Life Scieacea Vol. 22, pp . 1481-1484 Printed in the U.S .A .
Pergamon Prees
POLYAMINES AND PROTEIN KINASE II . EFFECT OF POLYAMINES ON CYCLIC AMP - DEPENDENT PROTEIN KINASE FROM RAT LIVER Jacob Hockiman, Aviva Katz and Uriel Bachrach Department of 7~oology, The Hebrew University, Jerusalem, and Department of Molecular Biology, Hebrew University - Hadassah Medical School, Jerusalem, Israel (Received in final form March 6, 1978) Summary The effect of polyamines on the activity of cAMP-dependent protein kinase from rat liver was determined . It has been shown that polyamines inactivate the enzyme in the decreasing order of activity : spermine > spermidine > putrescine . This effect is due to inhibition of the catalytic subunit . On the other hand, binding of cAMP to the regulatory subunit of the enzyme is not affected by polyamines . It is suggested that the inhibition of protein kinase by polyamines is a general phenomenon . The unequivocal importance of cyclic AMP-dependent protein kinase as the mediator of CAMP action has been suggested and established in various hormone-responsive systems (1-3) . Recent studies indicated that the induction of various enzymes is mediated by cAMP-dependent protein kinase . One of these enzymes is ornithine decarboxylase (ODC), which catalyzes the conversion of ornithine to putrescine . This reaction is the rate limiting step in the biosynthesis of the naturally occurring polyamines (4) . In the preceding paper (5), we described the induction of ODC in rat glioma cells and demonstrated that increases in CAMP-dependent protein kinase activities precede ODC induction. It has also been shown (5) that the poly- . amine spermine inhibits CAMP-dependent protein kinase, apparently by interfering with the activity of the catalytic subunit . The purpose of the present study is to extend these findings and to show that cAMP-dependent protein kinase from rat liver is also inhibited by polyamines, which inactivate the catalytic subunit of the enzyme . It thus appears that the regulation of CAMP-dependent protein kinase by polyamines is a general phenomenon, which may be of importance in controlling cellular growth . Methods Female albino rats from the Hebrew University strain, approximately Solu3 months old, were used . Animals were killed by cervical dislocation . ble extracts of the liver were prepared by homogenizing the tissue (20$, w/v) in an all-glass Potter-Elvehjem homogenizer in TD buffer (50mM Tris-HC1, 5mM DTT, pH 7 .5) . The homogenate was centrifuged at 30,000 g for 1 hr in a Sorvall RC 2-B refrigerated centrifuge and the supernatant was used for further studies Protein kinase and [3H]-CAMP binding studies . P~tein kinase activity was determine y measur ng t e traps er o ~[yP]-ATP [prepared as described by Glynn and Chappell (6)] to histone (Sigma) in the absence and 0300-9653/78/0501-1481$02 .00/0 Copyright Q 1978 Pergamon Press
1482
Polyaminea and Protein Kinase
Vol. 22, No . 17, 1978
presence of lOyM CAMP as previously described (7) . ~e unit of protein kinase activity is defined as the transfer of one nmole of P per 10 minutes from [Y-32p] -ATP to histone . Binding of [3H]-cA14P (2 x 10 -7M, New England Nuclear) was measured by a modification of the millipore filter technique (8) as will be described elsewhere (9) . Separation of the catalytic subunit . The catalytic subunit of the protein kinase was separate as previouslydescribed (10) . Essentially, the soluble liver extract was passed through a DEAE (Whatman DE-52) column and washed with TD buffer until O .D .2g0 was less than 0 .1 . The catalytic subunit of the enzyme was eluted from the column with 10 -5 M cAMP into test tubes containing 300ug/ml of egg albumin . Fractions containing catalytic subunits were dialyzed against TD buffer and were found to be devoid of any CAMP binding activity . Results and Discussion Figure 1 shows the effect of sparmine on the activity of both cAMPdependent and independent protein kinase activities in rat liver extracts . Reactions ware carried out at appropriate dilutions of a 20$ homogenate, so that enzyme activity was linearly related to protein concentration (usually a dilution of 1 :50 - 1 :100) . It may be seen (Fig . 