Isolation of a 50 000 dalton cAMP binding protein and its characterization as a regulatory subunit of protein kinase II

Vol. 104, No. 3, 1982 Februory 11, 1982

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ISOLATION OF A 50 000 DALTONCAMP BINDING PROTEIN AND ITS CHARACTERIZATION AS A REGULATORYSUBUNIT OF PROTEIN KINASE II

Institut

Wolfgang Weber, Gerhild Schwoch, and Helmuth Hilz fur Physiologische Chemie, Universitit Hamburg,

Germany

Received December30, 1981 An unusual CAMPbinding protein of 50 000 Da previously found in human tumors was isolated from HeLa cells in the presence of protease inhibitors. The protein was neutralized by anti-bovine RII antibodies but not by anti-RI. It was able to form a dimer, and to inhibit HeLa C kinase in a dose-dependent manner. The HeL*a RII 50 000 was also subject to limited proteolysis and it could be phosphorylated by C kinase. HeLa cells contain two RI proteins, a predominant 49 000 Da and a minor 51 000 Da isoprotein. In addition, large amounts of a protein ccnsistinq of 19 000 and 20 000 Da subunits were isolated by 8-thio-CAMP affinity chromatography that was not immunologically related to the R proteins. INTROCUCTION Mammalian tissues contain two types of CAMP-dependent protein kinases, which differ in their regulatory subunits while the catalytic subunits C appear to be identical in both isoenzymes. The RI subunit has uniform size in nearly all mammalian tissues studied (m.w. 49 000). In contrast RII exhibited a certain microheterogeneity , although the predominant form in most species is the 54 000 iscprotein [for reviews see 1,2]. Recently we have identified by immunological criteria a 50 000 Da CAMP binding protein as the principal or even the exclusive type II R protein in various human tumors [3,4]. This variant was not found in several non-human tumor cells. To determine whether the 50 000 RII protein of human tumor cells had lost some or all of its basic regulatory functions, the RII protein was isolated from a homogeneous population of human tumor cells by affinity chromatography, and its properties were compared with those of RII 54 000 from bovine heart. METHODS Regulatory subunits were purified from rabbit muscle (RI) and bovine heart (RII) [5]. Determination of total R proteins and immunotitration of regulatory subunits were performed as-previously described [5,61. Extracts were prepared by sonication of HeLa cells (6-8x107 cells/ml) in 30 m&lTrissacetate pH7.4, 20 mM benzamidine, 4 mM EDTA, 150 U/ml 50 ug/ml leupeptin, 10% glycerol followed by centrifuTrasylol(Bayer), gation (10 min; Hettich Rotosilenta). DEAE cellulose chranatography for separation of protein kinase isoto [7]. Extracts from 15x109 HeLa enzymes was performed according 0006-291X/82/031134-08$01.00/0 Copyrrghr 0 1982 by Academic Press, Inc. .-III righrs 01 reproducrion in an.r form reserved.

