Molecular and CeNular Endocrinology, 30 (1983) 241-266 Elsevier Scientific publishers Ireland, Ltd.

247

REVIEW

POL~A~INE-~E~~~~ PROTEIN F~OS~H~R~AT~~N~ A POSSIBLE TARGET FOR IN~CELLULAR ~LYA~INE ACTION

Claude COCHET and Edmond M. CHAMBAZ *

laboratoire de Biochimie Endocrinienne, INSERM Unite 244, CNRS ERA 942, WniversitP Scientifique et Mhdicale de Grenoble, 387C.W La Tronche (France)

Received 22 Februaqi 1983

Potyamines are well-known ubiquitous components of living ceils. Although these polycations have been implicated in the regulation of major c&,&r functions such as DNA. RNA and protein synthesis occurring during cellular proliferation and/or differenti- ation processes, their mecha~sm of action at the molecular level has remained obscure. On the other hand, protein pbospho~lat~on has emerged as a regulatory process of prime importance in cellular regulation. Data have recently been presented suggesting that polyamines may express at least part of their biological action through an effect upon selective protein phosphorylation systems. Two types of polyamine-sensitive protein kinases have been characterized in the last few years. The best known in molecular terms is the widespread casein kinase G (also termed casein kinase II), which represents a muitifunc” tional protein kinase, at present classified as a messenger-independent activity. The other is a polyamine-dependent nuclear omithine decarboxylase kinase characterized in Phpwum polycephalum and several mammalian tissues. Both protein kinases are activated by poly amines in vitro at concentrations compatible with a physiologica role, by a mechanism which most fikeIy a&o involves an effect through the protein substrate conformation. Prdiminary evidence suggests that both kinases may be implicated in the regulation of DNA-dependent RNA polymerase activities, although severai other potential substrates have been suggested for casein kinase G. Another suggestion is that these kinases may also participate in the post-translational regulation of ornithine decarboxyIase, the rate-limiting step in the polyamine biosynthetic pathway.

A novel class of protein kinase activities may thus be defined as polyamine-mediated phosphorylation systems for which polyamines may function as intracellular messenger. Although their biological significance remains to be fully established, especially with regard to the definition of their specific intra~l~~lar target(s) and subsequent biological functions, these systems will be interesting to consider in future studies aimed at understanding the role of polyamines in cell regulation.

Keyworbs: polyamines; protein kinases: protein phosphory~ation; adrenaf cortex.

* To whom correspondence should be addressed.

0303-7207j83/$03.00 0 1983 Elsevier Scientific Publishers Ireland, Ltd.

248 C. Cachet and E.M. Chambar

Reversible protein phosphorylation has emerged as one of the major mechanisms by which a living cell may regulate many of its metabolic

activities, especially in response to a number of humoral or neural effecters (Langan, 1973; Rubin and Rosen, 1975; Nimmo and Cohen,

1977; Weller, 1979; Krebs and Beavo, 1979; Cohen, 1982). The post-

translational covalent intr~uction of phosphate into proteins is cata- lysed by nucleoside triphosphate-protein : phosphotransferases (protein

kinases) and integrated in a reversible regulatory process by the comple- mentary action of phosphoprotein phosphatases. The different types of protein kinase activities characterized in various tissues may be classified

as proposed by Krebs and Beavo (1979) according to their specific positive effector( which represents an established or a potential in- tracellular messenger for the given phosphorylation system. Table 1 lists the major protein kinases known to date, according to this classification, which appears the most informative, especially in the field of endocrinol-

ogy. It depends on the general concept that protein kinase activities are

under the control of specific intracellular messenger(s) and integrated in

cascade reactions resulting in specific target protein phosphorylation(s)

and subsequent biological effect(s). When the definition of such an effector is still lacking, the corresponding protein kinase will be termed

messenger-independent (Krebs and Beavo, 1979). On the other hand, polyamines are ubiquitous components of living

cells and have been implicated as potential effecters of a number of

Table 1

Protein kinase activities characterized in mammalian tissues and classified according to

their positive effecters (Krebs and Beavo, 1979)

Type of protein kinase Effector Known entities

1. 2.

3.

4.

5.

6.

7.

Cyclic AMP dependent

Cyclic GMP dependent

Calmodulin-Ca’+ dependent

Virus dependent

Growth factor dependent

ds-RNA dependent

Phospholipid-Ca*+

dependent

8. Messenger independent

Cyclic AMP

Cyclic GMP

CaImodulin-Ca2 ’

Encoded by viral

genome

Growth factors, insulin

ds-RNA

Ca2+-diacyl-

glycerol, phos-

pholipids 1

Two isoenzymes

One

Several (substrate specific)

Several (retrovirus)

Associated with the plasma

membrane receptor

One (interferon-treated

cells)

One, with a Ca*‘-dependent

proteolysis related form

--Casein kinase A (I)

-Casein kinase G (II)

P&amine- mediated protein phosphorylations 249

major biological processes such as DNA, RNA and protein synthesis, although their mechanism of action is at present poorly defined (Morris and Fillingame, 1974; Tabor and Tabor, 1976; Williams-Ashman and Canellakis, 1979; Heby, 1981). The fact that activation of polyamine biosynthetic activity appears as a regular event concomitant with trigger- ing of cell growth and proliferation has stimulated an active search to understand the role and possibly control the intracellular metabolism of polyamines (Raina et al., 1980; Bachrach, 1980; Mamont et al., 1980).

