calmodulin-dependent protein kinase II using the baculovirus expression system

Vo1.173, No. 2,1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December14,1990

Pages 578-584

EXPRESSION AND CHARACTERIZATION OF THE ALPHA-SUBUNIT OF Ca2+/CALMODULIN-DEPENDENT PROTEIN KINASE II USING THE BACULOVIRUS EXPRESSION SYSTEM

Debra A. Brickey, Roger J. Colbran, Yiu-Lian Fong*, and Thomas R. Soderling Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232-0615 Received October 25, 1990

Sf9 cells infected with the recombinant mouse CaMKII-~ (Ca2+/calmodulin dependent kinase II) baculovirus expressed 12-15 mg of MCaMKII-c~ per liter of cells. Approximately 50% of the MCaMKII-~ activity could be purified using a CaM-Sepharose affinity column. The purified MCaMKII-o~ had a Mrap p of 50 kDa by SDS-PAGE and a native Mr of 600 kDa. MCaMKII-~, like rat braln CaMKII, had an A 0 5 for CaM of i00 nM, a K m for syntide-2 of 8 ~M, and was able to generate Ca2+-independent activity by autophosphorylation. The baculovirus system expressed large quantites of MCaMKII-~ with characteristics similar to the rat brain CaMKII, thus providing an expression system for the detailed biochemical analysis of MCaMKII-~. ©1990 Academic Press,

Inc.

Ca2+/calmodulin-dependent

protein kinase II (CaMKII) is widely distributed

and exhibits a broad substrate specificity. comprised of subunits of 49-62 and bind CaM.

CaMKII is

kDa,

all

(1) and has been implicated

in

including the maintenance of

comprise

the

long-term 10-12

with molecular weights 50/58/60

kDa

preparing large amounts of CaMKII

potentiation copies

(reviewed in 2).

The

of multiple types of subunits

(~,8',~) by SDS-PAGE which complicates

biochemical analysis of its function. reported

up to 2% of the total protein

regulation of several neural functions

purified brain CaMKII contains

been

of which are catalytically active

highly concentrated in neural tissues particularly

in the rat hippocampus where it can

of brain CaMKII have

The holoenzyme (300-700 kDa) is

Recently several cDNA's for subunits (3-6)

which

containing

*Current address-- The Johns Hopkins University, Biology and Genetics, Baltimore, MD 21205.

a

offer the possibility of single type of subunit. To

Department of Molecular

Abbreviations: CaMKII, Ca2+/calmodulin-dependent protein kinase II; MCaMKII~, ~-subunit of mouse brain Ca2+/calmodulin-dependent protein kinase II; Sf9 cells, Spodoptera frugiperda insect ovarian cell line; SDS-PAGE, Sodiumdodecyl sulfate polyacrylamide gel electrophoresis; CaM, calmodulin; FBS, Fetal bovine serum; EGTA, [ethylenebisoxyethylenenitrilo]tetraacetic acid; BSA, Bovine serum albumin; DTT, Dithiothreiotol; PMSF, phenylmethylsulfonyl fluoride; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; MOI, multiplicity of infection. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

578

Vol. 173, No. 2, 1990

this end we

have

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

expressed

baculovirus expression

the

system.

=-subunit This

detailed biochemical characterization of

of

method mutants

mouse should

brain CaMKII in the prove

valuable for

of CaMKII and large-scale

generation of kinase for crystallization and other purposes.