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cells were prepared in the presence of 2 n-Mphenylmethylsulfonyl fluoride in 5 mMTris.acetate/l mMEDTA/l mMDTTpH 7.6 and fractionated on a DEAE column (DE-32 Whatman, 200 ml). Fractions were analyzed for casein kinase and histone kinase activity +cAMP [8], and for CAMP binding [51. The peak I and peak II fractionswere used for affinity chromatography. Affinity chromatography on a 8-(2'-hydroxyethyl)-thio-CAMP matrix (2 ml) was performed as previously described [5]. After loading with extracts (corresponding to 4-15x109 HeLa cells) the gel was extensively washed with 2 M NaCl followed by 2 volumes each of 1 mM 5'-AMP and 1 mM CAMP (removal of a low affinity 35 kDa protein). The column was then eluted with two vol. 30 mMCAMPwithin 10 min yielding the protein composed of 19 and 20 kDa subunits. Further incubation with 30 mMCAMP at room terrtperature for at least 2 h and slow elution yielded the R proteins. Preparation of C subunits. HeLa cell extract (2-3x107 cells) was incubated with 12 pl each of anti-RI and anti-RII antiserum in a suspension (final volume 0.9 ml) containing 80 ul-(protein A)-Sepharose (Pharmacia) in 20 mM TriseHC1/2 mMDTT/80 ITM NaCl/lO% glycerol pH 7.4 for l-2 hrs at 5oC. The tubes were rotated. After washing the beads with 5 IIM TrissHC1/145 rrlvlNaCl pH 7.4 (2x1 ml), C subunits were released by 30 @l CAMPin 5 mMTris-HC1/2 nM DTT/lOO rrMNaC1/2 mg/ml serum albumin/ 30% glycerol pH 7.4. The supernatant was used directly or, if removal of CAMP was required, after treatment with half a volume of charcoal suspension (10% in 10 ITMTrissHCl pH 7.4/20 mg/ml serum albumin/lo% glycerol). In some experiments C from bovine heart protein kinase II (Sigma) obtained by an analogous procedure was used. RESULTS AND DISCUSSION HeLa cells as a source of RII 50 000. Selection of a suitable tumor cell line was based on the following criteria: The cells should have reasonable amounts of type II CAMP binding protein and this should be isolation also required that represented by the RII 50 000. Effective the variant had retained a high affinity to CAMP matrices. Immunotitration [6] served to analyze the amounts of RII isoproteins. When HeLa cell extracts (1.22 + 0.2 pmol CAMP binding sites per 40% of total R 106 cells) were titrated with anti-RII antibodies, proteins could be neutralized (Fig. 1). was performed by a proIdentification of RI and RII isoproteins cedure [6] that combines affinity labeling with [32P]n3cAMP [9], sedimentation of type I or type II proteins with specific antibodies + (protein A)-Sepharose, SDSgel electrophoresis and autoradiography (Fig. 2). Three major bands close to each other were found, one corresponding to the expected RII 50 000 as deduced from its affinity to anti-RII. Surprisingly, the anti-RI antibodies bound two polypeptides, the 'regular' RI 49 000 and a labeled component with higher apparent molecular weight. The existence of this RI isoprotein (about 51 000 Da) was confirmed together with RI 49 000, applying affinity chromatography by isolation, to the protein kinase I fractions (DEAE cellulose). Whether this component represents indeed a larger (precursor?) RIpolypeptide or is derived

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anti

-R,

3

60%.

\

20%

60%

T RI

60%

20%

0:5

lb

1.;

pl ant1-R,,

Figure 1.

Immunotitration of HeLa cell extract.- Crude HeLacell extract was titrated with anti-RI (upper curve) and anti-RII (lower inversed curve) in the presence of saturating [*IcAMp and (protein A)Sepharose [61. R.[&i]cAMPproteins not sedimentedbythe antibodies + (protein A)-Sepharose were quantified in the supernatant. Total CAMPbinding sites (1 pmol/sample) were set 100%. The intercept of both titration curves on the right indicates the percentage of each type of R protein present in the extract. from RI 49 000 by an endogenous modification enains to be determined. Binding of the RII 50 000 to immobilized CAMP was studied by application of total HeLa cell extracts to 8-thio%AMP Sepharose columns. The matrix was extensively washed with 2 M NaCl and 1 n-M 5'-AMP, followed by 1 mM CAMP to elute low affinity bit-ding proteins. When the concentration of CAMP was raised to 30 nM, large amounts of a pure protein were displaced from the CAMPmatrix prior to the Rproteins. It consisted of two subunits (19 K and 20 K; Fig. 3a). All criteria including innnunolcgical analyses showed that it is not related to the R proteins. The high affinity CAMP binding R proteins could be finally displaced by prolonged incubation of the column with 30 IIM CAMP, together with some contaminating 19/20 K polypeptides (Fig. 3, .ane b). Immunotitration showed that this fraction contained RI an3. RII isoproteins in the same ratio as the starting extract (cf. Fig. 1) indicating that RII 50 000 binds to the 8-thio-CAMP matrix with an affinity comparable to RII 54 000 and RI 49 000. Isolation of RII 50 000. In order to separate from RI and the 19 K/20 K contaminant, extracts 1136

the tpe II R protein of HeLa cells prepared

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Rstandards

-serum albumin -

54 K 49 K

I

a Figure

C

b

d

2.

