Membrane phosphorylation - Bioscience Reports

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University of Manchester Institute of Science and Technology,. P.O. Box 88, Manchester M60 IQD. (Received 4 December 1980). Insulin, epidermal growth factor ...
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Bioscience Reports I, 19-34 (1981) Printed in Great Britain

Membrane phosphorylation: a c r u c i a l r o l e in the a c t i o n of i n s u l i n , EGF, and pp6OSrC? Miles D. HOUSLAY Department of Biochemistry, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester M60 IQD (Received 4 December 1980)

Insulin, epidermal growth factor (EGF), and the membrane protein r e s p o n s i b l e for m a l i g n a n t t r a n s f o r m a t i o n by avian sarcoma virus, pp60 s r c , have at l e a s t two things in c o m m o n . The f i r s t is an involvement with cell growth and proliferation, and the second is an ability to regulate the phosphorylation of specific membrane proteins. Covalent modification by phosphorylation of soluble proteins has been demonstrated to be an i m p o r t a n t c o n t r o l m e c h a n i s m r e g u l a t i n g a v a r i e t y of cellular processes (Rubin & Rosen, 1975; Nimmo & Cohen, 1977; Krebs & Beavo, 1979; Weller, 1979). It is p r e s u m a b l y t h e n t h r o u g h a l t e r i n g the a c t i v i t y of t h e s e t a r g e t p r o t e i n s that these entities express certain of their biological effects. Insulin ( C u a t r e c a s a s , 1974; Czech, 1977) and EGF (Carpenter & Cohen, 1979) both bind to specific receptors i n t e g r a t e d into p l a s m a m e m b r a n e , and pp60 s r c is also found in t h e p l a s m a m e m b r a n e (Willingham e t a l . , 1979). This review explores some connections b e t w e e n t h e s e s y s t e m s and c o n s i d e r s spatial relationships between kinases and target proteins in the membrane. M e m b r a n e p r o t e i n s are free to undergo lateral diffusion in two dimensions of the lipid bilayer unless specifically c o n s t r a i n e d . Such proteins may b e divided into two categories: these are the integral proteins, which are firmly embedded in the bilayer, and the peripheral proteins, which are associated with the membrane through electrostatic interactions. All copies of a particular i n t e g r a l p r o t e i n e x h i b i t an a b s o l u t e a s y m m e t r y which is set up during b i o s y n t h e s i s , and the r e m o v a l of t h e s e p r o t e i n s n e c e s s i t a t e s t h e u s e of d e t e r g e n t s . P e r i p h e r a l p r o t e i n s , h o w e v e r , can be l i b e r a t e d by m e r e l y adding chelating a g e n t s or by i n c r e a s i n g t h e pH or ionic s t r e n g t h (see Warren & H o u s l a y , 1980). R e c e n t s t u d i e s have demonstrated that integral membrane proteins can be divided into a number of subclasses based upon the degree of penetration of their globular portions into the lipid bilayer. Peripheral p r o t e i n s , on t h e o t h e r hand, can be divided into two categories: those that interact predominantly with phospholipids and those that bind s p e c i f i c a l l y to i n t e g r a l p r o t e i n s . These are shown schematically in Fig. l. It would seem important, then, for any study of the phosphorylation of m e m b r a n e p r o t e i n s to assess: i) whether peripheral or integral proteins are involved, ii) to which integral proteins the peripheral proteins are attached, iii) the a s y m m e t r i c a l o r i e n t a t i o n of the proteins, iv) whether free lateral diffusion is necessary to propagate the response, v) if an activated m e m b r a n e - b o u n d kinase can act on both soluble and membrane-bound targets, and vi) whether soluble kinases can act on m e m b r a n e - b o u n d 9

The Biochemical

Society

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b)

c)