EXPERIMENTAL PROCEDURES Materials. [y-32p]ATP was purchased from Dupont-New England Nuclear. Restriction enzymes and DNA modifying enzymes were from Promega or Bethesda Research Laboratories. Grace's insect medium, lactalbumin hydrolysate, yeastolate, Pluronic F-68, and antibiotics were from Gibco/BRL. FBS was from Hyclone. Betaine was from Sigma. The Sf9 cells were a gift from Dr. Stanley Cohen's laboratory (Vanderbilt University). The wild type baculovirus (Autographica californica) and the pVLI393 transfer vector were gifts from Dr. Max Summers' laboratory (Dept. of Entomology, Texas A & M University, College Station, TX). All other materials were of the highest quality available from general suppliers. Proteins and Peptides. CaMKII was isolated from rat forebrain (7). CaM was purified from bovine brain ( 8 ) . Rabbit polyclonal antibody was provided by Dr. Koji Fukunaga (9). The CaMKII substrate peptide, syntide-2~ was synthesized and prepared as described (I0). Culture of Sf9 Cells. Sf9 cells were cultured in TNM-FH medium (Grace's insect medium containing 3.33 g/l lactalbumin hydrolysate and 3.33 g/l yeastolate) containing 10% FBS and 0.1% Pluronic F-68 at 27°C as attached cells in flasks and as suspended cells in Corning spinner flasks as described by Summers and Smith (ii). Antibiotics (50 ~g/ml gentamicin and 25 ~g/ml Fungizone) were used during protein expression and production of viral stocks. Construction of Recombinant Baculovirus. An EcoRl fragment containing the entire coding sequence of the MCaMKII-= cDNA (12) was inserted into the EcoRI site of pVLI393. The pVLI393-MCaMKII-= transfer vector was cotransfected with purified wild-type baculovirus DNA into Sf9 cells and the recombinant MCaMKII-= baculovirus purified by 2 rounds of plaque purification as described (Ii). The recombinant baculovirus plaques were visually distinguished from the wild type baculovirus plaques by the lack of occlusion bodies within the infected cells. Several recombinant baculovirus plaques were screened for protein expression by the CaMKII activity assay and Western blotting with polyclonal antibody specific for CaMKII. Large scale protein expression was done in 100-150ml Corning spinner flasks at an initial cell density of 3 x 106 cells/ml and a MOI of i0 (ii). Purification of MCaMKII-=. Cells infected with recombinant baculovirus were harvested 72-80 hr post-infection by centrifugation at 800 x g for I0 min and washed once with Buffer A (I0 mM Tris-HCl, pH 7.5 containing 5% betaine, I mM EGTA, i mM EDTA, 0.5 mM DTT, 0.i mM PMSF, 5 mg/l leupeptin, and 20 mg/l soybean trypsin inhibitor). The washed cell pellet was either frozen in liquid N 2 and stored at -70°C or immediately resuspended (I x 107 cells/ml) in Buffer A. The cells were sonicated on ice (Branson cell disruptor) from 3x10 sec bursts at i0 sec intervals. The cell homogenate was centrifuged at 30,000 x g for 30 min. The pellet was discarded and the supernatant centrifuged at i00,000 x g for I hr. The i00,000 x g pellet was rehomogenized in 0.5 volumes Buffer A using a Potter-Eveljheim homogenizer for 5 strokes and then centrifuged at i00,000 x g for i hr. The two I00,000 x g supernatants were combined and (NH4)2SO 4 added to 60%. The precipitated proteins were collected by centrifugation and the pellet resuspended in Buffer B (50 mM HEPES, pH 7.5 containing i00 mM NaCI, 3 mM Mg (Ac)2 , 0.5 mM CaCI2, and I mM DTT) and applied to a 20 ml CaM-Sepharose column equilibrated in Buffer B. The column was washed with 5 column volumes of IM NaCI in Buffer B and the kinase eluted with EGTA (7). Protein concentration was determined by the method of Bradford (13) using BSA as the standard. The purified expressed kinase was stable for at least 6 months when stored at -20°C.

579

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CaMKII Activity Assay. CaMKII was incubated at 30°C for 1-2 min in the presence of 50 mM HEPES, pH 7.5, l0 mM Mg(Ac)2, 400 UM [y-32p]ATP (sp. act. 400-500 cpm/pmol), 20 ~M syntide-2, and either 0.5 mM Ca2+/l ~M CaM or i mM EGTA in a final volume of 25 B1 unless indicated otherwise. All assays were initiated by the addition of kinase diluted appropriately in 50 mM MEPES, pH 7.5 containing 2 mg/ml BSA and 10% ethylene glycol. 32p-incorporation was determined by spotting 15 ~l aliquots onto Whatman P81 phosphocellulose papers (14). CaMKII autophosphorylation was carried out as previously described (15) unless indicated otherwise.