Autoradiography of affinity-labeled Rproteins in HeLa extract and of sedimented immune canplexes.Extracts (1.3xlob cells/sample) were photoaffinity-labeled [91 in the presence of 1.2 )JM [32P1n3cAMP; aliquots were treated with antisera as described previously [6]. Control extract (a), immune sediments (b,c) and Rprotein standards (d, rabbit muscle RI + bovine heart RII) were subjected to SDS electrcphoresis and autoradiography.

in the presence of protease inhibitors were separated by DEAR cellulose the peak fractions corresponding to protein chromatography and kinase isoenzymes I (60% of total activity) and II (40%), respectively, The pooled fractions were subjected to affinity chromawere collected. tography on thio-CAMP matrices, which adsorbed 75-80% of the CAMPbinding activities. Selective elution of R proteins was performed as described previously [5]. In this way, pure RII 50 000 was obtained frcxnthe DEAE cel(Fig. 3e). The yield was about25 ug per lOlo cells. lulose peak IIfractions Characterization

of RTT

50

0Of1.

From previous 1137

studies

it

was known

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R,, 54K \ R,, 50 K /-R,

49K

e

c-

19K 20K

b

a

C

d

SDS gel electrophoretic affinity chromatography.or [B] DEAE cellulose peak II fractions to affinity chromatography on the (b) and (e) (a) 19/20 kDa protein: muscle RI and (d,g) bovine heart RII Figure

3.

analysis of proteins isolated by [A] Total extract of 4x10YHeLa cells from 15x10g cells were subjected 8-thio-cAMF' matrix (see Methods). R protein fractions: (c,f) rabbit standards.

some of the R isoproteins of lower molecular weight had lost the ability to formdimers and to interact with the catalytic subunit [lo-131. When RI* 50 000 from HeLa cells was analyzed electrcphoretically under non-denaturing conditions, it migrated in a 9% gel to a position that was close to the dimer of RII 54 000 (nor shown). This indicated that RII 50 000 still contains the domains required for R2 formation.

that

The abilitytobind normal

range

CAMPwith high affinity

as judged

from

the

resistance

also appeared to be in the of

RIrbound

CAMP to char-

coal treatment [cf. 131, from the ready formation of covalent photoreactive n3cAPIP and from the high CAMP concentrations vated

temperature

required

to

displace 1138

the

RI1

50 000

from

bonds to the and the elethe

matrix.

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I

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.4 pmol CAMP bIndIng sites .I

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I

I

I/II

.0

Inhibition of C activity by recombination with R.- R proteins as eluted from the affinity columns were freed from excess CAMP by ammonium sulfate precipitation and Sephadex G 25 chromatography.

Figure

4.

Increasing amounts of R were added to preparations of C (activity = and the resulting kinase activities were 1.4 pool phosphate incorp./min), measured in a final volume of 200 ~1 as described 1131.

The most important property with respect to the regulatory function of R proteins is the ability to bind and inactivate the catalytic subunit C. To study this interaction, C protein was isolated by a new procedure. It is based on the previous observation [6] that anti-RI or anti-RI1 in the presence of (protein A)-Sepharose also sedimented the of the corresponding protein kinase holoenzymes. Subsequent treatment sediment with CAMP releases pure C into solution. When such preparations were freed fran CAMP and incubated with increasing amounts of HeLa RI1 50 000, kinase activity was progressively reduced (Fig. 4). Bovine heart RII 54 000 proved to be somewhat rrore effective than the HeLa RI1 50 000 as a regulator of C activity. RII proteins are further characterized by their ability to serve as substrates for autophosphorylation and RII proteins from [141. RI HeLa cells were incubated with HeLaC subunits in the presence of [Y-~~P]ATP and the reaction products were analyzed by SDS gel electrophoresis and autoradiography. As shown in Fig. 5, RII 50 000 from HeLa cells was intensively phosphorylated while HeLa RI 49 000 (lane a) was not a

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-RI

(bovine heart)

-h

(HeLa cells)

C

Figure 5.