In this brief presentation, we shall review some recent data which may suggest that intracellular polyamines could express at least part of their biological actions by a modulation of the activity of defined protein kinase systems. This proposal will be based upon two lines of observa- tion: (i) polyamine effects on the catalytic activity of a messenger-inde- pendent protein kinase (casein kinase G), which has been investigated in some detail in our laboratory and in others and appears to be of widespread occurrence; and (ii) the recent characterization by Kuehn’s group of a protein kinase whose activity appears to be dependent upon the presence of polyamines (Daniels et al., 198 1). Although many of these data require further confirmation, a working hypothesis may be put forward, suggesting that polyamines can represent specific messengers regulating certain intracellular protein kinase reactions, which may thus define a novel class of polyamine-mediated phosphorylation systems.

A. POLYAMINES, PROTEIN PHOSPHORYLATION AND CELLIJ- LAR METABOLIC ACTIVITIES

I. Polyamines Polyamines are aliphatic cationic small components of all living cells,

represented by three major structures (Fig. 1). Putrescine and spermidine are ubiquitous while spermine appears to be confined to nucleated eukaryotic cells (Tabor and Tabor, 1976; Williams-Ashman and Canel- lakis, 1979). The major biosynthetic route leading to polyamine genera- tion in mamma~an cells involves decarboxylation of L-ornithine cata- lysed by omithine decarboxylase (ODC), to yield putrescine in a reaction considered as the rate-limiting step in the pathway (Tabor and Tabor,

H; N-_(CH2)4-N+ H, Pulrescine

H; N-(CH,),-HN-(CH,),-N+ H, Spermidine

H;N-(CH,),-HN-~CH,)~~NH~(CH,)~-N+H, Spermine

Fig. 1. Structures of the three major polyamines.

250 C. Cocher and E.M. Chombar

1976; Canellakis et al., 1979). Decarboxylation of S-adenosylmethionine provides one or two propylamine radicals, which are condensed to putrescine to yield spermidine and spermine respectively. Ornithine

decarboxylase is a pyridoxal phosphate-requiring enzyme (half-life 7-20 min) whose activity in the resting cell is usually very low and is increased

manyfold in response to a number of cellular effecters, including hormones and growth factors (Canellakis et al., 1979; Bachrach, 1980).

This enzyme induction results in intracellular levels of spermidine and spermine in the millimolar range. ODC may be regulated by post-transla-

tional modifications, including phosphorylation (see section C). In addi- tion, an endogenous protein inhibitor (antizyme) has been described,

which is induced in cells exposed to high polyamine levels (Canellakis et al., 1981).

The wide interest in polyamine research has been stimulated by the observation that ornithine decarboxylase activity and polyamine levels

are strikingly increased in rapidly growing tissues (for reviews see Heby, 198 1; Williams-Ashman and Canellakis, 1979). A dramatic increase in ODC activity appears to be one of the very first changes taking place

when synchronized quiescent cells in the G, state enter the G, phase. In cycling cells, a peak of activity was observed in the G,-S phase, before DNA synthesis starts. Selective inhibition of genetic defects in ODC

activity (Heby, 1981; Mamont et al., 1980; Duperray et al., 1981) resulted in reduced proliferation rate in various cell systems whereas proliferation resumed at a normal rate upon addition of putrescine to the

cell medium (Mamont et al., 1980; Duperray et al., 1981). The implication of polyamines in cell differentiation processes is

suggested by the fact that a number of polypeptide hormones induce an

increase in ODC activity in their target cells (Williams-Ashman and

Canellakis, 1979; Bachrach, 1980; Heby, 1981). The ODC increase precedes the .rise in RNA and protein synthesis. This hormonal effect

may in some cases be clearly dissociated from a proliferative action; for example, ACTH markedly stimulates ODC activity in cultured adreno- cortical cells, while at the same time the hormone blocks cell proliferation (Kudlow et al., 1980; Gill et al., 1980; Duperray et al., 1981). Polyamines have also been implicated in the maturation process of amphibian oocyte upon progesterone stimulation (Sunkara et al., 1981) as well as in mammalian embryogenesis (Fozard et al., 1980). On the other hand, an increase in polyamine biosynthesis has also been observed as an early event in chemically induced carcinogenesis (Verma and Boutwell, 1980) and viral cell transformation, although polyamines were apparently not required to maintain the transformed cell phenotype (Heby, 198 1; Holtta et al., 1981).