RESULTS AND DISCUSSION

Expression and Purification of

MCaMKII-~--The EcoRl fragment containing the

coding sequence of the MCaMKII-= eDNA of

the

baculovirus

transfer

(12)

vector,

was ligated into the EcoRl site

pVLI393.

Cotransfection

of this

transfer vector with the purified

wild

resulted in in vivo recombination

producing a recombinant baculovirus whose

polyhedron gene has been

replaced

by

type baculovirus DNA into Sf9 cells

the

found to accumulate in spinner cultures 72-80 hr post-infection (data not shown). after 96 hr, apparently due to at

72

hr

post-infectlon

described in Methods. 7 x 108 cells.

cell

was

MCaMKII-=

of

MCaMKII-= was

Sf9 cells to a peak activity at Kinase activity decreased rapidly

lysis.

purified

eDNA.

MCaMKII-= from cells harvested

over

a

CaM-Sepharose

column as

Typically, 2-5 mg of ~-subunit could be purified from

Figure i illustrates the typical expression and purification

obtained under these conditions.

The antibody clearly detects the expressed

protein at 50 kDa, corresponding to but not in uninfected cells.

the

Mr

of the ~-subunit, in infected

Betaine was added to Buffer A, and the i00,000

x g pellet rehomogenized because

preliminary studies without betaine showed

that as much as 60% of the

~-subunit could be particulate (data not shown).

The addition of 5% betaine

consistently

released 90~ of the ~-subunit into

i

Figure i. SDS-PAGE and Western Blot of Expressed MCaMKII-=. A. Coomassiestained fOX polyacrylamide gel. Lane I, Purified rat brain CaMKII; Lane 2, Purified MCaMKII-=; Lane 3, Ist I00,000 x g pellet; Lane 4, Ist I00,000 x g supt.. B. Western Blot using anti-brain CaMKII polyclonal antibody with peroxidase-conjugated 2 o antibody. Lane i, purified MCaMKII-~; Lane 2, ist i00,000 x g pellet; Lane 3, Ist I00,000 x g supt.; Lane 4, i00,000 x g pellet of uninfected Sf9 cells; Lane 5, i00,000 x g supt. of uninfected Sf9 cells. The arrows indicate the position of the 50 kDa subunit of brain CaMKII. 580

Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I Purification of MCaMKII-= Expressed in Sf9 Cells

Total Protein (mg)

Total Activity (~mol/min)

Specific Activity (~mol/min/mg)

Homogenate

315

6.1

0.02

-

100%

30000 x g supt.

276

5.7

0.021

1.0

93%

1 s t 100000 x g supt.

201

5.3

0.026

1.3

87%

0.42

0.022

i.i

6.9%

Purification Step

2nd I00000 x g supt.

19.5

Purification Factor

Yield

(NH4)2SO 4 Pellet

173

4.4

0.025

1.25

72%

On CaM-Sepharose

170

6.7

0.039

1.95

110%

3.2

3.4

170

52.4%

Pooled CaM-Sepharose Fractions

the soluble

0.94

fraction

purification

in the combined

was obtained

using

i00,000

activity of the purified MCaMKII-= was prepared

by a similar

prepared

by this method

Table

2

method

summarizes

(7).

similar As

seen

several

biochemical

The holoenzyme

determined

on a Superose-6

comparable

by chromatography

holoenzyme

(Table

A 170-fold

i) and the specific

to rat or mouse brain CaMKII in

Figure i, the MCaMKII-a

is at least 95% pure.

MCaMKII-= and brain CaMKII.

after the thyroglobulin

x g supernatants.

CaM-Sepharose

standard

to rat

brain

and

structure

that

properties

Both eluted just

MCaMKII-=

containing

approx.

had formed a 12 subunits.

