Modulators of rat liver cytosol casein kinases 1 and 2

Vol. 109, No. 4, 1982 December 31, 1982

BIOCHEMICAL

MODULATORS OF RAT LIVER M. Plana, M.D. Departament de Bioqulmica, Autbnoma de Barcelona,

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

CYTOSOL CASEIN

KINASES

Guasch and E. Itarte Facultat de Ciencies, Bellaterra (Barcelona),

1284-1290

1 AND 2

Universitat Spain

Received November 16, 1982 Rat liver cytosol casein kinases 1 and 2 are similarly activated by spermine and inhibited by caffeine. On the contrary they are differently affected by heparin and basic proteins. Low concentrations of heparin inhibited selectivelv the phosphorylation of casein by casein kinase 2 whereas protamine and histones inhibited specifically casein kinase 1. On the contrary the basic proteins stimulated slightly the activity of casein kinase 2 and released its inhibition by heparin. Increasing the concentration of casein substrate reversed both the inhibition of casein kinase 1 by protamine as well as that of casein kinase 2 by heparin. The data suggest the existence of modulators having either similar or opposite effects on each type of casein kinase.

Rat liver cytosol contains two casein kinases which are different in their molecular weight and kinetic properties (1). The characteristics of these enzymes are similar to those of the casein kinases we have isolated from rabbit muscle (2,3) and pig polymorphonuclear leucocytes (41, and seem to correspond to the type I and II casein kinases present in other mammalian tissues (1). Over the past years it has become evident that the activity of the type II casein kinase, or casein kinase 2 (CK-2) is inhibited by heparin (5-11) and caffeine (12) and stimulated by polyamines (5-8) and basic proteins such as histones and protamine Less is known about the modulators of type I casein kinase, (6,131. or casein kinase 1 (CK-1) which is unaffected by heparin and basic polypeptides (6,7). Previous reports from our group have demonstrated the presence in rat liver cytosol of an inhibitor protein of casein kinases (141, but the inhibitor was unspecific, eaually inhibiting both types of casein kinases as well as the catalytic subunit of cyclic AMP-dependent protein kinase. Thus, the existence of specific effecters of CK-1 was unknown. As a part of our studies on the regulation of both types of casein kinases we have undertaken the screaning for effecters of 0006-291X/82/241284-07$01.00/0

Copyright 0 1982 by Academic Press, Inc. AN righfs of reproduction in any form reserved.

1284

BIOCHEMICAL

Vol. 109, No. 4, 1982

CK-1 and found bitors

for

this

that

protamine

AND BIOPHYSICAL

and histones

RESEARCH COMMUNICATIONS

are potent

specific

inhi-

enzyme. MATERIALS AND METHODS

Glucose-l-P, glucose-6-P, fructose-1,6-P2, cyclic GMP, spermine, caffeine, heparin, calf thymus histone (type IIA) and protamine from salmon were obtained from Sigma Chemical Co. Protamine was desalted by passage through an AG 1x2 (hidroxide form) ion exchange resin. Very lysin rich, lysin rich and arginine rich histones prepared from chicken erythrocytes were generously donated by Dr. Joan R. Daban from this laboratory. CK-1 and CK-2 were isolated from rat liver cytosol as described in (1). Protein kinase assay was done at 30°C as reported previousof CK-1 and CK-2 was 0.6 units/ml and that ly (1). The concentration of casein or phosvitin was 0.4 mg/ml except where indicated. One unit of casein kinase is the amount of enzyme that incorporated one nanomol of 32P from [v-~* P]ATP to casein (4 mg/ml) per min.

Effect

of

casein

kinases

both

RESULTS cyclic GMP, spermine

suqar-phosphates,

and caffeine

on

activity

Several metabolites were tested as potential effecters of casein kinases. Among them, glucose-l-P, glucose-6-P, fructose-

20(

x .-> z a 1oc ,P

I

Fig.

