TIBS -February 1983

whereas it formed a floating pellet without their addition, and phase separation into a detergent phase and an aqueous phase was also prevented.

Prospect and conclusions At least two of the three-dimensional

crystals of membrane proteins, namely porin and the photosynthetic reaction centres, are well ordered enough for high- resolution structural analysis. We should soon know whether the concepts summar-

'ized in this article are correct. The demon- stration that well-ordered three-dimensional crystals of membrane proteins can be obtained will hopefully lead to successful attempts with other membrane proteins.

Acknowledgements I thank Prof. R. Huber and Prof. D.

Oesterhelt for support and discussion, Dr

R . M . Garavito for communication of results prior to publication, and Karen. Duffy for reading the manuscript.

References 1 Helenius, A. and Simons, K. (1975) Biochim.

Biophys. Acta 376, 13-26 2 Tanford, C. and Reynolds, J. A. ( 1976 ) Biochim.

Biophys. Acta 457, 133-170 3 Helenius, A., McCaslin, D. R., Fries, E. and

Tanford, C. (1981) Methods Enzymol. 56, 734-749

4 Yonetani, T. (1960) J. Biol. Chem. 236, 1680-1687

50zawa, T., Suzuki, H. and Tanaka, M. (1980) Proc. Natl Acad. Sci. U.S.A. 77,928-930

6 Ozawa, T., Tanaka, M. and Shimomura, Y. (1980) Proc. Natl Acad. Sci. US.A. 77, 5084-5086

7 Michel, H. and Oesterhelt, D. (1980)Proc. Natl Acad. Sci. U.S.A. 77, 1283-1285

8 Henderson, R. and Shonon, D. (1981}) ,/. Mol. Biol. 139, 99-109

59

9 Garavito, R. M. and Rosenbusch, J. P. (1980) J. Cell Biol. 86, 327-329

10 Mathews, F. S., Argos, P. and Levine, M. (1972) Cold Spring Harbor Symp. Quant. Biol. 36, 387-397

11 Wilson, I. A., Skehel, J. J. and Wiley, D. C. (1981) Nature (London) 289,366-373

12 Henderson, R. and Unwin, P. N. T. (1975) Nature (London) 257, 28-32

13 Unwin, P. N. T. and Henderson, R. (1975) ,/. Mol. Biol. 94, 425-.440

14 Michel, H., Oesterhelt, D. and Henderson, R. (1980 ) Proc. Natl Acad. Sci. U.S.A. 77,338-342

15 Vanderkooi, G., Senior, A E., Capaldi, R. A. and Hayashi, H. (1972) Biochim. Biophys. Acta 274, 38-48

16 McDonnel, A. and Staehelin, L. A. (1980)J. Cell Biol. 84, 40-56

17 Li,J. andHollingshead,C. (1982)Biophys.,/. 37, 363-370

18 Michel, H. (1982)./. Mol. Biol. 158,567-572 19 Hendrickson, W. A. and Teeter, M. M. (1981)

Nature (London) 290, 107-113

20 Michel, H. (1982)EMBOJournal 1, 1267-1271

Control of the cytoskeleton by calmodulin and calmodulin-binding

proteins Shiro Kakiuchi and Kenji Sobue

(_almodulin controls the contractile apparatus and cytoskeleton through two groups o f calmodulin-binding proteins. Spectrin (erythrocyte) and spectrin-like proteins (brain and other tissues), both associated with membranes as the major member o f the submembranous cytoskeleton, constitute one group. 7he other consists o f r protein (brain microtubules), caldesmon (smooth muscle) and a M, 135 ~ )~. Po protein (smooth muscle), collectively referred to as flip-flop switch proteins, lhese proteins interact with calmodulin and cytoskeletal protein (either tubulin or actin) alternately

depending upon the concentration o f (~ a 2 .

In 1970, Kakiuchi and associates dis- covered cyclic nucleotide phosphodiesterase which can be activated by Ca ~+ (Ref. 1) and a protein factoi '2 (calmodulin) which con- fers Ca 2+ sensitivity on the enzyme. This coincided with the discovery of protein activator of brain phosphodiesterase by Cheung ~. The latter report, however, lacked information on the effect of Ca 2÷ and it took 3 years before these two inde- pendent lines of research merged when Wang 4 carried out an extensive study on the protein.