Po@antine . ~~diaied protein Fhosp~o~lal~o~s 251

Numerous studies have considered either protein kinase activities and/or protein phospho~lation patterns in relation to cell activity. Although more than 30 enzymes are known to be phosphorylated in vitro, a limited knowledge is available as to the nature and functions of the multiple protein phosphorylations occurring in the intact cell (Weller, 1979; Krebs and Beavo, 1979; Cohen, 1982). In addition to its well- established role in the regulation of carbohydrate and lipid metabolism, protein phospho~lation has been implicated in the regulation of major cellular functions such as DNA, RNA (Rubin and Rosen, 1975; Weller, 1979; Cohen, 1982; Jungmann and Kranias, 1977) and protein synthesis (Hunt, 1980; Leader, 1980). Increased nuclear histone phosphorylation (especialy H, ) has been correlated with cell growth, and appears maxim- ally active during the G, phase. Histone phospho~lation was suggested to be required for chromosome condensation and initiation of mitosis (Matthews, 1980). On the other hand, non&stone proteins (NHP) associ- ated with chromatin contain protein kinase activities, and changes in the NHP phospho~lation pattern have been correlated with gene activation processes occurring after hormonal stimulation (Jungmann and Kranias, 1977). Tyrosine phospho~lation in specific proteins has recently shed a new light on the mechanisms involved in viral cell tr~sformation (Collet and Erikson, 1978) and cellular response to several growth factors (Cohen et al.. 1980).

3. P~~~a~~~ine~ and ~r5tein kinase activities

A possible regulatory function of polyamines on protein kinase activi- ties has been suggested by experiments showing that the nuclear protein phospho~lation pattern resulting from endogenous protein kinase activi- ties was influenced by the presence of these agents. The phosphorylation rate of several non&stone proteins was stimulated by polyamines (and other basic components such as histones) in nuclear extracts (Kaplowitz et ai.. 1971; Johnson et al., 1973; Imai et al., 1975; Kuroda et al., 1977; Ahmed et al., 1978; Hara et al., 1982) as well as in isolated rat liver nuclei (Fa~on-Furstenthal and Li~tholder, 1978). The endogenous pro- tein kinase(s) involved in these observations have usually been char- acterized as cyclic nucleotide independent.

The effect of polycationic structures on various types of protein kinase preparations has been reported by several research groups. The CAMP- dependent protein kinase was i~ibit~ by polyamines (Murray et al., 1976; Ho&man et al., 1978) whereas crude cyclic nucleotide independent (casein kinase) activities were stimulated (Murray et al., 1976). Spermi- dine was shown to inhibit the free CAMP-dependent protein kinase

252 C. Cachet and E.M. Chamba;

catalytic subunit, whereas CAMP interaction with the holoenzyme was not modified (Hochman et al., 1978). A messenger-independent protein kinase (PK-380) isolated from an adrenocortical tumor and phosphory- lating eIF, was inhibited by spermine (Kuroda et al., 1982). The phos- pholipid-Ca*+-dependent p rotein kinase recently obtained in a highly purified form was inhibited by spermine (Wise et al., 1982). Polycationic

structures such as polyarginine were found to stimulate the purified

cGMP-dependent protein kinase basal activity whereas the polycation abolished the cGMP sensitivity of the enzyme (Walton and Gill, 198 1). A

polycation binding site was characterized on the enzyme and the protein

kinase modulator protein was shown to release the polycation effect and to maintain the cGMP dependence of the enzyme.

These considerations suggest that polyamines (and possibly other polycationic structures) may exhibit a general inhibitory action on cyclic

nucleotide (and Ca2+ -phospholipid-dependent protein kinase) whose preferred substrates in vitro are basic proteins (histone, protamines). By contrast, polyamine activation has been observed with cyclic nucleotide independent protein kinase reactions, which exhibit a general preference

for acidic substrates (casein, phosvitin, non&stone proteins).

B. CASEIN KINASE G: A UBIQUITOUS, MULTIPOTENTIAL POLYAMINE-MODULATED CASEIN KINASE (CK II)

1. Adrenal cortex casein kinase G

Besides its well-characterized CAMP-dependent protein (histone)-

kinase system (Garren et al., 1971; Saez et al., 1981) with two isoen- zymatic forms, adrenocortical tissue extracts display a quantitatively-

important cyclic nucleotide independent protein kinase activity easily detected when acidic proteins (casein, phosvitin) are used as exogenous

substrates (Cachet et al., 1977a). The tissue also contains endogenous phosphate acceptors for this activity (Cachet et al., 1977b). When analysed

by phosphocellulose chromatography, this activity could be resolved into two different moieties: (i) a casein kinase of the A type (CKA), using only ATP as a phosphate source; and (ii) a casein kinase G (CKG) so termed since it uses GTP (K, 18 pm) as well as ATP (K, 8 pm) as phosphate donor (Cachet et al., 1980). Both casein kinases were char- acterized as messenger-independent enzymes, being insensitive to cyclic nucleotides and Ca’+-calmodulin. Casein kinase G was found to be markedly activated by Mg*+ at supraphysiological (25-50 mM) con- centrations in the assay. The search for potential intracellular effecters revealed that polyamines in the millimolar range had a striking 5-IO-fold

Po!tomine - mediated protein phosphotyiations 253

stimulating effect on CKG activity in vitro. Spermine was the most active

and the polyamine effect was mimicked by other polycations such as polyor~t~ne and polylysine (4- and 2-fold activation, respectively).