Fig. i. C l a s s e s of integral and peripheral proteins. Schematic representations of the various relationships of integral (I-IV) and peripheral (V-VI) proteins with the lipid bilayer. I~ transmembrane globular integral protein with functioning region inserted in the bilayer~ e.g. erythrocyte anion transport protein (Band 3)~ Ca 2+ ATPase, and bacteriorhodopsin; II~ t r a n s m e m b r a n e f i b r o u s i n t e g r a l p r o t e i n with globular mass in aqueous compartment~ e.g. glycophorin and aminopeptidase; III~ globular portion in aqueous phase but anchored by an integral hydrophobic pedicle that does not span the bilayer~ e.g. cytochome b 5 and cytochrome b 5 reductase; IV~ globular integral protein associated with essentially one or the other half of the bilayer. A fibrous tail may span the bilayer, e.g. adenylate cyclase (see e.g. Rothman & Lenard, 1977; Kenny & Booth~ 1978; Warren & Houslay~ 1980; Houslay et al., ]980); V~ p e r i p h e r a l protein associated essentially through interactions with phospholipids and therefore able to partition between membranes~ e.g. cytochrome c and pyruvate oxidase; VI~ p e r i p h e r a l protein binding to specific integral transmembrane proteins and therefore displaying a single location~ e.g. erythrocyte glucose-6-phosphate dehydrogenase and ankyrin (Rothman & Lenard 9 1977; Kant & Steck~ 1973; Bennett & Stenbuck~ 1980). Fibrous tails or anchors are shown as twists of e-helix integrated into the phospholipid bilayer. Carbohydrate attached to proteins shown as (~).

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targets. For such questions to be answered) phosphorylation studies need to be carried out both on purified membranes and in whole ceils which can subsequently be fractionated. Both types of studies need careful evaluation because of the many inherent problems.

pp60 src The RNA-containing retroviruses are natural agents of oncogenesis found widely distributed in nature. Those that induce transformation of fibroblasts in culture or sarcomas in animals insert a single gene ( s r c ) , produced by the action of reverse transcriptase, into the host chromosomal DNA (Bishop, 1978). In chicken fibroblasts infected with Rous s a r c o m a virus ( R S V ) , t h e e x p r e s s i o n of s r c leads to t h e s y n t h e s i s of a single p r o d u c t , pp60 s r c , which is a phosphorylated polypeptide, /~r 60 000 (Collett & Erikson, 1978). This is r e l a t e d to t h e n o r m a l cellular protein pp60 s a r c which is found only in minute amounts (Collett e t a l . , 1976, 1979a; Oppermann e t a l . , 1979; Bishop, 1978). pp60 s r c is l o c a l i z e d to the c y t o s o l surface of the cell plasma membrane (Willingham e t a l . , 1979) and expresses an unusual kinase 1978), in that it activity (Collett & Erikson, 1978; Levinson e t a l . , specifically t r a n s f e r s p h o s p h a t e to t y r o s i n e r e s i d u e s on s u b s t r a t e proteins (Hunter & Sefton, 1980). It is presumably through this kinase activity that RSV is a b l e to have such d r a m a t i c e f f e c t s on c e l l f u n c t i o n , a d h e s i o n , and shape (Pastan & Willingham, 1978). Indeed, pp60 s r c can i n v i t r o phosphorylate tyrosine residues on a spectrum of proteins associated with the cell cytoskeleton as well as causing its own p h o s p h o r y l a t i o n ( C o l l e t t e t a I . , 1980). A soluble c y c l i c A M P - d e p e n d e n t p r o t e i n kinase can also a c t on pp60 s r c , but this phosphorylates at a distinct site supplied by a serine residue (Collett et al., 1978, 1979b).