Contractile elements such as actin and myosin are ubiquitously distributed among eukaryotic cells. In non-muscle tissues, these and other filamentous proteins that

S. Kakiuchi and K. Sobue are at the Department of Neurochemistry and Neuropharmacology, Institute o f Higher Nervous Activity, Osaka University Med- ical School, Nakanoshima, Kita-ku, Osaka 530, Japan.

include microtubules and intermediate filaments constitute the cytoskeleton. While the actin-myosin interaction (i.e. contraction) in vertebrate skeletal and car- diac muscles is regulated by troponin-Cs, in a Ca ~ -dependent manner, no such analogy exists in the contractile elements of smooth muscle and non-muscle tissues as troponin has not been detected in these tissues. In- stead, calmodulin was found in these tissues in abundance. Therefore, it is reasonable to assume that calmodulin mediates the actions of Ca ~ on the regulation of contrac- tile elements and cytoskeleton in these tis- sues. However, since the discovery of cal- modulin, major efforts in this research field have been focused upon its Ca 2+- dependent activation of intracellular enzymes, and surprisingly little work has been done on the direct interaction of cal- modulin with contractile elements and cytoskeleton. This situation is rapidly

changing since the recent discoveries of calmodulin-binding proteins that can interact with actin or tubulin directly. Moreover, spectrin and spectrin-like pro- teins, which are attached to the inner sur- face of the plasma membranes as major components of cytoskeletons, have been found to be calmodulin-binding proteins. Here we review these calmodulin-binding proteins as they have given us new insights into the Ca 2 - and calmodulin-dependent control of cytoskeleton.

Caldesmon - a ealmodulin-binding protein of smooth muscle able to bind to F-actin in a flip-flop fashion

As in the case of striated muscle, contrac- tion of smooth muscle is controlled by micromolar concentrations of intmcellular Ca 2 . However, its detailed regulatory mechanism has not been fully understood. Several groups of investigators (Adelstein el al., Hartshorne et al., Sobieszek et al., Stull et al. and Watanabe et al. ; for review see Ref. 5) found that activation of actomyosin ATPase activity or the contrac- tion of smooth muscle is accompanied by the Ca 2 - and calmodulin-dependent phos- phorylation of two of the mol. wt 2<~ <J<~0 light chains of myosin. They have claimed that a specific Ca ~ - and calmodulin- dependent myosin light-chain kinase is re- sponsible for the regulation of smooth- muscle contraction. On the other hand, Ebashi and associates have claimed the pre- sence of a control via thin filaments. The controversy between the two theories has not been solved.

Recently, Sobue et al.' discovered in chicken gizzard smooth muscle a Ca 2 - dependent calmodulin-binding protein that is able to bind F-actin. The purified protein,

C Elsevier Biomedical Press 1983 0376 - 5067/83/0000 - 0000/$01.00

60

termed 'caldesmon' (a combination of cal- modulin and the Greek d e s m o s , binding), consists of two polypeptide chains having estimated molecular weights of 150 000 and 147 000 (Ref. 7). Although caldesmon can bind to both calmodulin and F-actin filaments, the binding between caldesmon and F-actin is controlled by Ca2+-calmodu - lin in a flip-flop fashion, i.e. caldesmon can interact with F-actin when caldesmon is not bound to calmodulin and the Ca 2+- dependent association of calmodulin with caldesmon eliminates the interaction be- tween caldesmon and F-actin. Therefore, in the presence of calmodulin, caidesmon and F-actin, the concentration of Ca 2~ regulates the formation of two types of protein com- plex: calmodulin-caldesmon at high Ca ~+ concentrations and caldesmon-F-actin at low Ca 2÷ concentrations. At 1 /zM Ca 2+, equal amounts of both complexes are formed ~. Then the suggestion has been made that this caldesmon-linked flip-flop mechanism may control the actin-myosin interaction of smooth muscle. Subse- quently, Sobue et al. ~ showed that the activ- ity of the actin-myosin interaction of smooth muscle, as determined by super- precipitation and actomyosin ATPase activ- ity, is controlled by a dual mechanism linked to both myosin and thin filaments., While Ca 2÷- and calmodulin-dependent phosphorylation of myosin light chain is a prerequisite for the actin-myosin interac- tion, the calmodulin- and caldesmon-linked flip-flop mechanism acts as a direct on-off switch for the actin-myosin interaction, i.e. association of caldesmon with actin fila- ments at decreased Ca 2~ levels abolishes the actin-myosin interaction, and the Ca2÷-dependent association of calmodulin with caldesmon at increased Ca 2~ levels relieves the caldesmon-induced inhibition of the actin-myosin interaction (Fig. 1). Presence of tropomyosin is essential for the system. Caldesmon was also detected in smooth muscle of bovine aorta. Thus, Ca 2~ controls the actin-myosin interaction of smooth muscle through both myosin- and thin filament-linked regulatory systems and the effects of Ca 2÷ on both the systems are mediated by calmodulin.