Casein kinase G was obtained in highly purified form (Cachet et al., 1981). The purified enzyme, which may present two isoenzymatic moie- ties, indistinguishable by their functional properties in adrenocortical

tissue, exhibits an apparent molecular weight of 140000 upon gel filtra- tion and appears as an oligomeric structure made up of an (Y (M, 38000) and a p (M, 27000) subunit with a most likely IY~& ~mbination (C. Cachet et al., in press). Affinity labelling with fluorosulphonylbe~oyl-

adenosine showed that the a-subunit bears the catalytic site (J.J. Feige et

al., in press) whereas the /3-component is self-phosphorylatable in the native enzyme. Recent data showed that isolated casein kinase G a-sub-

unit displays a limited catalytic activity and that addition of the /3-com- ponent leads to recovery of a maximal activity (C. Cachet et al., in press).

The purified casein kinase was highly sensitive to spermine, but detailed study in vitro revealed that a complex interplay apparently takes place

between the enzyme, its substrates (protein and nucleotide) and poly- an-tines. The major observations were that (i) spermine did not stimulate

CKG in the absence of Mg’+, thus it did not substitute entirely for the ion. However, the polyamine effect was optimal below 5 mM Mg2+ and

progressively faded at higher Mg” concentrations, which exhibited a marked activating action on CKG in the absence of polyamine. A polyamine-activated (low Mg2+) and a Mg’+-activated state of the

enzyme may thus be suggested, the two types of activation being non-ad-

Fig. 2. Effect of polyamines on casein kinase G activity using either casein (A) or phosvitin

(B) as the protein substrate. Assays were carried out (Cachet et al., 1977) in the presence of

5 mM MgCl,, 0.1 mM [ y-3ZP]ATP, 2.5 mg/ml of substrate and increasing concentrations

of spermine (O), spermidine (0) or putrescine (A), as indicated.

254 C. Cocher and E.M. Chamba:

ditive. (ii) Spermine did not influence the K, of the enzyme for ATP but induced a 5-fold increase in the I’,,,,. In the presence of spermine. the relationship between phosphorylation rate and casein concentration be-

came sigmoidal, suggesting a cooperative effect either through the en- zyme and/or the substrate molecular organizations. (iii) The polyamine effect was obviously dependent upon the nature of the protein substrate:

phosvitin phosphorylation (Fig. 2) was weakly stimulated or even partly

blunted, depending upon the Mg2+ concentration and the spermine/

phosvitin ratio. Self-phosphorylation of CKG was activated in the pres- ence of polyamines, but spermidine was more effective than spermine in

this case. (iv) A direct effect of polyamines on the enzyme was suggested by the strong affinity exhibited by CKG for a spermine-Sepharose support and blotting experiments showing that labelled spermine was

selectively bound to the a-subunit of CKG after its transfer on to a

nitrocellulose support (C. Cachet, unpublished). In addition, the velocity sedimentation behaviour of CKG was shifted to a heavier form in the presence of spermine, as will be discussed later.

An observation of potential interest with regard to the regulation of

CKG activity in vivo was that an apparent activation of the enzyme

occurred upon the first steps of purification from crude adrenocortical extracts. This suggested the presence in the tissue of an endogenous inhibitory factor, which was indeed isolated and characterized in various

bovine tissues (Job et al., 1979a). This heat-stable factor displayed a selective inhibitory activity towards casein kinase G, whereas CKA and CAMP-dependent protein kinase were not affected, and was thus termed casein kinase G inhibitor (CKG I). Kinetic studies showed that CKG I acted as a competitive inhibitor of casein in the reaction (Job et al.. 1979a).

It was then observed that CKG inhibition by CKG I could be released in a dose-dependent manner when polyamines were added to a CKG-CKG I mixture in vitro (Job et al., 1979b). As seen in Fig. 3, a 50-60% CKG I-inhibited CKG (which is approximately the case in crude

bovine adrenocortical cytosol) underwent a progressive activation when spermine was added to the assay medium. At low concentration (< 0.1 mM), the polyamine released CKG inhibition; in the millimolar range of spermine, the CKG I effect was lost and a direct effect on the casein kinase reaction took place, resulting in further increase in CKG activity.

The interaction between CKG, CKG I and polyamines was examined using a velocity sedimentation study (Fig. 4). Purified CKG alone sedi- mented at 7s; CKG I added to the preparation partly co-sedimented with the enzyme, suggesting that a CKG-CKG I complex was generated. corresponding to the inhibited form of the enzyme. Addition of spermine

Polyamine - mediared protein phosphotylations 255

l-2

FRACfiON EJ;MESER

Fig. 3. Release of CKG i~bition by CKG I, and CKG activation by polyamines. Purified

casein kinase G activity was assayed either in the absence (0) or the presence (0) of CKG I

and increasing concentrations of spermine, as indicated. The corresponding percentage

CKG inhibition given by CKG I was plotted (A).