Table 2 Characteristics of MCaMKII-= vs Rat Brain CaMKII Percent Ca2+-Independent Activity 2 +

Kinase

Holoenzyme Structure I

Rat Brain

650 kDa

<2%

40-50%

I00 nM

8 ~M

MCaMKII-=

600 kDa

<2%

40-50%

ii0 nM

8 ~M

Ao 5 Calmodulin 3

Km Syntide-24

]Determined by S u p e r o s e - 6 chromatography u s i n g t h y r o g l o b u l i n (669 kDa), BSA (67 kDa), and r i b o n u c l e a s e a (13.7 kDa) as s t a n d a r d s . 2Ratio of activity +EGTA/activity + Ca2+/CaM determined before (-) and after (+) autophosphorylation under optimal conditions (0.5 mM ATP, l0 mM Mg(Ac)2 , 2 mM CaCl2, 3 ~M CaM) (14). 3Determined with 250 ~M syntide-2, 400 ~M ATP with CaM concentrations ranging from 5 nM to 25 ~M. 4Determined by Lineweaver-Burke analysis. 581

of

of the two kinases was

FPLC column.

indicating CaMKII

physical

Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

30

25

O

J

>~ >

O

2O

T ~

5 Oi 2

w~ 6

¢

J 8

10

12

TIME (min)

Figure 2. Time Course for Generation of Ca2+-Independent Activity. Rat r a ~ C a M K I I (0) or MCaMKII-~ (&) was preincubated at 4°C (i pM subunit) under conditions of limiting autophosphorylation (7 ~M ATP, 0.5 mM Mg(Ac)2, 2 mM CaCI2, 3 ~M CaM) (22) for the indicated times. Controls (e,A) lacked ATP. After the indicated time, aliquots were diluted 10-fold in Stopping Buffer (50 mM HEPES, pH 7.5, 10% ethylene glycol, 1 mg/ml BSA, 5 mM EDTA) and stored at 4°C until assayed in the presence of Ca2+/CaM and in the presence of EGTA. The percentage Ca2+-independent activity is shown. Total kinase activity (+Ca2+/CaM) decreased by l e s s than I0~ during the preincubation. The data shown are representative of 2 similar experiments.

CaM-activation

curves

were

determined

syntide-2 and [y-32p]ATP (0.25 that the interaction

of

the

mM

and

two

at

saturating

concentrations

0.4 mM, respectively)

kinases

of

and indicated

with CaM was indistinguishable.

Both kinases also exhibited similar kinetics for phosphorylation

of syntide-

2. To determine the

similarity

several biochemical Ca2+-independent a unique

and

in

CaMKII

and

exhibited

similar

rates

under

limiting

Under

autophosphorylation the

Figure for

with

the

both

to approximately

1.6 mol

32p

could

be

incorporated

under optimal conditions with both MCaMKII-~ shown). This study

has

demonstrated

the

system to express the ~-subunit of

CaMKII.

conditions.

maximum

percent In

reaction, up

per mol of a-subunit

and rat brain CaMKII (data not

feasibility

similar in enzymatic and physicochemical

generation of

kinases (Table 2).

similar experiments using [y-32p]ATP in the autophosphorylation

is

2 shows that

autophosphorylation

conditions,

same

Generation of

of threonine-286

CaMKII,

activity

optimum

CaMKII, we compared

of

Ca2+-independent

independent activity was also

and

experiments.

autophosphorylation

characteristic

brain

MCaMKII-~

parallel

kinase activity by

regulatory

MCaMKII-~

between

properties

of

using the baculovirus

The expressed MCaMKII-~ is very

properties to native brain CaMKII.

The ~-subunit had previously been

expressed using several different systems

including in vitro expression

a

and in vivo

in

E.coli

in

(16-18),

rabbit reticulocyte lysate system (12)

CHO 582

cells

(19),

and COS cells (20,21).

Vol. 173, No. 2, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

However, the baculovirus system has several advantages over these expression systems.