It is clearly of some importance to identify the natural targets for phosphorylation by pp60 s r c , as alterations in the t a r g e t proteins can lead to morphological revertants, even though pp60 s r c remains active (Coliett et al., 1976). Recently a protein (Mr 34 000-36 000) has been identified which becomes rapidly phosphorylated after exposure of fibrobiasts to RSV (Radke e t a l . , 1980; Erikson & E r i k s o n , 1980). The functional significance of this has yet to be elucidated. However, it appears to exist in two phosphorylated populations, one of which e x h i b i t s a p h o s p h o s e r i n e and the o t h e r a p h o s p h o t y r o s i n e residue (Radke e t a l . , 1980). This suggests an interplay between the cyclic AMP-dependent and - i n d e p e n d e n t phosphorylation systems. Furthermore, two plasma-membrane proteins (Mr 55 000 and 57 000) become dephosphorylated upon expression of the transformed phenotype (Witt & Gordon, 1980), suggesting that pp60 s r c can a c t i v a t e either a specific phosphatase or a specific phosphotransferase. Spector e t a l . (1980) have just demonstrated that the 8-subunit (Mr 53 000) of Na+/K+-ATPase in ascites tumour cells is phosphorylated on a single tyrosine residue by a membrane-bound kinase. This l e a d s to a d e c r e a s e in the e f f i c i e n c y of the pump, causing more molecules of ATP to be hydrolysed to maintain a p a r t i c u l a r r a t e of ion t r a n s p o r t . As Na+/K+-ATPase provides a futile cycle, then this decrease in efficiency will lead to an increase in ATP utilization. The

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n e t e f i e c t of this will be to i n c r e a s e both g l y c o l y s i s and h e a t production, observations that are often made ol transformed cells. As it is r a t h e r u n c o m m o n to find phosphotyrosine residues in proteins, then such modifications may well be related to processes that lead to cell transformation. Indeed a number of transformation proteins - the Harvey murine sarcoma virus p 2 1 s r e protein (Shih e t a l . , 19801 Willingham e e e l . , 1980), polyoma middle T antigen (Ito ee a l . , 1977)I Abelson MuLV p120 protein (Witte e t e l . , 1980), and the simian virus gO (SV40) p90 protein (Tijan & Robbin% 1979; GriIfin ee a 2 . , 1979) - exhibit similar properties. They are all associated with t h e cell p l a s m a m e m b r a n e , a r e exposed at its cytosol surlace, are autophosphorylated, and express kinase or phosphotransferase a c t i v i t y . Furthermore, a transformation-dependent protein kinase (Mr 18 000) is induced (Summerhayes & C h e n , 1980) in epithelial cells exposed to the c a r c i n o g e n 7,12-dimethylbenz[c~]anthracene. This is also phosphorylated, although it is not known whether it is membrane-bound. Indeed, the levels of plasma-membrane cyclic AMP-independent protein kinase activity appears to be higher in proliferating cells than in resting ones, w h e r e a s the o p p o s i t e is t r u e of the c y c l i c AMP-dependent kinase activity (Branton, 1980). Thus i n v e s t i g a t i o n s on t h e n a t u r e and targets of cyclic AMP-independent kinases, especially those modifying tyrosine residues, should prove rewarding in understanding cell growth, prolileration, and transformation. EGF

Mouse EGF is a single p o l y p e p t i d e (Mr 6 000) containing three intrachain disulphide bridges that are essential for a c t i v i t y , w h e r e a s human EGF appears to consist of two non-identical polypeptide chains linked by a disulphide bridge ( C a r p e n t e r & Cohen, 1979). These mitogenic peptides bind to a single class os receptor glycoproteins on the cell plasma m e m b r a n e , w h e r e u p o n t h e y s t i m u l a t e cell g r o w t h ( H o l l e n b e r g & C u a t r e c a s a s , 1975; Carpenter e t a l . , 1975). EGF receptors are free to diffuse over the surface of the cell, but upon occupancy they rapidly form clusters and are subsequently internalized by r e c e p t o r - m e d i a t e d e n d o c y t o s i s ( S c h e c h t e r et al., i978; Schlessinger e t a l . , 1978). The formation of endocytotic vesicles is believed to require Ca,+ and to involve the activity of a t r a n s g l u t a m i n a s e e n z y m e (Davies e t a 2 . , 1980) and the cell cytoskeleton (Salisbury e t a 2 . , 19g0). An analogue (CNBr-EGF) of EGF, made by cleavage with cyanogen bromide, although able to bind with reduced affinity to EGF receptors, is unable to cause their internalization and is devoid of biological activity. However, by cross-linking CNBr-EGF with anti-EGF antibodies, receptor a g g r e g a t i o n can be i n d u c e d and biological activity restored. Thus the cross-linking of EGF receptors may be connected with cell activation (Schechter e t a 2 . , 1979). EGF causes the rapid, cyclic-nucleotide-independent phosphorylation of a number of membrane proteins when added to p u r i f i e d p l a s m a m e m b r a n e s from an e p i d e r m a l carcinoma cell line (King e t a l . , 19g0). Both ATP and GTP can a c t as the p h o s p h a t e donor in a system which attains maximal levels at 120 nM EGF, requiring Mn ~+ or Mg 2+ to optimize the activity. The major species phosphorylated