Phcephorylation of R proteins.R subunits (0.4 ug RLL 50 000 or RTT 54 000. 0.7 ua HeLa RT 49 000) were incubated (15 min, 37oC) with C i&uivalent to 106 HeLa cells) I in 100 ul containing 20 ITMTris.RCl pH 7.4, 1 W DTT, 8 nM Mg2+.acetate, 1 mMmethylisobutylxanthine, 8 uM [y-j2P]-ATP (7 Ci/mmol), and excess CAMP(>0.2 RM). Sampleswere subjected tc SDSgelelectrcphoresis and autoradiography (61. (a) HeLa RI 49 000; (b) HeLa RII 50 000; (c) bovine heart RII 54 000 substrate. Compared to bovine heart RII 54 000, however, the tumor RII accepted significantly less phosphate. Finally a comparative analysis of HeLa RII and bovine heart RII was performed with respect to limited proteolysis. While the bovine subunit is known to form a rather stable R' product of 37 000 Da when treated with various proteases [lo-131, RII 50 000 was degraded to a 36 000 Da protein which proved to be less resistant to further proteolytic attack than the R' 37 000 (not shown). Thus, the 50 000 Dalton protein from HeLa cells proved to exhibit all the fundamental properties that are characteristicof protein kinase regulatory subunits: it can bind CAMPwith high affinity, forma dimer and inactivate the protein is to be classified as the catalytic subunit C. In addition, a type II subunit because of its affinity to anti-RI1 and its substrate properties for C kinase. 0-1 a quantitative basis, however, the tumor RII can be differentiated fran the 'regular' RII 54 000 isoprotein by an impaired interaction with the catalytic subunit and an altered sensitivity to proteolytic degradation. The RII 54 000 isoprotein has beendetected so far ina number of human tumors, but not in non-human tumor cell lines like mouse Ehrlich ascites tumor and rat AH 130 hepatama. Whether the protein is restricted to human tumor tissues is not yet clear. 1140

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ACKNOWLEDGEMENTS Our thanks to G. Jarmers who provided the HeIa cells. This supported by the Deutsche Forschungsgemeinschaft, SFB 34.

work was

ADDENDUM During the preparation of the manuscript, a paper by Friedman and Strittholt [Biochim. Biophys. Acta 675, 334-343 (1981)] appeared that showed by way of affinity labeling the presence in HeLa cells of the same RI and RII proteins that we have isolated. REFERENCES 1. Nimmo, H.G. & Cohen, P. (1977) Adv. Cyclic Nucleotide Res. 8, 145-266 2. Doskelard, S.G. & Ogreid, D. (1981) Int. J. Biochem. l3, l-19 3. Weber, W., Schwcch, G., Schroder, H. & Hilz, H. (1981) Cold Spring Harbor Conf. on Cell Proliferation S, 125-140 4. Weber, W., Schwoch, G., Wielckens, K., Gartemann, A. & Hilz, H. (1981) Eur. J. Biochem., in press 5. Weber, W., Vogel, C.W. & Hilz, H. (1979) FEBS Lett. 2, 62-66 6. Weber, W., Schrijder, H. & Hilz, H. (1981) Biochem. Biophys. Res. Ccmmun. 29# 475-403 7. Corbin, J.D., Keely, S.L. & Park, C.R. (1975) J. Biol. Chem. 250, 218-225 8. Schwoch, G. (1978) Biochem. J. 170, 469-477 9. Haley, B.E. (1975) Biochemistry 14, 3852-3857 10. Corbin, J.D., Sugden, P.H., West, L., Flockhart, D.A., Lincoln, T.R. & McCarthy, D. (1978) J. Biol. Chem. 253, 3997-4003 11. Weber, W. & Hilz, H. (1978) Eur. J. Biochem. 83, 215-225 12. Potter, R.L. & Taylor, S.S. (1979) J. Biol. Chem. 254, 2413-2418 13. Weber, W. & Hilz, H. (1979) Biochem. Biophys. Res. Commun. 90, 1073-1081 14. Erlichman, J., Fosenfeld, R. & Rosen, O.M. (1974) J. Biol. Chem. 249, 5000-5003

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