1) that spermine 2mM caused a 40$ inhibition of cAMP-dependent protein kinase activity . Cyclic-AMP binding activity was not affected by spermine even at a concentration of 20mM (Fig . 1, insert) . This experiment thus implies that the catalytic and not the regulatory subimit of the enzyme is affected by spermine . This assumption was verified by separating the enzyme subunits and testing the effect of polyamines on the various components . It is obvious from Fig. 2 that polyamines inhibited the activity of the catalytic subiutit and that spermine was the most effective inhibitor . The effect of spermine was reversible ; dialysis restored the activity cf catalytic subunits previously inactivated by 20mM spermine, Similarly, speralne did not interfere with the recombination of the regulatory and catalytic subunits of the enzyme (data not shown) . The Mg ++ dose response curve for CAMP-dependent protein kinase activity (Fig . 3A) shows a characteristic biphasic curve with maximal activation at 3-lOmM . In the presence of 5mM spermine, the curve is reduced (Fig . 3A) . The inhibitory effect of spermine was most pron~~mced at 2-lOmM Mg ++ (Fig . 3B) . Takai et al . (11) and Murray et aZ . (12) have recently reported the inhibition of cAMP-dependent protein kinase by polyamines . However, the generality of this effect was questioned by the same authors (12), who failed to detect a significant effect of polyamines on rat liver protein kinase . This observation is somewhat surprising, since the liver is known to be rich in polyamines (13) . Our data demonstrate that the liver enzyme is also susceptible to the inhibitory effect of polyamines . The physiological significance of the polyamine effects are therefore strengthened . The same conclusion holds true also for the enzyme in tissue cultures and in neoplastic cells (5, 14) . In conclusion, we suggest that the intracellular increase in cAMP by various hormones activates the cAMP-dependent protein kinase, which in turn activates the rate limiting step in polyamine biosynthesis - namely, ornithine decarboxylase . The subsequent intracellular increase in polyamine concentration can regulate further polyamine formation by either inducing the synthesis of a protein which specifically inhibits ornithine decarboxylase (15) or by inhibiting the activity of cAMP-dependent protein kinase . The induction of the inhibitor protein requires a concentration of lOmM spermine (15) . This is compatible with the concentration of spermine,required to inhibit cANIP-dependent protein kinase (see above) . The interrelation between these two negative feed-back mechanisms in in vivo systems may turn out to be of importance to the regulation of cellular growth and division in normal and neoplastic cells .
Polyaminea and Protein Rinase
Vol. 22, No . 17, 1978
1483
._. w ..
r
f-
~100,-0~
H C~
Q 1.0
c ~p
W
!n Q
Z_ Y Z
a
0 0 5 10 15 20 Spermine(mM) cAMP-Dependent
0.5
W O OC
50~
o b~o--~~ cAM P- Independent
o.o
0
5
1
10
15
SPERMINE (mM)
.
20
'
FIG . 1 Effect of spermine on cAMP-dependent and independent protein kinase activities in rat liver homogenates . The insert shows the effect of spermine on cAMP binding in the same preparation .
É
v >FFC~
a
W
a
Z Y Z
W !O tY tl
FIG . 2 Effect of polyamines on the catalytic subunit of cAMP-dependent protein kinase from rat liver .
1484
Polyamises and Proteia Risase
O
2
IF 60
B
F- ~. ~
m z
Vol. 22, No, 17, 1978
20
l
0
.
v
10
~
v
.