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has molecular weights (Mr ) of 170 000, 150 000, 80 000, and 22 500, with less reproducible e f f e c t s on some other components (Mr 60 000, 55 000, 30-40 000). The two most heavily phosphorylated species (Mr 170 000 and 150 000) are glycoproteins and, like the EGF-stimulated kinase, are also integral proteins. The kinase can, however, act on soluble proteins such as histones and ribonuclease. Very r e c e n t l y Ushiro and Cohen (1980) have shown t h a t the EGF-stimulated kinase s p e c i f i c a l l y p h o s p h o r y l a t e s p r o t e i n t y r o s i n e residues. Furthermore the major target for phosphorylation is believed to be the EGF receptor itself (King e t a l . , 1580; Ushiro & Cohen, 1980). This bears analogy with p p B 0 s r c ; however, in this instance the EGF r e c e p t o r does not i t s e l f express a p r o t e i n k i n a s e a c t i v i t y (Carpenter e t a l . , 1979; King e t a l . , 1980). As the mobility of the EGF receptor and its ability to cluster in the presence o5 hormone are crucial to its biological activity, it is likely that the EGF receptor and the kinase are normally independent entities, free to m i g r a t e in the bilayer. Addition of EGF might then trigger their association and the activation o5 the kinase as suggested by the Mobile Receptor and Collision Coupling Models (Fig. 2) which are obeyed by glucagon or 8-adrenergic receptors when a c t i v a t i n g a d e n y l a t e c y c l a s e (see e.g. Houslay et a Z . , 1577, 1980; Schramm e t a l . , 1977; Tolkovsky & Levitzki, 1978; Martin e t a l . , 1979). In view of this it would be of interest t o e x a m i n e t h e e f f e c t s of C N B r - E G F on p r o t e i n phosphorylation.

Mobile Receptor Model

'~

Collision Coupling Model Fig. 2. R e c e p t o r - t a r g e t protein interaction. This model suggests that both the receptor (R) and the kinase catalytic unit (C) are integral membrane proteins able to undergo free lateral diffusion in the plane of the bilayer. When effector (e) occupies the receptor, this complex interacts with the kinase~ activating it and forming a transmembrane complex (Mobile Receptor Model). The kinase may in some instances be released in an activated (C*) state from this complex (Collision Coupling Model).