1 2 3 4 5 spermine (mM) 10 20 0 caffeine (mM) I - Effects of spermine and caffeine on casein kinases 1 and 2. The activity of purified CK-1 (0 ,A) or CK-2 (e A) was determined in the presence of the indicated amoun; of spermine (0 ,a) or caffeine (A ,A ) using 4.0 mg/ml casein as substrate. 1285

Vol. 109, No. 4, 1982 1,6-P2

(all

at

BIOCHEMICAL

1 mM)

and

AND BIOPHYSICAL

cyclic

GMP

(50

PM)

RESEARCH COMMUNICATIONS were

without

significant

effect. casein (Fig. effect

On the other hand spermine stimulated the phosphorylation of by both casein kinases up to a maximum of 1.9 to 2.1-fold 1). The concentration of spermine required for half-maximal was 0.5 mM for G-1 and 1.5 mM for CK-2. Caffeine also affected both casein kinases, inhibiting their activity, although CK-1 was more sensitive to this methylxanthine than (X-2. The effects of spermine and caffeine were also observed when casein was substituted by phosvitin in the assay (not shown). Effect

of heparin Heparin

and protamine was a potent inhibitor of CK-2, with an 150=0.5 ug/ml (Fig. 2A). CK-1 was unaffected by concentrations of heparin up to 1 ug/ml but progressively inhibited by higher concentrations. Protamine showed a biphasic effect on CK-1 (Fig. 2B). Low concentrations of this protein stimulated slightly its activity but concentrations of protamine higher than 20 !Jg/ml were inhibitory, with an 150=60 ug/ml. On the contrary, CK-2 was stimulated by protamine at concentrations above 10 ug/ml. Furthermore, protamine coun teracted the inhibition of CK-2 by heparin and this effect was al-

loo- ~0-0-0-0, A “1 A

0 \

\

0

A

.$50a

0

\

0

* -2

Fig.

-

-1 0 1 log [heparin] (pglml)

2 - Effect

log [protamine] (mgknl)

of heparin and protamine on casein kinases 1 and 2. The activity of purified CK-1 (0 l ) or CK-2 (A A ) was measured in the presence of the i;dicated amounts'of hepaof CK-1 was rin (A) or protamine (B). In A the activity assayed both in the absence (0) and in the presence of of CK-2 was 0.3 mg/ml of protamine (0 ). In B the activity determined both in the absence (A) and in the presence of 10 pg/ml of heparin (A). In all cases, casein (0.4 mg/ml) was used as substrate. 1286

Vol. 109, No. 4, 1982

BIOCHEMICAL

3-

AND BIOPHYSICAL

- 0.5

RESEARCH COMMUNICATIONS

05

0

log [easer] (mgiml) Fig.

ready range.

3 - Effect of substrate (casein) concentration on the inhibition of casein kinase 1 by protamine and of casein kinase 2 by heparin. The inhibition of CK-1 by protamine (0.3 mg/ml) (0 ) and that of CK-2 by heparin (1 vg/ml) (A ) was determined using the indicated amount of casein as substrate.

observed

at concentrations heparin did In contrast,

by protamine. rin

ratio

However,

(w/w)

it

necessary

of protamine below the stimulatory not release the inhibition of CK-I

has to be noticed to overcome

the

that

the

inhibition

glycosaminoglycan was 1:l to 2:l whereas the highest with CK-1 was 3O:l since increasing the concentration strongly inhibitory. Control experiments in the showed no phosphorylation of protamine by either any of the conditions tested. Increasing progressive release

protamine/hepaof CK-2 by the ratio tested of heparin was

absence of casein CK-1 or CK-2 under

the amount of casein used in the assay of the inhibition of CK-1 by protamine

caused a and of

CK-2 by heparin (Fig. 3). These data suggest that protamine and heparin were competitive'inhibitors with respect to casein. On the con trary, protamine did not affect the Km for ATP of CK-1 (11 uM) and heparin did not vary that of CK-2 (13 uM). The extent of inhibition of CK-1 by 0.3 mg/ml protamine (90%) and that of CK-2 by 0.4 pg/ml heparin (38%) were not affected by varying the amount of casein kinase used in the assay in the range of 0.1 to 1.2 units/ml with 0.4 mg/ml casein as substrate. 1287

Vol. 109, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

Influence of the substrate on the effect parin on the activity of W-1 and CK-2 Other phosphorylation

RESEARCH COMMUNICATIONS

of basic

proteins

and he-

basic proteins such as histones also affected the of casein by CK-1 and CK-2 (Table I), although they

were somehow less effective and releasing the inhibition Differences

than protamine in both of CK-2 by heparin.