Fig. 4. Velocity sedimentation analysis of casein kinase G interaction with CKG I and the

effect of polyamines. Purified CKG was analysed by sucrose (S-208) density gradient

centrifugation after incubation with its purified inhibitor CKG I either in the absence of

polyamine (panel b) or in the presence of 0.5 mM spermine (panel c). CKG (0) and CKG I

(0) activities were assayed along the gradient. Panel a illustrates the sedimentation

behaviour of CKG I alone. Glucose oxidase (G) and bovine serum albumin (B) were used

as sedimentation markers (Job et al., 1979). In all cases the medium contained 0.5 M NaCI.

(i) dissociated the complex and (ii) resulted in a faster-sedimenting CKG, which should represent the polyamine-activated enzyme. Independent experiments showed that labelled spermine was strongly bound by iso- lated CKG I. These data obtained in vitro with purified components suggested a working hypothesis, which might apply to the regulation of CKG activity in the intact cell, according to the following scheme (Job et al.. 1979b):

CKG-CKG I + Polyamine + CKG I-polyamine + CKG (inac:ive enzyme- (active enzyme) inhibitor complex)

CKG + Polyamine s CKG-polyamine (activated enzyme)

256 C. Cocher and E.M. Chamba:

This hypothesis would integrate CKG as part of a protein phosphory- lation system modulated by the cellular content of polyamines. In this

view, CKG I is not an obligatory factor but its presence.would amplify the range of polya~ne-modulated CKG activity.

Further study resulted in the identification of CKG I as a proteo- glycan mixture in which a heparan sulphate-like structure represented the

active moiety (Pirollet et al., 1981). In fact, heparin mimicked the effects of CKG I on CKG at micromolar concentration in vitro (Feige et al.. 1980). A polyamine effect on CKG I might have been expected due to anionic neutralization by cationic structures: however. acid-base titra- tion of various glycosaminoglycan structures active as selective CKG

inhibitors suggested that additional specific molecular determinants were involved (Feige et al., 1980).

The proposed model has not yet been established as operating in the intact cell. Although ornithine decarboxylase activity is induced in ACTH-treated adrenocortical cells both in vivo (Levine et al., 1973) and in vitro (Kudlow et al., 1980). assay of the state of activation of intracellular CKG is not directly accessible experimentally. Whereas CKG, CKG I and polyamines are both found in the soluble tissue extract, it remains to be established whether the intact cell compartmen-

talization permits the free interplay of these components. After subceiiu- lar analysis under isotonic conditions, bovine adrenocortical CKG was

found distributed mostly between cytosol (about (60%) and the nuclear

fraction (Cachet et al., unpublished). The intracellular distribution of polyamines is not precisely known owing to the strong interaction of these polycations with many cell components (Tabor and Tabor, 1976).

Glycosa~noglycans are mostly known as pericellular matrix components (Lindahl and Heok, 1978). However, newly synthesized glycosaminogly-

cans have been found partly (about 15%) associated with the cell pellet in cultured bovine adrenocortical cells. Interestingly. in this model, ACTH induced an increase in heparane sulphate biosynthesis. most of the material being, however, excreted in the culture medium (Feige et al..

1982). The fact that a polyamine-activated protein kinase system is present in

adrenocortical tissue extract is shown by the st~ulation of “P incorpo- ration in a limited number of endogenous proteins, in the presence of [y-32P]GTP and polyamines (Fig. 5). Attribution of this activity to CKG was assessed by the use of GTP as phosphate source and selective in~bition by added CKG I or heparin. The nature of the co~espond~g target proteins, especially the major 50-5s K dalton phosphorylated moiety, are currently under investigation as briefly discussed below.

Polyamine - mediated protein phosphorylations 257

2. Widespread occurrence of the G-type casein kinase Casein/phosvitin kinases of the A and G types, as described in

adrenal cortex, have emerged as ubiquitous components in mammalian

DISTANCE (cm,

Fig. 5. Polya~ne-dependent phospho~iation pattern in bovine adrenocortical cell cytosol.