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EGF stimulates glycotysis and the transport of a variety of solutes into the cell (Carpenter & Cohen, 1979). These may well be e f f e c t e d by the p h o s p h o r y l a t i o n of p l a s m a - m e m b r a n e proteins. Indeed, the phosphorylation of a component (Mr 55 000) of s i m i l a r size to t h e 8-subunit of the Na+/K+-ATPase has been noted. It is also possible that the internalization events which lead to the d o w n r e g u l a t i o n of EGF r e c e p t o r s ( C a t t er a 2 . , t979) depend upon phosphorylation. This could trigger a Ca2+-flux essential Ior the a c t i v a t i o n of e i t h e r transglutaminase (Davies e t a / . , 1980) or myosin light-chain kinase (Nishikawa e t a 2 . , t 9 8 0 ) , or it may d i r e c t l y i n f l u e n c e c y t o s k e l e t o n - a s s o c i a t e d internalization events (Salisbury er a / . , 1980), perhaps by p h o s p h o r y l a t i n g myosin. In this r e s p e c t it is r a t h e r i n t e r e s t i n g t h a t the phosphorylation of a small, membrane-associated protein (Mr 22 500) is stimulated by EGF. As to why EGF triggers the endocytosis of its receptors, one can speculate that the trigger may be related to the p h o s p h o r y l a t i o n of the receptor. This could explain why transformation by RNA sarcoma viruses,~ which leads to the expression of a plasma-membrane protein kinase phosphorylating tyrosine residues ( p p 6 0 S r c ) , actualty eliminates or greatly reduces the a m o u n t of EGF r e c e p t o r in t h e m e m b r a n e (Todaro e t a 2 . , 1977). Indeed the downregulation of B-receptors is a s s o c i a t e d with the a p p e a r a n c e of two s p e c i f i c p h o s p h o p r o t e i n s (Chuang & Costa, 1979), one of which is similar in size to a subunit of the :B-receptor itself (Stagni e t a 2 . , 1977; S t r o s b e r g e t a / . , 1980). Furthermore, pp60 s r C , which is autophosphorylated, has been identified in smatl a m o u n t s within the cell (Willingham e t a l . , 1979). This m i g h t well r e f l e c t its a s s o c i a t i o n with endocytotic vesicles t h a t are either destined for d e g r a d a t i o n in the l y s o s o m e s (Anderson e t a 2 . , 1977) or involved in membrane recycling (Stanley e t a l . , 1980). The ability of EGF receptors, then, to interact with and stimulate a cyclic AMP-independent protein kinase which specifically phosphorylates tyrosine residues may well be exploited by a variety of agents. Examples of these are tumour promoters, such as phorbol esters and diterpenes, and also growth-stimulating peptides released from various sarcoma cell lines, all of which bind to EGF r e c e p t o r s ( T o d a r o e t a/., 1980). This evidence again suggests that kinases connected with t h e p h o s p h o r y l a t i o n of t y r o s i n e r e s i d u e s on t a r g e t p r o t e i n s a r e connected with processes modulating cell growth and proliferation. Insulin

This p o l y p e p t i d e h o r m o n e p l a y s a k e y r o l e in r e g u l a t i n g blood-glucose levels and metabolic e v e n t s within the cell. In this respect its central targets for action are the liver, fat, and muscle. However, most cells possess insulin receptors which may r e f l e c t t h e i m p o r t a n c e of this h o r m o n e in m a i n t a i n i n g the growth of cells in culture, in stimulating tissue regeneration, and in p r o m o t i n g n o r m a l growth in mammals. Insulin binds with affinity to receptor glycoproteins exposed at the e x t e r n a l s u r f a c e of the plasma m e m b r a n e ( C u a t r e c a s a s , t 9 7 4 ) . Solubilized r e c e p t o r s have an o v e r a l l m o l e c u l a r w e i g h t of about 300 000 ( C u a t r e c a s a s , 1972; Pilch & C z e c h , t980) and c o n s i s t of