The E.coli system produces

specific activity (16), and with a

a monomeric (17,18) ~-subunit of lower variable K a for CaM (ranging from 95-600

riM) (16-18). In addition, a considerable be insoluble (17) and

However, as seen in Figure of

expression

appears

The rabbit reticulocyte systems produce an

amount of the expressed kinase can

proteolysis/premature

termination

can be a problem.

i, neither proteolysis nor premature termination

to

be

a

lysate

problem

system

oligomeric

and

=-subunit

in

the

the

baculovirus

system.

mammalian cell expression

(12,19-21) of comparable activity

and enzymatic properties although the amount of kinase generated is limited. Sf9 cells produce a greater quantity using the rabbit reticulocyte systems and

also

baculovirus system

have

lysate,

little,

offers

of

if

several

kinase than can be easily obtained CHO

any,

cells,

or COS cells expression

endogenous

significant

kinase.

Thus, the

advantages over previously

used expression systems and will be useful for detailed biochemical analyses of wild-type and mutant CaMKII proteins. Acknowledgments-- We would like to thank Martha Bass and Elizabeth Wolf for their excellent technical assistance. This work was supported by National Institutes of Health Grants GM41292 and DK17808. REFERENCES i. Erondu, N.E. and Kennedy, M.B. (1985) J. Neurosci. 5, 3270-3277. 2. Colbran, R,J., and Soderling, T.R. (1990) In Current Topics in Cellular Regulation (B.L. Horecker, E.R. Stadtman, P.B. Chock, and A. Levitzki, Ed.) Vol. 31, pp. 181-215. Academic Press, San Diego, CA. 3. Hanley, R.M., Means, A.R., Ono, T., Kemp, B.E., Burgin, K.E., Waxham, N., and Kelly, P.T. (1987) Science 237, 293-297. 4. Lin, C.R.~ Kapiloff, M.S., Durgerian, S., Tatemoto, K., Russo, A.F., Hanson, P., Schulman, H., and Rosenfeld, M.G. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5962-5966. 5. Bulleit, R.F., Bennett, M.K., Molloy, S.S., Hurley, J.B., and Kennedy, M.B. (1988) Neuron I, 63-72. 6. Bennett, M.K. and Kennedy, M.B. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1794-1798. 7. Hashimoto, Y., Schworer, C.M., Colbran, R.J., and Soderling, T.R. (1987) J.Biol. Chem. 262,8051-8055. 8. Gopalakrishna, R. and Anderson, W.B. (1982) Bioehem. Biopys. Res. Commun. 104, 830-836. 9. Ohta, Y., Ohba, T., Fukunaga, K., and Miyamoto, E. (1988) J. Biol. Chem. 263, 11540-11547. I0. Hashimoto, Y. and Soderling, T.R. (1987) Arch. Biochem. Biophys. 252, 418-425. II. Summers, M.D. and Smith, G.E. A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agricultural Experiment Station Bulletin No. 1555, 1988. 12. PonE, Y.L., Taylor, W.L., Means, A.R., Soderling, T.R. (1989) J. Biol. Chem. 264, 830-836. 13. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 14. Colbran, R. J., Fong, Y.L., Schworer, C.M., & Soderling, T. R. (1988) J. Biol. Chem. 263, 18145-18151. 15. Roskoski, R., Jr., (1983) Methods Enzymol. 99, 3-6. 16. Ohsako, S., Watanabe, A., Sekihara, S., Ikai, A., and Yamauchi,T. (1990) Biochem. Biophys. Res. Commun. 170, 705-712. 583

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

17. Waxham, M.N., Aronowski, J., and Kelly P.T. (1989) J. Biol. Chem. 264, 7477-7482.

18. Waxham, M.N., Aronowski, J., gestgate, S.A., and Kelly~ P.T. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1273-1277. 19. Yamauchi, T., Ohsako, S., and Deguchi, T. (1989) J. Biol. Chem. 264, 19108-19116. 20. Hanson, P.I., Kapiloff, M.S., Lou, L.L., Rosenfeld, M.G., and Schulman, H. (1989) Neuron 3, 59-70. 21. galdmann, R., Hanson, P.I., Schulman, H. (1990) Biochemistry 29, 16791684. 22. Schworer, C.M., Colbran, R.J., Keefer, J.R., and Soderllng, T.R. (1988) J. Biol. Chem. 263, 13486-13489.

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