Ahquots of cultured adrenocortical cell cyotosol were labelled by incubation with [y- s2P]GTP in the absence ( -) or the presence f- - - - - -) of 2.5 mM spermine. The

samples were subjected to polyacrylamide gradient (S- 188) slab gel electrophoresis in the

presence of sodium dodecyl sulphate. followed by autoradiography. The corresponding

autoradiogram scannings are illustrated. The arrows indicate the location of marker

proteins of known molecular weight.

tissue (for review see Hathaway and Traugh, 1981). Although often termed casein kinase I and II, respectively, according to their elution behaviour from ion exchange resins, we propose the A and G nomencla-

ture since it implies a distinctive functional parameter (use of GTP as a phosphate source). Casein kinase G is similar to casein kinase II from reticulocytes (Hathaway and Traugh, 1979), calf thymus (Dahmus, 1981a),

skeletal muscle (Huang et al., 1982); to nuclear casein kinase N II (Thornburg and Lindell, 1977; Rose et al., 198 l), liver casein kinase TS

(active on threonine and serine in casein) (Meggio et al., 1977), muscle

glycogen synthase PC 0.7 (DePaoli-Roach and Roach, 1982) and GSK-5

(Cohen et al., 1982). Although some discrepancy occurs concerning the subunit composition of the enzyme, a general agreement emerges as to an oligomeric structure with most probably an CQ& stoichiometry, in which the a-subunit bears the catalytic site (Hathaway and Traugh, 1981). Heparin inhibition has been recognized as one of the characteristic

features of CKG and used as a tool for the characterization of this type of kinase (Hathaway and Traugh, 1981; Rose et al., 1981; Hara et al., 198 1; DePaoli-Roach and Roach, 1982; Cohen et al., 1982). Polyamine activation of CKG has been documented as well as the reversal of the inhibition by heparin (Hara et al., 1981; DePaoli-Roach and Roach,

258 C. Cachet and E. M. Chambo:

1982) or other polyanions such as t-RNA (MBenpBa, 1977). Endogenous selective CKG inhibitory factors different from CKG I have been iso-

lated from rat liver nuclear extracts (Farron-Furstenthal, 1980).

The mechanism by which Glyamines (or other polycationic struc-

tures) stimulate casein kinase G activity has been discussed in several recent reports; since the polyamine potency is dependent upon the nature

of the substrate protein, a general suggestion is that the polycation may act through a conformational modification of the substrate (Ahmed et al., 1978; Farron-Furstenthal and Lightholder, 1978; Yamamoto et al..

1979; Hara et al., 1982; DePaoli-Roach and Roach. 1982). This was confirmed by circular dichroism study in the case of phosvitin, but does not dismiss a concomitant effect upon the casein kinase itself (Hara and Endo, 1982), as mentioned above in the case of the adrenal cortex

enzyme. Polyamines have been shown to interact with a number of proteins (Tabor and Tabor, 1976) and possibly result in intra- or inter- molecular non-covalent bridges (Oriol-Audit. 1978; Williams-Ashman

and Canellakis, 1979). The complex interaction between the enzyme. its protein substrate, Mg2+ and polyamine has recently been stressed in the case of muscle glycogen synthase PC 0.7 (DePaoli-Roach and Roach. 1982).

3. Possible physiological significance of casein kinase G Casein kinase of the G type (CK II) appears as a multipotential

protein kinase (Hathaway and Traugh, 1981); however, its physiological

implications remain to be defined. A limited number of soluble endoge- nous substrates of potential interest have been identified following in

vitro studies: muscle and liver glycogen synthase (DePaoli-Roach and Roach, 1982; Cohen et al., 1982; Meggio et al., 1981); calsequestrin, troponin T and the phosphatase inhibitor I (Meggio et al., 1981): protein synthesis initiation factor eIF, (Rittschof and Traugh, 1981; DePaoli- Roach et al., 1981); acetyl CoA-carboxylase (Cohen et al.. 1982) and the regulatory subunit of the CAMP-dependent protein kinase type II isoen- zyme (Carmichael et al., 1982; Hemmings et al., 1983). However, the role of these phosphorylations has not yet been clearly established. The phosphorylation of glycogen synthase by the enzyme does not alter its activity (Cohen et al., 1982); dephosphorylation of the R II subunit does not affect its affinity for cyclic AMP or its ability to recombine with the

C subunit (Rymond and Hofmann, 1982). Phosphorylation of eIF, has been reported to cause a slight stimulation of eIF,-dependent binding of met-tRNA, to the 40s ribosomal subunit, but the physiological signifi- cance of this effect has not yet been determined (DePaoli-Roach et al.,

1981).

i’o@amine - mediated protein phosphoty!ations 259

On the other hand, nuclear targets for casein kinase G have recently been suggested. The high mobility group (HMG) chromosomal proteins are subject to a number of postsynthetic modifications including phos- phorylation. Among the four HMGs identified, only HMG 14 and 17 are phosphorylated in vivo. The phosphorylation of HMG 14 in synthesized HeLa cells is increased 2.5-fold during metaphase. It has been shown that in vitro only casein kinase G specifically catalysed phosphorylation of

HMG 14 at a site which is phosphorylated in vivo (Walton and Gill, 1983). Whether this phosphorylation of HMG 14 by casein kinase G

plays some role in the association with chromatin is at present unknown.