Calmodulin-binding proteins of microtubules and control of microtubule assembly-disassembly

Cytoplasmic microtubules are composed mainly of tubulin (Mr 55 000) and of sev- eral accessory proteins called MAPs (microtubule-associated proteins). High molecular weight MAPs of approximately Mr 3(X) 000 and a family of four closely related lower M~ (55 000-62 000) proteins collectively termed tau (T) factor have recently been characterized (for review, see

Fig. l. Control o f actin-myosin interaction o f giz- zard smooth muscle by a caldesmon-linked flip- flop mechanism 7. CaM = calmodulin; CaD - cal- desmon; A = F-actin; TM - tropomyosin; M = myosin (phosphorylated form).

Ref. 8). Microtubules are implicated in mitosis and in the maintenance of cell shape and cell membrane functions. During these cellular activities, they exist either in polymerized or depolymerized form. Therefore, an important question is how microtubule assembly-disassembly is con- trolled in the living cells.

in 1972, Weisenberg assembled micro- tubules by chelating Ca 2+ with EGTA, suggesting that the concentration of Ca 2+ determines the state of the assembly-dis- assembly. Later, the hypothesis was advanced to include the fact that the effect of Ca 2~ may be mediated by calmodulin. This idea is supported by the inhibitory effect of calmodulin (+ Ca ~ ) on micro- tubule assembly in vitro as well as by the characteristic location of calmodulin in the chromosome-to-pole region of the mitotic apparatus seen by the immunofluoresence staining of dividing cells (for review, see Ref. 9). In subsequent experiments using brain microtubules, Kumagai and Nishida demonstrated the formation, in the pres- ence of Ca 2÷, of a calmodulin--tubulin complex which is incapable of polymeriz- ing into microtubules 9.

Recently, another line of evidence was obtained by Kakiuchi and associates 19,n with regard to the mechanism by which calmodulin produces the Ca2~-dependent disassembly or inhibition of assembly. They found r factor to be a calmodulin- binding protein capable of forming a revers- ible complex with calmodulin in the pres- ence of micromolar concentrations of Ca 2~ . Subsequently, using calmodulin, ~- factor and tubulin purified from the brain, they. reconstituted a Ca2÷-sensitive microtubule assembly system in which the assembly of tubulin was dependent on the presence of r factor. In this system, raising the Ca 2+ con- centration inhibited the assembly by form- ing a calmodulin-~- factor complex. Thus, 7- protein acts as a Ca2~-dependent flip-flop switch between calmodulin at the increased Ca 2~ concentrations and tubulin at lower Ca 2~ concentrations, thereby leading to disassembly and assembly, respectively, of microtubules. Further work is needed to evaluate the roles of both systems (with tubulin and r protein as the target of cal- modulin action) in vivo and to determine

T I B S - February 1983

whether the two systems are independent or complementary.

Ca 2 +- and calmodulin-dependent flip-flop mechanism in regulation of cytoskeleton

We have discussed two examples of the flip-flop switch protein, namely caldesmon and ~- factor. In addition, the Mr 135 000 calmodulin-binding protein recently puri- fied from chicken gizzard smooth muscle was also identified as a flip-flop switch, i.e. in the presence or absence of Ca 2~ , the Mr 135 000 protein bound to either cal- modulin or F-actin, respectively, in a flip- flop fashion TM. This Mr 135 000 protein had myosin light chain kinase activity and, judged from its substrate specificity, it is probably this enzyme itselP 2. Thus, besides its well-known role in regulating the myosin function by catalysing phosphory- lation of myosin light chains, the smooth- muscle light-chain kinase can interact with actin filaments directly. Further study on this calmodulin-binding enzyme is urgently needed to re-evaluate its role in the regula- tion of smooth-muscle contraction.