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non-identical subunits held together by disulphide bridges. This complex appears to consist of two identical glycosylated subunits (Mr 125-135 000) together with another glycosylated component (Mr 90 000)9 which itself may consist of two identical subunits (Harmon et aI., 1980; Heinrich e t a 2 . , 1980; 3acobs e t a 2 . , 1980; Pilch& Czech, 1979, 1980; Yeung et a l . , 1980; Yip et a l . , 1980). However) there are indications that two other components (M r 56 000 and 34 000) m a y also be associated with, or form part of, the receptor (Lang et al., 1980). As with EGF, insulin induces its mobile receptors (Schechter et al., 1978) to cluster and be internalized (Schlessinger et a l . , 1978)) although the rates of internalization vary with cell type (see Catt et al., 1979). This clustering of insulin receptors may well be of importance in mediating the action of insulin~ as it has been shown that bivalent anti-insulin-receptor antibodies can mimic both the short-term antilipolytic action of insulin on adipocytes (Kahn et al., 1978) and its long-term effects on adipocyte lipoprotein lipase (Van Obberghen et al., 1979). In contrast) univalent fragments of these antibodies) whilst still binding to the receptor) were ineffective unless cross-linked together to form a bivalent reagent capable of aggregating the receptors. The action of insulin on the phosphorylation of adipocyte plasma-membrane proteins has been studied by using either isolated membranes or whole cells that were subsequently fractionated. These studies have undoubtedly been confused owing to difficulties in o b t a i n i n g pure p l a s m a - m e m b r a n e p r e p a r a t i o n s and) in w h o l e - c e l l studies) by the t i m e t a k e n to r e s o l v e t h e c o m p o n e n t m e m b r a n e s . H o w e v e r , a r e c e n t l y devised method (Belsham e t a l . , 1980) for preparing subcellular fractions looks very promising provided that lower EGTA c o n c e n t r a t i o n s are used in order to avoid s t r i p p i n g off peripheral proteins. Avruch e t a l . (1976) d e m o n s t r a t e d that in intact adipocytes, insulin decreased the c y c l i c A M P - m e d i a t e d p h o s p h o r y l a t i o n of one p e p t i d e (Mr 26 000). However) in the presence of adrenaline and presumably elevated intracellular cyclic AMP levels) it t r i g g e r e d t h e p h o s p h o r y l a t i o n of two other plasma-membrane peptides (Mr 62 000 and 79 000). More r e c e n t l y Belsham e t a l . (1980) have added either insulin or adrenaline alone to fat cells and observed increases in the phosphorylation of a single component (Mr 61 000). This has also been noted by Benjamin and Clayton (1978), who also observed that adrenaline decreased the phosphorylation of this band9 whereas Belsham et al., (1980) found that adrenaline increased its phosphorylation. However) Benjamin and C l a y t o n (1978) had not i s o l a t e d a pure p l a s m a - m e m b r a n e f r a c t i o n ) so it is possible that these workers are looking at different bands. The addition of insulin alone to isolated a d i p o c y t e p l a s m a membranes (Seals e t a l . , 1979a) has also been shown to cause a small c y c l i c A M P - i n d e p e n d e n t d e c r e a s e in t h e phosphorylation of a Mr-120 000 component. Insulin also a f f e c t s the p h o s p h o r y l a t i o n of o t h e r p r o t e i n s ; for e x a m p l e it inhibits the a d r e n a l i n e - s t i m u l a t e d phosphorylation of a peptide (Mr 69 000) in the endoplasmic reticulum. However) insulin or adrenaline) either together or alone) both stimulate the phosphorylation (Benjamin & Clayton) 1978; Hughes e t a l . , 1980; Avruch e t a l . ,