Nuclear targets for casein kinase G have also been suggested in studies of DNA-dependent RNA polymerase regulation. RNA polymerase I was shown to be stimulated after phosphorylation on both serine and

threonine residues in rat liver nuclear preparations, although the protein kinase(s) involved were not identified (Hirsch and Martelo, 1976). RNA

polymerase II from calf thymus was shown to be phosphorylated by a highly purified preparation of a soluble casein kinase G obtained from

the same tissue (Dahmus, 198 la, b). However, the polymerase activity was not modified by this phosphorylation in assays using native calf thymus DNA as template (Dahmus, 198lb). In contrast, calf thymus RNA polymerase II was found to be phosphorylated and concomitantly activated by a purified nuclear cyclic AMP independent protein kinase very similar to casein kinase G (Kranias and Jungmann, 1978). Similarly,

purified Morris hepatoma RNA polymerase I was found to be activated by a homologous G-type nuclear casein kinase (N II kinase); the activa- tion resulted in the synthesis of RNA chains of increased length (Duce-

man et al., 1981). The same nuclear kinase was also shown to be able to phosphorylate hepatoma RNA polymerase II with a concomitant in- crease in the number of RNA chains synthesized in the in vitro assay

(Stetler and Rose, 1982). A conflicting point remains as to whether casein kinase G (N II kinase) is a component of RNA polymerase (Rose et al.,

1981) or an associated co-purified moiety (Dahmus, 1981~). Further

evidence that CKG may be implicated in the regulation of nuclear activity and RNA metabolism is that a 5-fold increase in the enzyme activity was found in hepatoma as compared to normal liver nuclei (Rose et al., 1981) and that casein kinase G was found associated with m-RNP particles in reticulocyte extracts (Rittschof and Traugh, 1982). Recently, the phosphorylation and concomitant activation of Morris hepatoma RNA polymerase II by the homologous nuclear N II kinase were shown to be stimulated in the presence of millimolar concentration of spermine. When the direct effect of the polyamine upon RNA polymerase activity was taken into account, it was concluded that RNA polymerase was

260 C. Cachet and E. M. Chambar

simultaneously activated by a polyamine-stimulated phosphorylation mechanism (Jacob et al., 1983).

The endogenous polyamine-activated protein phosphorylations

exhibited by adrenal cortex cytosol (Fig. 5) disclosed that, in addition to

glycogen synthase (tentatively identified as the 85 000-90000 M, moiety),

a major target was a protein band of M, 50000-55000. This substrate has been partially purified (Cachet et al., unpublished) and found to

contain ornithine decarboxylase activity, which has been separated from the phosphorylatable cyclic AMP dependent protein kinase R II subunit.

Further study is in progress to examine whether soluble ODC is a possible target for CKG in this tissue. This possibility would represent a striking line of similarity with the polyamine-dependent protein kinase system described by Kuehn et al. (see next section).

C. NUCLEAR POLYAMINE-DEPENDENT PROTEIN KINASE IN

Physarum polycephalum

In a recent series of investigations, Kuehn and colleagues have ex- amined the role of non&stone protein phosphorylation in the regulation of nuclear functions of the slime mould Physarum pofycephalum. Isolated desoxyribonucleoprotein (r-DNP) particles exhibiting DNA-dependent RNA polymerase I activity and able to synthesize r-RNA in vitro were shown to incorporate phosphate in the presence of ATP-Mg*+ in several endogenous non&stone protein components (Atmar et al., 1978). In the presence of spermine or spermidine, at sub-millimolar concentrations,

phosphorylation of an endogenous polypeptide (M, 70000 daltons upon SDS polyacrylamide gel electrophoresis) was dramatically increased in r-DNP particles as well as in nucleolar and nuclear extracts. Phosphory-

lation was correlated with a 5-fold increase of in vitro RNA synthesis whereas treatment of the preparations with alkaline phosphatase led to a

decrease in the transcriptional activity. The newly synthesized RNA species were identified as r-RNA following hybridization experiments

(Kuehn et al., 1979). A protein kinase activity was then purified from nucleolar non-histone protein extracts, strongly associated with the 70 K substrate and shown to require the presence of polyamines to be de- tected. An equimolar mixture (0.5 mM each) of spermine and spermidine was the most effective (Daniels et al., 1981). The polyamine-dependent Physarum protein kinase was insensitive to cyclic nucleotides and was inactive upon exogenously added substrates such as casein and histones. Polycations such as polylysine and polyarginine somewhat mimicked the polyamine effect. Since the polyamine activation appeared dependent

Po&amme - mediated protein phosphovlations 261

upon the substrate concentration in the assay, it was suggested that the polyamines acted through an interaction with the 70 K peptide rather than directly on the kinase. The catalytic activity was attributed to a 26000 dalton component of the preparation and appeared strikingly sensitive to NaCl with a narrow 40-50 mM optimal concentration range.

