Naunyn-Schmiedeberg's Arch Pharmacol (1981) 317:100-102

Naunyn-Schmiedeberg's

Archives of Pharmacology �9 Springer-Verlag 1981

Quercelin Inhibils Tyrosine Phosphorylalion by the Cyclic Nucleolide-Independenl, Transforming Prolein Kinase, pp60 src

H. Glossmann 1 , P. PreseI~ 1 , and E. Eigenbrodt 2

1 Rudolf-Buchheim-Institut fiir Pharmakologie and

2 Institut for Biochemie und Endoktinologie, Frankfurter Strasse 107, D-6300 Giessen, Federal Republic of Germany

Summary. The bioflavonoid quercetin is a potent inhibitor of

a cyclic nucleotide-independent, tumor virus-coded protein kinase which phosphorylates tyrosine residues and acts as a

cellular transforming protein. Half-maximal inhibition of the

protein kinase occurred at 3-4 /aM quercetin whereas rutin

was much less effective. The finding, that quercetin inhibits a cyclic nucleotide-independent protein kinase activity, may provide clues to the diverse pharmacological effects of the bioflavonoids.

Material and Methods

Materials

[3,-3~P] ATP (2000 Ci/mMol) was from Amersham, Braunschweig, FRG; Blue Sepharose and protein-A Sepharose were from Pharmacia, Freiburg, FRG. Quercetin, rutin, histone type IIa and a-casein were from Sigma, Mtinchen, FRG. Galangin, morin and myrecitin were kind gifts from Dr. v. Bruchhausen, Berlin and the purified catalytic subunit of the cyclic AMP dependent protein kinase (type II) from beef heart was donated by Dr. F. Hofmann, Heidelberg, FRG.

Key Words: Quercetin - Tyrosine Phosphorylation - Pro-

tein kinase activity - Tumor virus - Flavonoids Methods

Introduction

Certain bioflavonoids, like quercetin, inhibit growth of cells in culture. Quercetin, at low concentrations, also inhibits

glycotysis and lactate transport in Ehrlich ascites tumor cells

(Soulinna et al. 1975). Since mitochondrial ATPase and the plasma membrane-bound Na+-K + ATPase are inhibited by

the flavonoid (Spector et al. 1980), it was suggested that the

high aerobic glycolysis in cancer cells is sustained by aberrant

ATPases (susceptible to quercetin inhibition) which supply

ADP and Pi as a driving force. This concept has been con- tested by Bustamante and Pedersen (1977) who found an un- usual, highly active mitochondria-bound hexokinase in hepa-

toma cells which was inhibited by quercetin as well. In ad- dition it has been recently observed that quercetin inhibits the (Ca 2+ + Mg~+)-ATPase of sarcoplasmatic reticulum

(Shoshan and MacLennan 1981). We will report in the following that quercetin acts as an

inhibitor of the cAMP-independent protein kinase which is

the src-gene product of Rous sarcoma vires, pp60 src. This

protein Linase is necessary for in vitro transformation and formation of sarcomas in birds, pp60 src is a 60.000 dalton phosphoprotein, indicating that it is part of a protein kinase cascade and is an unusual protein kinase since substrates are exclusively phosphorylated in tyrosine residues (Hunter and Sefton 1980; Collett et al. 1980; Levinson et al. 1980).

Send offprint requests to H. Glossmann at the above address

Preparation of the Cyclic Nucleotide-Independent Protein Kinase, pp60 sre. The vitally coded protein kinase was partially purified by means of successive blue-sepharose and casein-affinity column chromatography from Rous sarcoma virus induced mouse tumors (Glossmann et al. 1981). The final preparation, which was between 300-500-fold purified with respect to the transforming Protein ki- nase, was stored in 50% glycerol at -25~ Prior to use the protein kinase preparation was dialysed for 2 h at 4~ against a 2000-fold excess of 50 mM TRIS-HC1 bffffer, pH 7.4, containing 0.1 mM phenylmethylsulfonylfiuoride.

Phosphorylation Experiments. Phosphorylation experiments were performed with protein-A Sepharose purified IgG obtained from antisera of tumor bearing rabbits (TBR-sera). The tumors were in- duced by the Rous sarcoma virus. The TBR-IgG will serve as arti- ficial but highly specific phosphate acceptor (CoUett et al. 1980; Levinson et ai. 1980). Phosphorylation by the pp60 src kinase will occur in the heavy chain, exclusively in tyrosine residues. 7.5 ~g of affinity-purified TBR-IgG were incubated with 5 #1 of the dialysed pp60 src kinase preparation in the presence of 0.03 I~M ['r -32p] ATP (2000 Ci/mMol), 10 mM MgC12 and 50 mM TRIS-HC1 buffer, pH MgC12 7.4, in a final volume of 40 ~1, with and without additions. phosphorytation was allowed for 60 rain at 4 ? C and the entire reaction mixture after addition of sample buffer and boiling applied onto 11% polyacrylamide slab gels as described (Presek et al. 1980). The radio- activity incorporated in the TBR-IgG heavy chain region was quanti- tared by Cerenkov counting.

The two-dimensional separation and identification of a 2 P-labelled aminoacids after acid hydrolysis of the TBR-IgG heavy chain was per- formed as described by Hunter and Sefton (1980).

Results and Discussion

Our pp60 src protein kinase preparation was free from cAMP- dependent protein kinase activity and phosphorylated TBR- IgG exclusively in tyrosine residues free in solution. This proves the pp60 src specific nature of the phosphorylation.