, ~-, ~1_._I
20 ~ 30 i0 Mg" (mM)
~
100
FIG . 3 A. B,
Effect of Mg ++ and spermine on cataly~~c subimit activity . Activity in the presence of increasing Mg concentrations . Relative inhibiti~~ ($) of kinase activity by spermine at various concentrations of Mg . activity + spermine $ inhibition = (1 activlt T - spermlâe ) X 100
Y
Acknowledgement This work was supported by a grant from the United States-Israel Binational Science Foundation to J .H . (No . 1470) . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
J .F, Kuo and P . Greengard, Proc . Nat . Aced . Sci . USA 64 :1349-1355 (1969) . T .R . Soderling, J . P . Hicken~ottom,E .M . Reimânn,F .L.Hunkeler, D .A . lYalsh and E.G . Krebs, J . Biol . Chem . 245 :6317-6328 (1970) . J .K . Huttunen, D. Steinberg and S .E . Mâyer, Proc . _Nat . Aced . _Sci . _USA 67 :290-295 (1970) . D .H . Russell, C.V . Byus and C.A . Manen, Life Sci . 19 :1297-1306 (1976) . U. Bachrach, A . Katz and J . Hockiman, Life Sci~accépted for publication) . I .M . Glynn and J . B . Chappell, Biochem. J . 90 :147-149 (1964) . P. A . Insel, H.R . Bourne, P . Cof l~andG.I~.- Tomkins, Science _190 :896898 (1975) . A.G . Gilman, Proc . Nat . Aced . Sci . USA _67 :305-312 (1970) . J . Hockiman and A. Katz In prepâratlô) J. Hockiman, P .A . Insel, H.R . Bourne, P. Coffino and G .M . Tomkins, Proc . Nat . Aced . Sci . USA 72 :5051-5055 (1975) . ~~akâl S. Nkeys, M. Înoûe, A. Kishimoto, K . Nishiyama, H. Yamnm,~ra, and Y. Nishizuka, J . Biol . Chem . 251 :1481-1487 (1976) . A.W . Money, M. Frôsclo nd~Rogérs, Biochem . Biophys . _Res . Commun . 71 :1175-1181 (1976) . U . Bachrach, Function of Naturally Occurring Polyamines, Academic Press New York (1973 . J. Hockiman, A. Katz and U. Bachrach (in preparation) . J.S . Heller, W.F . Fong and E .S . Canellakis, Proc . _Nat . Aced . _Sci . _USA 73 :1858-1862 (1976) .
Pergamon Prees
POLYAMINES AND PROTEIN KINASE II . EFFECT OF POLYAMINES ON CYCLIC AMP - DEPENDENT PROTEIN KINASE FROM RAT LIVER Jacob Hockiman, Aviva Katz and Uriel Bachrach Department of 7~oology, The Hebrew University, Jerusalem, and Department of Molecular Biology, Hebrew University - Hadassah Medical School, Jerusalem, Israel (Received in final form March 6, 1978) Summary The effect of polyamines on the activity of cAMP-dependent protein kinase from rat liver was determined . It has been shown that polyamines inactivate the enzyme in the decreasing order of activity : spermine > spermidine > putrescine . This effect is due to inhibition of the catalytic subunit . On the other hand, binding of cAMP to the regulatory subunit of the enzyme is not affected by polyamines . It is suggested that the inhibition of protein kinase by polyamines is a general phenomenon . The unequivocal importance of cyclic AMP-dependent protein kinase as the mediator of CAMP action has been suggested and established in various hormone-responsive systems (1-3) . Recent studies indicated that the induction of various enzymes is mediated by cAMP-dependent protein kinase . One of these enzymes is ornithine decarboxylase (ODC), which catalyzes the conversion of ornithine to putrescine . This reaction is the rate limiting step in the biosynthesis of the naturally occurring polyamines (4) . In the preceding paper (5), we described the induction of ODC in rat glioma cells and demonstrated that increases in CAMP-dependent protein kinase activities precede ODC induction. It has also been shown (5) that the poly- . amine spermine inhibits CAMP-dependent protein kinase, apparently by interfering with the activity of the catalytic subunit . The purpose of the present study is to extend these findings and to show that cAMP-dependent protein kinase from rat liver is also inhibited by polyamines, which inactivate the catalytic subunit of the enzyme . It thus appears that the regulation of CAMP-dependent protein kinase by polyamines is a general phenomenon, which may be of importance in controlling cellular growth . Methods Female albino rats from the Hebrew University strain, approximately Solu3 months old, were used . Animals were killed by cervical dislocation . ble extracts of the liver were prepared by homogenizing the tissue (20$, w/v) in an all-glass Potter-Elvehjem homogenizer in TD buffer (50mM Tris-HC1, 5mM DTT, pH 7 .5) . The homogenate was centrifuged at 30,000 g for 1 hr in a Sorvall RC 2-B refrigerated centrifuge and the supernatant was used for further studies Protein kinase and [3H]-CAMP binding studies . P~tein kinase activity was determine y measur ng t e traps er o ~[yP]-ATP [prepared as described by Glynn and Chappell (6)] to histone (Sigma) in the absence and 0300-9653/78/0501-1481$02 .00/0 Copyright Q 1978 Pergamon Press
