Activation of partially purified rat liver lipid methyltransferase by phosphorylation

Vol.

122,

August

No.

16,

BIOCHEMICAL

3, 1984

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BIOPHYSICAL

RESEARCH

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1984

A~'l'TV.A'l'TI\N

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PWIFIED RA T LTVER FH?SPfIORYLATION

OF PARTIALLY

June

19,

LIPID

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Tncllhation of partially purified rat liver lipid methvltransferase with MrAT? ,a.~~~! the cat,alytic subunit of the cyclic AM ? (dependent protein kinase r12sults irl u. to '+-fold activation of the nethylation reaction. When (Y-32?) ClrA'TP is inrl:l:trd in the assay mixture, the analvsis caf the phosphoprotein products b.,r a sinplc Drotein tland oil clectrnnhcresis shows the incorporCation of ?'P intn 3buut E>l?k: .inri p1 4.75. It is concluded that rat liver li;i'd methvltr
The klecn

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1 ,: 1 . The hr>rm:>nt?s

nf to

flow and

sipnal from

,powth

phosphatidvlethanolamine transduction PtdFtn f.dctors.

to

(Pt.iEtn) in

PtXhol The

exact

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modulated

relitionshir‘

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Vol.

Blue

122,

No.

3, 1984

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BIOCHEMICAL

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r>rcs+znt

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BIOPHYSICAL

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RESEARCH

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COMMUNICATIONS

phosphnrylation

and

r~c-

Materials. iMeth\Tl'~)-c-ad~nq~ylmethionine (7.8 mCi/mmol) was from New England 1Iuclea.r snd adenosine-5'('Y-F)-triphosphate (3000 Si/mmol) was from Amersham. Ultrasnhere-Si colu-in (5 pm, 250 x 4.6 mm) for HFLC was from Beckman. Products otein) in ,a Firlil volcre I+ 443 p1. The reaction was initiated by the addition of $-a~~pnnavl~et~innine and terminated bv pir;ettinp IOr) pl assay m'xture into 2 ml chlornrorm/rn~thano~/2N HCl (Fa:3:1, v/v) (9). The chloroform phase wis washed three times with 1 ml 0.: "1 k'C1 in iO'i methanol. After washin,c, the chloroform nhase WI's drip11 at C)O'C under Nq ;Ind counted. Products of the reaction were analvzed bv IIPL? usine' ,an Illtr-asphere-Si column (11). The column was eluteJ with a linear \:ratdient over a 15 min. r'eriod startine with 96% (isopropato 939; (isnpropanollhexane (1:l)) and 7% wsiter. nollhexane (1:l)) and "4 water The flow rate was 1 -il/min. Fractions were collected it 1 min intervals and counted. ?ecover,v of the raied out by 10% acry(30 units/ml). Se:laration lcf ~-F-labelled buffer in a similar lImi,ie slab eels in the TBresence of SDt in Tris-HCl-CDS (Hio-Fad laboratories hullemanner to that describer! bv Bio-F iti laboratories acrylamide gels using 1 non-equilibrium pH-gradient tin 1024) or h~v TEF in 10" (12). F roteins were stained with Coomasie Blue. from 3.5 to 10.n as described Stand;irds for molecular Weipht determination were: lysoz;/me (14400), soyhean trypsin inhibitor 121rOn), c.arbnnir ;inhydrar,e (:ilOOl?), nvalbumin (45000), boviStandards for l>T deterne serum albumIn (Ci;?O!‘i) ,ar~d yhnr,phr~r~vl~~sP YI (92100). Foybean trxjpsin inhibitor (4.55), mination were: =~mvln~l~~cnsidase 0.5Q), lactoelcbulin ( :.:o), Lovine ,c,a~~h~r~ni- anhI.-dra;,? P (Cj.Bi), horse mynFlobinac& lectin-acidic band ,Jic b,,an,j (F,. a[,) , l,crse rvnvlnbir-hasi~z t~.irl~l ( 7. 35) , lentil , lentil lectin-basic hand (ri.6S) and (!?.lC), lentil lectin-miiidlo 3intl (8.4") 1066

Vol.

122,

No.

3, 1984

BIOCHEMICAL

AND

l'A3LF EFFFI‘T

0'

THE ADDITION

?b- ?POTfIN I,IW:P

I,TPTD

PINArE

BIOPHYSKAL

COMMUNICATIONS

1 AN3 MEATY

YETWLTPAN'F'E'ASC

1067

RESEARCH

0?1 TX:

4CTIVITY

OF FA'I

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No.

3, 1984

BIOCHEMICAL

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400 1500

92ooo 500 I-A----J

l?

0

0

E

i

:I000

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Fig.1.

Analysis

phorylation and

Methods.

Fig.2. Gwas indicate

by reaction Arrows

200

fL

SDS-PAGE of phosphorylated lipid methyltransferase. Phoswas carried out for 30 min. as described under Materials indicate the molecular weight of the protein standards.