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Table 1 Effects of bioflavonoids on the activity of the cyclic nucleo- tide-independent, virally coded protein kinase, pp60 src. The activity of pp60 src kinase with IgG as phosphate acceptor was determined as described in Materials and Methods. Bioflavonoids were present at the indicated final concentrations. The activity of the protein kinase is given in fmol 32p per assay incorporated (as phosphotyrosine) into the heavy chain region of IgG obtained from sera of Rous sarcoma tumor bearing rabbits. All assays contained 0.25% (v/v) of dimethyl- sulfoxyde, which was used as solvent for the bioflavonoids. Results are means from triplicate experiments -+ S.E.M.

Additions [/~M] Activity of pp60 src kinase %-Inhibition

None 12 -+ 1,4 0

Quercetin 1 3

10 30

100 300

Rutin 3

10 30

300 9O0

Myrecetin 2O

Morin 2O

Galangin 20

7,7 -+ 0,3 6,1 -+ 0,4 2,7 +- 0,5 1,5 -+ 0,3 1,7 -+ 0,2 2,7 + 0,7

11,6 +- 1,9 12,3 +- 1,7 12,0 -+ 0,8 11,7 -+ 2,0 5,8 -+ 1,2

5,3 -+ 0,4

6,9 -+ 1,2

8,1 + 0,9

36 49 78 88 86 78

3 0 0 3

52

56

43

33

Table 1 shows that the bioflavonoid quercetin at low concen- trations was able to inhibit the tyrosine phosphorylat ion by the pp60 src kinase. 50% inhibition was observed 3-4/xM

quercetin. Dimethylsulfoxyde, used as a solvent for the bio- fiavonoids at a final concentration of 0.025% (v/v), had no effect on pp60 src kinase activity. However, as can be seen from Table 1, the inhibition was not complete and even part- ially reversed at quercetin concentrations above 100 /aM. Rutin was much less effective than quercetin to inhibit the pp60 src kinase. Among other bioflavonoids which were tested, myrecet in was the most active followed by morin and galangin. Our pp60 src kinase preparat ion also phosphory-

lated a-casein although less efficient than TBR-IgG. This phosphorylat ion was even more sensitive than the TBR-IgG phosphorylat ion with respect to quercetin inhibition (50% inhibition at 0.3/aM quercetin).

In order to investigate whether the effects of the bio- flavonoid is seen with other protein kinases we have tested it on the purified catalytic subunit of the cAMP-dependent protein kinase and histone type IIa as substrate. Quercetin, in concentrations up to 0.1 raM, was without effect on the act- ivity of this protein kinase. However, we have isolated and

purified about 2000-fold by means of a three step affinity chromatography a heparin-sensitive cAMP-independent pro- tein kinase from chicken Rous sarcoma tumors (similar to the casein kinase II, Hathaway et al 1980) which we found to be highly sensitive to quercetin.

This protein kinase phosphorylated serine residues in casein. Thus it appears that quercetin (and perhaps other bio- flavonoids) can act as inhibitors of cAMP-independent pro- tein kinases. Our findings, although observed in.an in vitro assay system with an artificial (but highly specific) phos- phate acceptor and a protein kinase coded by an RNA tumor virus may nevertheless provide some clues to the diverse pharmacological effects of the bioflavoids: E.g. pp60 src is closely related to a normal cellular protein kinase (termed pp60 sarc) which also phosphorylates tyrosine residues (Hunter and Sefton 1980.)

The role of this protein kinase in cellular metabolism is ye t not known. However, it can be safely stated that this act- ivity is of fundamental importance since an increase of its activity by means of the transforming Rous sarcoma virus leads to tumor formation and cellular transformation. I t is likely that the normal cellular enzyme (as shown here for virally coded activity) is inhibited by bioflavonoids as well. On the other hand, although our results suggest that quer- cetin-sensitive protein kinases should be looked for in cell- ular systems where pharmacological effects of the bioflavo-

noids are seen, the diversity of actions of the bioflavo- holds on energy conserving reactions and transport pheno- mena is perplexing. Recent evidence obtained with the (Ca 2+ + Mg2*)-ATPase of sarcoplasmatic reticulum indicates that the bioflavonoid may compete at the nucleotide binding site. The inhibition of ATPase was nevertheless noncom- petive with respect to ATP and the bioflavonoid apparently stablized the phosphorylated form of the enzyme which was unable to transport Ca 2+ (Shoshan and MacLenna 1981). I t will be necessary to determine the kinetics of inhibit ion of the protein kinases susceptible to quercetin as well as the structure-activity relationship for different flavonoids to clarify if a common mechanism is involved.

Acknowledgements The authors would like to thank our collegues at the Virology department, especially Drs. H. Bauer and R.R. Friis, for stimulating discussions arid supply of tumors and antisera. I. Seidel and A. MiJneh are thanked for competent assistance.

This work was supported by Stiftung Volkswagenwerk and Son- derforschungsbereich 47 of Deutsche Forsehungsgemeinschaft.

References

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Glossmann H, Presek P, Eigenbrodt E (1981) Association of the src- gene product of Rous sarcoma virus with a pyruvate kinase inacti- vating factor. Mol Cell Endocrinol (in press)

Hathaway GM, Lubben TH, Traugh JA (1980) Inhibition of casein kinase II by heparin. J Biol Chem 255: 8038-8041.

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Received April 23/Accepted May 26, 1981