1482
Polyaminea and Protein Kinase
Vol. 22, No . 17, 1978
presence of lOyM CAMP as previously described (7) . ~e unit of protein kinase activity is defined as the transfer of one nmole of P per 10 minutes from [Y-32p] -ATP to histone . Binding of [3H]-cA14P (2 x 10 -7M, New England Nuclear) was measured by a modification of the millipore filter technique (8) as will be described elsewhere (9) . Separation of the catalytic subunit . The catalytic subunit of the protein kinase was separate as previouslydescribed (10) . Essentially, the soluble liver extract was passed through a DEAE (Whatman DE-52) column and washed with TD buffer until O .D .2g0 was less than 0 .1 . The catalytic subunit of the enzyme was eluted from the column with 10 -5 M cAMP into test tubes containing 300ug/ml of egg albumin . Fractions containing catalytic subunits were dialyzed against TD buffer and were found to be devoid of any CAMP binding activity . Results and Discussion Figure 1 shows the effect of sparmine on the activity of both cAMPdependent and independent protein kinase activities in rat liver extracts . Reactions ware carried out at appropriate dilutions of a 20$ homogenate, so that enzyme activity was linearly related to protein concentration (usually a dilution of 1 :50 - 1 :100) . It may be seen (Fig . 1) that spermine 2mM caused a 40$ inhibition of cAMP-dependent protein kinase activity . Cyclic-AMP binding activity was not affected by spermine even at a concentration of 20mM (Fig . 1, insert) . This experiment thus implies that the catalytic and not the regulatory subimit of the enzyme is affected by spermine . This assumption was verified by separating the enzyme subunits and testing the effect of polyamines on the various components . It is obvious from Fig. 2 that polyamines inhibited the activity of the catalytic subiutit and that spermine was the most effective inhibitor . The effect of spermine was reversible ; dialysis restored the activity cf catalytic subunits previously inactivated by 20mM spermine, Similarly, speralne did not interfere with the recombination of the regulatory and catalytic subunits of the enzyme (data not shown) . The Mg ++ dose response curve for CAMP-dependent protein kinase activity (Fig . 3A) shows a characteristic biphasic curve with maximal activation at 3-lOmM . In the presence of 5mM spermine, the curve is reduced (Fig . 3A) . The inhibitory effect of spermine was most pron~~mced at 2-lOmM Mg ++ (Fig . 3B) . Takai et al . (11) and Murray et aZ . (12) have recently reported the inhibition of cAMP-dependent protein kinase by polyamines . However, the generality of this effect was questioned by the same authors (12), who failed to detect a significant effect of polyamines on rat liver protein kinase . This observation is somewhat surprising, since the liver is known to be rich in polyamines (13) . Our data demonstrate that the liver enzyme is also susceptible to the inhibitory effect of polyamines . The physiological significance of the polyamine effects are therefore strengthened . The same conclusion holds true also for the enzyme in tissue cultures and in neoplastic cells (5, 14) . In conclusion, we suggest that the intracellular increase in cAMP by various hormones activates the cAMP-dependent protein kinase, which in turn activates the rate limiting step in polyamine biosynthesis - namely, ornithine decarboxylase . The subsequent intracellular increase in polyamine concentration can regulate further polyamine formation by either inducing the synthesis of a protein which specifically inhibits ornithine decarboxylase (15) or by inhibiting the activity of cAMP-dependent protein kinase . The induction of the inhibitor protein requires a concentration of lOmM spermine (15) . This is compatible with the concentration of spermine,required to inhibit cANIP-dependent protein kinase (see above) . The interrelation between these two negative feed-back mechanisms in in vivo systems may turn out to be of importance to the regulation of cellular growth and division in normal and neoplastic cells .
