SS-P-K-QSP-SSSP-T-H-E-R-S-P-S-P-~F-R-W-

(*) * *** * * P-F-SG-K-T-SP-S-S-SP-A-S-L-S-R-GK-A-V-T-G

* D-I-S-E-DE-E-D

FIG. 8. Sequence of phosphorylated region of cytidylyltrans-

asterisk. ferase. Serine residues identified as phosphorylated appear with an

Possibly these residues are phosphorylated by a kinase that is not a member of the protein kinase C family, or they are already phosphorylated in normal, growing HeLa cells and so are not affected by phorbol ester treatment.

A very striking feature of the phosphorylation pattern in CT is that seven of the phosphorylated sites are Ser residues fol- lowed by Pro. This strongly suggests that a proline-directed kinase is involved in the regulation of CT activity. Two such kinases, p34cdc2 kinase and MAP kinase, have been well stud- ied in recent years, The former kinase is involved in control of cell cycle progression, and the latter kinase functions in signal transduction pathways. The consensus phosphorylation se- quence for p34cdc2 kinase is S/T-P-X-wR (33); the presence of the lysine or arginine is optimal (331, but the spacing between the SP sequence and the basic residue may vary (34-36). Of the phosphorylated serine residues in CT, none lie within se- quences matching S-P-X-K/R, although residues S315, S323, S331, and S347 all lie within less optimal p34cdc2 phosphorylation sites. The optimal primary sequence for MAP kinase phospho- rylation is PX(S/T)P (37-391, but the minimal recognition se- quence for MAP kinase, (S/l')P (377, is present in CT. Prelimi- nary evidence indicates that CT is phosphorylated by MAP kinase in vitro, but we have not identified the in vitro phos- phorylation sites.

The SerPro-rich sequences encompassing Ser315-SeP33 and Ser343-Pro348 are also similar to the Ser-Pro repeats found in the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase I1 (40) and which is highly phosphorylated during the transition from preinitiation complex assembly to elonga- tion (41, 42). A kinase responsible for phosphorylating these repeats has recently been purified and characterized (43). Like RNA polymerase and other enzymes involved in transcription, CT is also a nuclear protein (22, 44, 45), and as such it may come under the control of kinases involved in transcriptional regulation.

CT contains several possible glycogen synthase kinase 3 phosphorylation sites (S-X-X-X-S(P)) where S(P) indicates phosphoserine (46). Phosphorylation of Ser319, Ser323, Ser333, Ser343, and Ser347 identifies Ser315, SeP19, SeP9, SeP9, and Ser343 as potential sites for glycogen synthase kinase 111. Since some of these sites are contiguous, it is possible that phospho- rylation on only three sites, Ser323, SeP3 , and Ser357, allows phosphorylation of the other four. The final serine in the car- boxyl-terminal sequence is S e P 2 , which is within a casein ki- nase I1 consensus site. This site is conserved between yeast and mammals, despite an otherwise low degree of similarity in this region, suggesting the site is important for the function of CT. None of the serine residues identified to be phosphorylated could be placed within consensus sequences for CAMP-depend- ent protein kinase (RXS, RRXS, RXXS or KRXXS (30)). This result agrees with previous results indicating that CT is not a target for CAMP-dependent protein kinase in cultured hepato- cytes (47, 48).

Although CT is highly phosphorylated at the carboxyl termi- nus, the molecular role of phosphorylation on CT activity and location is not yet clear. CT activity is modulated considerably

by certain lipids (12, 15, 49, 501, and phosphorylation plays a vital role in vivo in the translocation of CT to a lipidic environ- ment where the enzyme is active. CT is a hydrophobic protein, and as such it binds very strongly to hydrophobic surfaces. I t is possible that phosphorylation operates as a mechanism to make CT more hydrophilic and, therefore, more soluble at times when membrane biogenesis is not needed. Previous data suggest, however, that translocation of CT may depend only on the phosphorylation of a few key sites because dissociation of CT from the membrane upon removal of the stimulus is not accompanied by complete phosphorylation (6). Conversely, membrane-associated CT is not completely dephosphorylated (5, 6). The interaction of CT at the membrane interface could involve associations with both lipid and protein, and phospho- rylation at key sites could play an important role in regulating any putative interaction between CT and a target membrane protein or proteins. Establishment of an in vitro assay system for studying the interaction between CT and target mem- branes, and how these interactions are governed by phospho- rylation would be highly advantageous. The diminished ability to bind target membranes in response to phosphorylation has been observed in vitro for synapsin I, one of a family of multiply phosphorylated proteins resident to synaptic vesicles (51, 52). Phosphorylation of synapsin I at Ser9 by Ca2+/calmodulin-de- pendent protein kinase results in a &fold decrease in the af- finity of this protein for purified synaptic vesicles whereas phosphorylation at Se1.556 results in only a negligible change in affmity (52).

Recently, CT has been shown to bind to a 112-kDa soluble protein from rat liver (53). Several possibilities were presented to explain this interaction, including the idea that the 112-kDa protein is a chaperone protecting CT from non-specific hydro- phobic interactions during translocation or that the binding protein itself acts as an inhibitor by binding to CT under cer- tain conditions and preventing translocation (53). Whatever the role of the 112-kDa protein it is possible that the interac- tions between it and CT are governed by reversible phospho- rylation. Identification of the sites at which CT is phospho- rylated sets the stage for investigations to assess the functional significance of these phosphorylation sites with respect to en- zyme activity and translocation.

2. 1.

3.

4.

6. 5.

7. 8.

10. 9.

11.

12.

13. 14.

15. 16.

17.

19. 18.

20. 21. 22.

23.

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