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1976; Crapo e t a 2 . , 1980) of a soluble peptide (Mr 123-130 000) which has been shown to be ATP=citrate lyase (Alexander e e a 2 . , 1979). In o t h e r s t u d i e s ( B e n j a m i n & Singer 1974, 1975), insulin apparently enhanced the phosphorylation of two proteins (V/r 140 000 a n d 50 0 0 0 ) , w h i l s t it i n h i b i t e d t h e c y c l i c A M P - d e p e n d e n t phosphorylation of another (Mr 60 000). However, the localization of t h e s e p e p t i d e s was u n f o r t u n a t e l y not a s c e r t a i n e d . Insulin also stimulates the phosphorylation (Hughes e e a 2 . , 19g0) of a n o t h e r protein which may well be the ribosomal $6 protein (Smith e e a l . , 1979). Indeed, the phosphorylation of ribosomal $6 p r o t e i n is also enhanced by glucagon (Gressner & Wool, 1976). One of t h e m o s t i n t e r e s t i n g e f f e c t s o f i n s u l i n on t h e p h o s p h o r y l a t i o n of a d i p o c y t e p r o t e i n s is t h a t it c a u s e s the dephosphorylation of t h e ~=subunit (~/r 42 000) of m i t o c h o n d r i a l p y r u v a t e dehydrogenase (PDH) (Seals e e a 2 . , 1979a,b; Hughes e t a 2 . , 1980). This e f f e c t can be seen in intact ceils and in a mixture o f m i t o c h o n d r i a and p l a s m a m e m b r a n e s , but not in a p u r i f i e d mitochondrial fraction (Seals e t a l . , 1979b). It has been suggested that this dephosphorylation, which leads to the activation of PDH, is achieved by insulin through the r e l e a s e of a ' p e p t i d e = l i k e ' second messenger from the plasma membrane ( J a r e t t & Seals, 1979; Larner e t a 2 . , 1979; Seals & 3arett, 1980). This is a p p a r e n t l y p r o d u c e d by stimulation of an arginine=specific intrinsic plasma-membrane protease, and may be derived from the insulin receptor itself (Seals & C z e c h , 1980). The a g g r e g a t i o n of insulin r e c e p t o r s could well be of importance in this event as the action of insulin in activating PDH in t h i s f a s h i o n is m i m i c k e d by a n t i - i n s u l i n - r e c e p t o r a n t i b o d i e s and concanavalin A acting on adipocyte plasma membranes (Seals & 3arett, 1980). These e f f e c t s are extremely interesting, although it must be kept in mind t h a t the m i t o c h o n d r i a used in t h e s e s t u d i e s w e r e u n d o u b t e d l y uncoupled. F u r t h e r m o r e , the stimulation of PDH was relatively small (25~), especially as ATP was present~ which i t s e l f resulted in up to a 75~ inhibition of the basal activity (Seals & 3arett, 1980). This factor does, however, appear to activate a phosphoprotein phosphatase and inhibit a cyclic AMP-dependent protein kinase (Larner et al., 1979)) implying that it could well r e f l e c t the means by which insulin c o n t r o l s p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n reactions within the c e l l . Indeed insulin can in vivo decrease the phosphorylation of protein phosphatase-I inhibitor-1 (Foulkes e t a i . , 1980), presumably either by activating a p r o t e i n p h o s p h a t a s e or by i n h i b i t i n g a p r o t e i n kinase. The net e f f e c t of this is t h a t the dephosphorylation of inhibitor 1 leads to the a c t i v a t i o n of p r o t e i n phosphatase l) w h i c h c a n t h e n a c t on b o t h s o l u b l e and membrane=bound phosphoproteins. Such events could well c o m p l i c a t e c o m p a r i s o n s b e t w e e n s t u d i e s using purified membranes and studies using whole cells where the m e m b r a n e s are i s o l a t e d a f t e r insulin challenge. Purified rat-liver plasma membranes have been used to investigate the effects of insulin on protein phosphorylation (Marchmont & Houslay, 1980c). H e r e , insulin blocked the phosphorylation of two integral m e m b r a n e p r o t e i n s , I 1 (Mr 1#0 000) and I 2 (Mr g0 0 0 0 ) ) by inhibiting a cyclic AMP=dependent protein kinase. At the same time, it t r i g g e r e d the c y c l i c A M P = d e p e n d e n t p h o s p h o r y l a t i o n of t h r e e