Analysis by IEF of phosphorylated lipid methyltransferase. carried out for 30 min as described under Materials the p1 of protein standards.

pelling

evidence

that

least

in vitro,

from

phosphorilated

form.

liver

lipid

a low activity These

methyltransferase

results

agree

microsomes

MgATP

in the presence

addition

of glucagon

activate

lipid

of cyclic

it

OF PHOSPHORYLATED

AMINOACIDS

description

induce

be important

TABLE ANALYSIS

previous

the

in rat

methyltransferase

which

by whether

are known to

phosphorylation

to know if

HCl

HYDROLYSIS

OF PHOSPHORYLATED

METHYLTRANSFERASE 32

Phosphoserine Phosphothreonine Phosphotyrosine

p, cpm 400 67

11

Purified lipid methyltransferase was phosphorylated for 30 min. as described the enzyme was precipitated under Materials and Methods. After phosphorylation, with acetone, washed, redisolved in 0.15 ml HCl 6N and incubated at llO°C for phosphoaminoacid standards were added and separated by 2h. After hydrolysis, high voltage electrophoresis on a cellulose plate with acetic acid/pyridine/ water (50:5:945, v/v) ,as described (15). After electrophoresis, the phosphoaminoacids were visualized with ninhydrine, scrapped from the plate and counted.

1068

of the

the activation

2

AFTER

at

activity

to be determined

hepatocytes,

also

would

to a high

remains

to rat (3,4),

form

of lipid

It

AMP (10).

metnyltransferase

our

activation

or isoproterenol

Furthermore,

with

PhosphorylaMethods. Arrows

can be converted,

dephosphorylated

of a time-dependent

liver

same 50X protein.

rat

and

the

Vol.

122,

No.

3, 1984

BIOCHEMICAL

AND

BlOPHYSiCAL

RESEARCH

COMMUNICATIONS

$k:: 0

q__n____

20

60

h

100

flllnUteS

Fig.3. Time course of lipid methyitransferase activation and phosphorylation. -ally purified lipid methyltransferase was mixed with MgATP and protein kinase as described under Materials and Methods. At various times a sample was taken and the activity lipid methyltransferase measured or the phosphorylation products analyzed by SDS-PAGE or‘ IEF. (0) time course of phosphorylation of the protein band of p1 4.75; (0) time course of phosphorylation of time course of lipid methyltransferase the protein band of Ms 5OK; and (A) activation. Methyltransferase Activity Ratio is the ratio of the activities measured in the presence and absence of protein kinase and MgATP. Results are the average of three independent experiments in triplicate.

1069

Vol.

122,

No.

3, 1984

BIOCHEMICAL

AND

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1 . Hirata,F. and Axelro~i,~J. (1980) S,cience 209,1082-1090. ?. Matca,,T.M. and Alemanv,?. (19S?) Riochem. #I. 113,1-10. Alemany,?., Nietn,A. and Mato,(T.M. (1980) ,T. Binl. Chem. ?. rastaio,J.f:., 255,9041-9Ob1. '1 . Marin Cac,D., Alvarez Chiva,V. snd Mato,J.M. (1983) Biochem. J. 216,675-680. 5. Alemany,c., Varela,T. and Mato,,T.M. (1981) FEBS letters 160,101-104. 6 . .4lvarez Chiva,V., Marin Cao,D. and Mato,J.M. (1993) FFBS letters 160,101-104. and Bremer,,T. (13Eh) Lipid Res. 7,38-45. 7. Fjornstad,P. P. Skurda1,D.N. and Cnrnatzer,W.C. (1375) Int. J. Biochem. 6,579-583. r . Alemany,S., Varela,I., Harper,J.F. and Mato,J.Y. (1382) J. Biol. [Chem. ?17 ,- 9249-9251. ..10. Mato,J.M., Alenany,S., Gwcia lGil.,M., Mar'in Cao,D., Varela,I. and Castaiio, ,J.C. (1982) in "Biochenistrv of S-adenosylmethionine and related compounds" (Llsdin,F., Borchardt,".T. and Creveling,C.R. eds.) pp 187-194. McMillan Press, London. 11. Pajares,M.A., Alcmany,?., Varela,T., Marin Cao,D. and Mat0,J.M. (1'384) Biochem. J. In press. l?. Thomis,G., Thorn&;. and Luther,fI. (1981) Proc. TJatl. Acad. Sci. USA 78, 5712-5716. Sci USA 76,6106-6109. 13. Matn,J.II. and Marin Cao,D. (1973) Proc. N,atl. Acad. 14. Bradf0rd.M. (1975) Anal. Biochem. 72,248-254. li,. Hi!ntcr,T. and Safton,B. (1990) Proc. Natl. Acad. Sci. USA 77,1331-1315. 16. Cohen,F. (19P1) Nature 296,613-619.

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