were observed

on the

effect

inhibiting

CK-1

of histones

on CK-1

when casein was substituted by phosvitin as substrate. As can be observed, in this case, the inhibition of CK-1 by very lysine rich histones (Hl+H5) was less pronounced and the lysine rich (H21HH2B) and arginine of this

rich histones enzyme. Control

(H%H4)

experiments

showed no incorporation or CK-2.

were not

in the

As may be observed

into

from Table with with

but

absence of casein

of phosphate

heparin on CK-2 also varied nounced with phosvitin than

inhibitors

histones

I the

the substrate casein.

activators or phosvitin

by either

inhibitory used,

CK-1

effect being

of

more pro-

DISCUSSION The results CK-2 is

stimulated

reported by spermine

herein

show that

and inhibited

rat

liver

by caffeine,

cytosol as descri-

bed for

the type II casein kinase from other sources (5-8, 12). Furthe effect of these modulators is also exerted on CK-1, thermore, what indicates the possible existence of a co-ordinated control of interesting in the case of spermine, these enzymes. This is specially which

has been postulated

of cellular

as a physiological

modulator

in a number

processes. On the other

hand, CK-1 and CK-2 are differently affected The basic proteins are specific by protamine, histones and heparin. inhibitors of the phosphorylation of casein by CK-1, whereas CK-2 is much more sensitive.to inhibition by heparin than CK-1. In both cases the inhibition is reverted by increasing the concentration of casein, suggesting that they act through a competitive mechanism. Nonetheless, the effect of histones on CK-1 was clearly different when phosvitin was used as a substrate instead of casein. In this case, the lysine rich and arginine rich of the phosphorylation

histones were not inhibitors but of phosvitin by this enzyme.Thus,

ble

activators it is possi-

that the effect of these‘basic proteins is exerted, at least in through an interaction with the substrate which would induce a part, conformational change in such a way as to render more or less phosphate acceptor sites accesible to the action of the casein kinase, 1288

43.8

H&H4

of

93.2

133.8

50.8

76.8

8.0

74.6

phosvitin

CK-la

effects

basic

90.1

96.0

103.4

102.6

109.3

100

control

casein

73.4

36.6

14.5

--

97.5

24.6

2.

in (b). in the

70.7

119.8

89.4

102.2

93.9

117.7

control

phosvitin

1 and

20 ug/ml on casein

CK-2b

kinases

+ heparin

on casein

%Activity

proteins

I

The concentration of effector protein was 0.3 mg/ml in (a) and was 10 pg/ml. Data are referred to the activity of each kinase of basic proteins to which a value of 100 was given.

74.4

87.0

H2A+H2B

A

40.3

II

type:

15.0

100

casein

of

Hl+HS

Sigma

histone

protamine

none

Additions

Comparison

TABLE

Heparin absence

80.7

27.9

89.0

--

89.5

4.4

+ heparin

Vol. 109, No. 4, 1982

BIOCHEMKAL

AND 8lOPHYSlCAL

RESEARCH COMMUNICATIONS

as indicated previously for the effect of histones on the phosphorylation of non-histone proteins by rat liver nuclear protein kinases (15). The dual effect of protamine, inhibiting CK-1 and reverting the inhibition of CK-2 by heparin may provide a mechanism for enhancing the activity of CK-2 while decreasing that of CK-1. Despite of the interest of this observation from the point of view of regulation it has to be indicated that CK-1 is also inhibited by high concentrations of heparin (16, and this report), fact that questions the role of heparin as an specific effector of CK-2 under physiological conditions. On the other hand, the presence of cytosolic proteins affecting both casein kinases in a way similar to protamine is unknown and thus, the involvement of this mechanism in the physiological control of these enzymes is only speculative. the selective inhibition of the phosphorylation Nonetheless, of casein by each type of casein kinases by heparin and protamine may be used as a tool in the accurate determination of the ratio of casein kinases 1 and 2 in mammalian tissues. Acknowledgments This work was supported Asesora de Investigacidn Cientifica

1. 2. 3. 4. 5. ;: a. 9. 10. 11. 12. 13. 14. 15. 16.

by grant 0339/81 from the "Comisi6n y TBcnica" of M.E.C. Spain.

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