Fig. 2 summarizes three examples of flip-flop switch protein. A general view from these examples is that the regulating action of Ca 2 ~ and calmodulin is transmit- ted to the contractile apparatus of smooth muscle and cytoskeleton of non-muscle tis- sues through specific calmodulin-binding proteins. However, there is another group of calmodulin-binding proteins capable of interacting with actin filaments, namely spectrin and spectrin-like proteins, which do not act as flip-flop switches (see below).

Spectrin as a calmodulin-binding protein The cytoplasmic surface of the erythro-

cyte membrane is lined with a mesh-work conventionally called erythrocyte cyto- skeleton. This structure remains after the membrane is extracted with non-ionic detergents such as Triton X-100 (Triton shell). Spectrin, together with actin and Band 4. l, is the major constituent of this Triton shell (for review, see Ref. 13). Recently, Sobue et al. 14 found that the human erythrocyte Triton shell, dissolved in 6 M urea, binds calmodulin in a Ca 2÷- dependent manner. The agent responsible for the binding activity was purified from the Triton shell and identified as spectrin. The Ca2+-pump ATPase of the erythrocyte membrane is known to require calmodulin for its activity and, therefore, binds cal- modulin in a Ca2+-dependent manner. However, it was calculated that there are 4 500 molecules of this enzyme per celP 5 - less than 3% of the number of the cyto- skeletal calmodulin-binding proteins (spectrin), that is about 200 000 per cell ~.

TIBS - February 1983

CALMODULIN BINDING PROTEIN-LINKED FLIP-FLOP MECHANISM

CaM-BINDING PROTEIN'~ fOaM-BINDING PROTEIN'~ (REGULATORY PROTEIN)J z, +Ca~ ~ \ (REG,ULATORY PROTEIN)//

TARGET PROTEIN "TARGET PROTEIN CYTOSKELETON) 1 [ ,(CYTOSKELETON) I

CaM-BP Tissue Target Function

7 Protein Brain MT Tubulin Control of tubulin polymerization

Gizzard SMM ~ Control of actin- Caldesmon ~ Aorta SMM ) F-actin myosin interaction 135 K protein Gizzard SMM F-actin ?

Fig. 2. Ca~+-dependent and calmodulin*binding protein-linked flip-flop mechanism in the regulation o f cytoskeleton. MT = microtubules; SMM - smooth muscle. For explanation, see text.

The Kd value of CaZ+-pump ATPase for calmodulin, 3 x 10 -9 M (Ref. 16), is three orders of magnitude less than that of spectrin (2.8 × 10 4 M) 14. Therefore, the Ca2+-pump ATPase seems to be sat- urated with calmodulin whenever Ca =÷ is present. On the other hand, since the concen- tration of calmodulin in human erythrocytes is 2.5 x 10 -6 M (Ref. 14), the formation of spectrin--calmodulin complexes may be determined by the local concentration of calmodulin and other intraceUular conditions.

Spectrin, together with actin filaments, is thought to be responsible for the control of cell shape, cell deformability and lateral mobility of transmembrane particles of the mammalian erythrocytes (for review, see Ref. 13). Spectrin binds to other cyto- skeletal proteins including actin fdaments, Band 2.1 (ankyrin, syndeins) and Band 4. l, and through the latter two proteins, it is attached to the cytoplasmic surface of the erythrocyte membrane (Reviewed in Ref. 13). It will be interesting to see if the Ca ~+- dependent spectrin-calmodulin interaction affects the binding of spectrin to these pro- teins thereby influencing the functions of spectrin.

Spectrin-like calmodulin-binding proteins of non-erythroid cells

In view of the central position of spectrin in the submembranous cytoskeleton of mammalian erythrocytes, where it medi- ates to couple actin f'darnents to the plasma membrane and maintains the shape and deformability of the cell, it is reasonable to assume that this protein may have a ubi- quitous distribution among eukaryotic cells. However, earlier attempts to detect such protein in different cell types by immunological means were not successful, and spectrin has been thought to be limited

to eryihrocytes (reviewed in Ref. 13). It is only recently that high molecular weight actin- and calmodulin-binding proteins which are structurally and functionally homologous to erythrocyte spectrin were discovered in brain and other tissues in several laboratories including ours (see below). Coincided with these discoveries, reports by Goodman et al. and Lazarides and associates appeared, demonstrating a wide distribution of proteins capable of reacting with antibodies to erythrocyte spectrin in a variety of cell types. Taken together it is now evident that there exists in a variety of cells the spectrin family, whose members share common features with ery- throcyte spectrin. We will review briefly, in the following section, new aspects of these spectrin-like caimodulin-binding proteins.