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p e r i p h e r a l membrane proteins, Pl (Mr 52 000), P2 (M r 2g 000), and P3 (Mr 14 000). The K a for i n s u l i n in p r o m o t i n g both of these processes was about 2 x I0 - I ~ M, which reflects the high a f f i n i t y component for insulin binding specifically to its receptor (Cuatrecasa% 1974). H o w e v e r , the K a values for the cyclic AMP dependence o f these two processes were significantly different, being 1.6 x 10-6 M for its e f f e c t on the peripheral proteins and 2.5 x 10-6 M for its effect on the integral proteins. This i m p l i e s t h a t d i f f e r e n t c y c l i c AMP-dependent protein kinases are involved. The magnitude of the K a values for the cyclic AMP-dependence of these events indicates that r e s t i n g levels of cyclic AMP (0.3-0.5 pM) in the hepatocyte (Exton et al., 1973; Smith ee a l . , 197g) would be too low e i t h e r to a l l o w the i n t e g r a l p r o t e i n s to be phosphorylated or for insulin to trigger the phosphorylation of the peripheral proteins. However, if the i n t r a c e l l u l a r c y c l i c AMP c o n c e n t r a t i o n were to rise, as it does to 2-4 pM after exposure to glucagon or adrenaline (3efferson ee a 2 . , 1968; B l a c k m o r e e t a l . , 1979), then this would be sufficient to allow these reactions to occur. Presumably the effects of insulin on these phosphorylation processes reflect in part the ability of insulin to antagonize the action of glucagon. These results bear comparison with those of Avruch and coworkers (1976), who noted that in adipocytes insulin could either antagonize the e f f e c t s of noradrenaline-stimulated p h o s p h o r y l a t i o n or p r o m o t e , in the p r e s e n c e of noradrenaline, the phosphorylation of c e r t a i n p l a s m a - m e m b r a n e p r o t e i n s . Disparate e f f e c t s of i n s u l i n on p r o t e i n p h o s p h o r y l a t i o n are l i k e l y to be encountered, dependent upon the levels of cyclic AMP within the cell preparations and whether or not peripheral proteins have been removed during the preparation of the membranes. T h e P I c o m p o n e n t has b e e n i d e n t i f i e d as a p e r i p h e r a l plasma-membrane cyclic AMP' p h o s p h o d i e s t e r a s e , whose a c t i v i t y at p h y s i o l o g i c a l c o n c e n t r a t i o n s of c y c l i c AMP is doubled upon its phosphorylation (Marchmont & Houslay, 19g0a,h). Upon activation this e n z y m e incorporates 1 tool of alkali-labile phosphate into each mole (~/r 52 000) of protein. This phosphate can be removed using either a p u r i f i e d a l k a l i n e p h o s p h a t a s e or the protein phosphatase activity in rat-liver cytoso% whereupon the activity of the enzyme reverts to its o r i g i n a l level. The most s i g n i f i c a n t kinetic parameter altered by phosphorylation (Fig. 3) is the Hill coefficient, which is reduced from 0.6 to 0.45. The net e f f e c t of this is to provide a greater activation at physiological (low) substrate concentrations, and indeed theoretical studies have shown that intracellular cyclic AMP levels are extremely sensitive to small changes in t h e Hill c o e f f i c i e n t s of c y c l i c AMP p h o s p h o d i e s t e r a s e ( E r n e u x e t a.Z., 1980). The activation of this enzyme, which can only occur in the h e p a t o c y t e a f t e r i n t r a c e l l u l a r c y c l i c AMP levels have been elevated above basal, may, at least in part, explain why insulin does not a f f e c t basal cyclic AMP levels, yet can antagonize the e f f e c t of glucagon on raising cyclic AMP levels. The release of the peripheral cyclic AMP phosphodiesterase f r o m t h e m e m b r a n e is usually a c h i e v e d by increasing the ionic strength (Marchmont & Houslay, 1979) of t h e m e d i u m , but r e l e a s e is also extremely sensitive to the presence of EGTA (Fig. 4). Thus methods of preparing plasma membranes which involve chelating a g e n t s could well remove peripheral proteins.

28

M. D. HOUSLAY

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~~c-AMP Fig. 3. C y c l i c A M P p h o s p h o d i e s t e r a s e a c t i v i t y in i n t a c t rat-liver plasma membranes . Lineweaver-Burk plots of the cyclic AMP phosphodiesterase activity (pmol.min-l.mg -~) in native rat-liver plasma membranes and those pretreated with insulin (i0 ruM) together with ATP (3 ~M) and cyclic AMP (0.I mM). This plot represents the sum of the activities of the integral enzyme (high K m) and the insulin-activated peripheral enzyme (low Km).

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