Polyaminea and Protein Rinase
Vol. 22, No . 17, 1978
1483
._. w ..
r
f-
~100,-0~
H C~
Q 1.0
c ~p
W
!n Q
Z_ Y Z
a
0 0 5 10 15 20 Spermine(mM) cAMP-Dependent
0.5
W O OC
50~
o b~o--~~ cAM P- Independent
o.o
0
5
1
10
15
SPERMINE (mM)
.
20
'
FIG . 1 Effect of spermine on cAMP-dependent and independent protein kinase activities in rat liver homogenates . The insert shows the effect of spermine on cAMP binding in the same preparation .
É
v >FFC~
a
W
a
Z Y Z
W !O tY tl
FIG . 2 Effect of polyamines on the catalytic subunit of cAMP-dependent protein kinase from rat liver .
1484
Polyamises and Proteia Risase
O
2
IF 60
B
F- ~. ~
m z
Vol. 22, No, 17, 1978
20
l
0
.
v
10
~
v
.
, ~-, ~1_._I
20 ~ 30 i0 Mg" (mM)
~
100
FIG . 3 A. B,
Effect of Mg ++ and spermine on cataly~~c subimit activity . Activity in the presence of increasing Mg concentrations . Relative inhibiti~~ ($) of kinase activity by spermine at various concentrations of Mg . activity + spermine $ inhibition = (1 activlt T - spermlâe ) X 100
Y
Acknowledgement This work was supported by a grant from the United States-Israel Binational Science Foundation to J .H . (No . 1470) . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
J .F, Kuo and P . Greengard, Proc . Nat . Aced . Sci . USA 64 :1349-1355 (1969) . T .R . Soderling, J . P . Hicken~ottom,E .M . Reimânn,F .L.Hunkeler, D .A . lYalsh and E.G . Krebs, J . Biol . Chem . 245 :6317-6328 (1970) . J .K . Huttunen, D. Steinberg and S .E . Mâyer, Proc . _Nat . Aced . _Sci . _USA 67 :290-295 (1970) . D .H . Russell, C.V . Byus and C.A . Manen, Life Sci . 19 :1297-1306 (1976) . U. Bachrach, A . Katz and J . Hockiman, Life Sci~accépted for publication) . I .M . Glynn and J . B . Chappell, Biochem. J . 90 :147-149 (1964) . P. A . Insel, H.R . Bourne, P . Cof l~andG.I~.- Tomkins, Science _190 :896898 (1975) . A.G . Gilman, Proc . Nat . Aced . Sci . USA _67 :305-312 (1970) . J . Hockiman and A. Katz In prepâratlô) J. Hockiman, P .A . Insel, H.R . Bourne, P. Coffino and G .M . Tomkins, Proc . Nat . Aced . Sci . USA 72 :5051-5055 (1975) . ~~akâl S. Nkeys, M. Înoûe, A. Kishimoto, K . Nishiyama, H. Yamnm,~ra, and Y. Nishizuka, J . Biol . Chem . 251 :1481-1487 (1976) . A.W . Money, M. Frôsclo nd~Rogérs, Biochem . Biophys . _Res . Commun . 71 :1175-1181 (1976) . U . Bachrach, Function of Naturally Occurring Polyamines, Academic Press New York (1973 . J. Hockiman, A. Katz and U. Bachrach (in preparation) . J.S . Heller, W.F . Fong and E .S . Canellakis, Proc . _Nat . Aced . _Sci . _USA 73 :1858-1862 (1976) .