On the basis of its molecular properties, amino acid composition, com- mon antigenicity and enzymatic activity, the isolated 70 K phosphopep-

tide was then identified as the monomer of ornithine decarboxylase

(Atmar and Kuehn, 1981). Phosphorylation by the polyamine-dependent

associated protein kinase resulted in inactivation of ODC activity of the preparation. ODC and the protein kinase appeared to associate strongly in an inactive complex at low ionic strength whereas above 150 mM NaCl, both enzymes were fully active and could be separated upon gel filtration. From these data, Kuehn’s group suggested that Physarum po~eephuIu~ nuclear non&stone proteins contain a polyamine-depen- dent protein kinase tightly associated with a specific substrate, ornithine decarboxylase. Polyamines activate the kinase resulting in ODC phos- phorylation. In its phosphorylated form, ODC is turned off as a poly- amine biosynthetic enzyme and possibly turned on as a selective activator

of r-RNA transcription. This hypothesis provides a mechanism for an

effect of polyamines on their own biosynthesis by a feedback action at

the ODC level. In this view, possible analogy between the 26 K protein kinase and the ODC antizyme has been suggested (Atmar and Kuehn,

1981) although not yet proved. The hypothesis would be in line with that presented by Manen and Russell (1977), suggesting that ODC by itself

may act as a regulatory protein of DNA expression.

Recent reports from the same research group (Atmar et al., 1981) suggest that this polyamine-dependent protein kinase system may be of wide distribution in eukaryotes. It has recently been characterized in

bovine epididymis spermatozoa and rat liver extracts as a 19000 dalton

protein strongly associated with a 70 K endogenous substrate (Atmar et al., 1981). The kinase used ATP but not GTP as phosphate donor and

was inactive on exogenous substrates. A similar polyamine-activated protein kinase activity was recently characterized in interferon-treated Ehrlich ascites cells and shown to be stimulated in the presence of ds-RNA (Sekar et al., 1982). These findings were discussed with regard to a possible similarity with the known protein kinase induced in interferon-treated cells. The latter is also ds-RNA sensitive, and increased phosphorylation of a 70 K endogenous substrate has been associated with the response of target cells to interferon treatment (Lebleu et al., 1976). Although of great interest, these data have been reported as yet by only one research group and must await further confirmation to be fully demonstrated and generally accepted.

262 C. Cachet and EM. Chambar

CONCLUSION AND PERSPECTIVE

The characterization of polyamine-mediated phosphorylation systems appears of wide potential interest as a possible link between the poly- amines, as ubiquitous cell components, and major cellular activities whose regulation implies phosphorylation processes. These may concern the control of DNA, RNA and protein synthesis in which these polycat- ions have been repeatedly implicated without a clear clue as to their mechanism of action in these processes.

To date, the activities of two protein kinases (the widespread casein kinase G and a polya~ne-dependent ornithine decarboxylase kinase, although the latter has as yet been described by only one research group) have been recognized as markedly activated by polyamines in vitro at concentrations compatible with a physiological role. These two examples suggest the definition of a novel class of polyamine-dependent protein kinases. However, since the polyamine effect may involve an interaction with the substrate and result in selective phosphorylation of a set of substrates, one may better suggest the term of polyamine-mediated phosphorylation systems. It remains to be assessed whether polyamines indeed function as intracellular messenger for these systems in the intact cell. This is a very attractive possibility, since polyamine generation is known to be dramatically activated by a number of effecters triggering cell proliferation and/or differentiation.

The best-defined polya~ne-sensitive protein kinase is casein kinase G (CK II), which represents a widespread mu~tipotential protein kinase found in both the soluble and nuclear cell compartments. Although several soluble cellular components have been demonstrated to be phos- phorylated by this casein kinase, the biological significance of these phosphorylations has not been established. On the other hand, recent data suggest that nuclear casein kinase G (N II) may regulate RNA polymerases I and II activities through a phosphorylation process. Al- though the polyamine-dependent nuclear protein kinase described by Kuehn and co-workers exhibits several different functional characteristics (e.g. it does not use GTP), it is also suggested as a regulator of RNA synthesis. Ornithine decarboxylase might appear as a substrate for both protein kinases and it will be of interest to examine whether casein kinase G might represent an equivalent of Kuehn’s enzyme in the mammalian cell.

Although much more work is clearly needed to confirm the pre- liminary evidence at present available, correlation between polyamine biosynthesis, protein phosphorylations and cellular activities would sug- gest the nuclear machinery as a major potential target for polyamine-

P&amine. mediated protein phosphorylakms 263

mediated phosphorylation. On the other hand, polyamine modulation of protein phosphorylation may not be limited to selective activation of certain as yet messenger-independent protein kinase activities but may be more complex in the intact cell, since polyamines have been shown to inhibit cyclic AMP dependent phosphorylation reactions in vitro and possibly specific protein phosphatase activities (Killilea et al., 1978). These observations open the possibility of a subtle balance between various protein kinase activities, which may result in polyamine-depen- dent shifts in intra~llular protein phosphorylation patterns with concom- itant biological consequences.

ACKNOWLEDGEMENTS

This work was made possible by the support of the INSERM (U-244; ATP 77 84 and 79 114), the CNRS (ERA 942) and the Fondation pour la Recherche Medicale Fraqaise. We are indebted to Dr. P. Cohen, Dr. S.T. Jacob and Dr. G.N. Gill for kind communication of material in press.

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