In 1978, using a rat brain homogenate, Teshima and KakiuchP demonstrated a Ca=~-dependent calmodulin-binding activ-

61

ity of particulate nature which was able to associate with 100 mg (6/zmoi) calmodulin per kg of brain. This particle-associated calmodulin-binding activity was present in all rat tissues examined, with the highest concentration in brain followed by adrenal gland IS. Subsequently, Kakiuchi and associates 19,2° solubilized this binding activity from brain membranes with 6 M urea 1~ or a low ionic strength medium 2° and then purified to homogeneity by successive column chromatographies. The purified protein appeared to be an extended, flexible rod by low-angle rotary-shadowing elec- tron microscopy (Fig. 3) 21 . It existed in the tetrameric form of molecule being com- posed of two head-to-head associated heterodimers with subunit molecular weights of 240 K (o~ subunit) and 235 K (/3 subunit) 2°'21. While the dimers bound to F-actin filaments, only the tetramers were able to cross-link them to produce a highly viscous gel 2°.21. This protein is a major con- stituent of brain, its concentration being more than 1 mg per g of brain wet weight, of which 70% was associated with mem- branes 2°. In another experiment, the protein accounted for about 3% of the membrane proteinlL Thus, the concentration of this protein in brain is comparable to that of spectrin in erythrocyte (1.4 mg spectrin per g of packed cells). These workers called the protein 'calspectin' from calmodulin- binding spectrin-like nature z°. 21.

The same protein has been studied by several groups. The same protein recog- nized as a calmodulin-binding protein, an actin-binding protein able to activate actomyosin ATPase and an axonally trans- ported protein, was partially purified from brain homogenates by Davies and Klee 22, Shimo-oka and Watanabe 23, and Levine and Willard za, respectively. The latter

Fig. 3. Electron micrographs o f rotary-shadowed brain spectrin-like protein ( tetramers)2,.

62

group termed the protein 'fodrin' as it was identified as a specific component of the conical cytoplasma of many cells including neurons 24. Subsequently Glenney et al. z,.2,, and Bennett et al. s7 purified the protein from brains and carried out detailed biochemical and morphological studies (see below). In addition, a similar but distinct protein (TW 260/240) was purified from the brush bor- ders of chicken intestinal epithelial cells 2' . The presence of these proteins in various tissues and cultured cells was demonstrated by using immunological methods 24.26.27 and ~25I-labeled calmodulin gel overlay technique2L

Comparison of properties of spectrin and spectrin-like proteins is summarized as fol- lows. (1) Each of these proteins is com- posed of a pair of non-identical polypeptide subunits having one common (24~J K) and one variable molecular weights, i.e. 220 K for spectrin, 235 K for the brain protein s~,2~' and 260 K for the brush border protein s' . (2) They all undergo dimer-tetramer inter- conversion in vitro depending upon condi- tions (pH, ionic strength, temperature, etc.) of the medium. However, the brain protein remains in the tetrameric form under condi- tions where spectrin tetramers are con- verted to dimers sl,27, indicating that tetra- mers of spectrin analogues are more stable than tetramers of spectrin. (3) They all bind to F-actin filaments2O,2t.s2 s7 but only tetramers can cross-link actin filaments to form a viscous ga121.2:'.2L (4) Like spectrin, spectrin analogues are capable of inducing the polymerization of G-actin to actin fila- ments by increasing nucleation under con- ditions where G-actin alone polymerizes at much slower rate. This was proved using the brain protein 2". (5) Both spectrin ~4 and spectrin analogues~.,, sl.ss.2.,.s, are Ca ~ - dependent ealmodulin-binding proteins. While calmodulin binding of spectrin can be seen only when 8 M urea was present '4, the brain 1:' ~ and brush bordeP" proteins do not require the presence of urea for their calmodulin binding. The calmodulin bind- ing ability of these proteins appear to be attributed to their c~ (240 K) subunits because the isolated ~ subunits of spectrin and the brain protein showed the calmodu- lin binding activity while/3 subunits failed to bind to calmodulin 21. (6) The molecular forms of these proteins revealed by a low angle rotary-shadowing technique appear to be extended, flexible rods of about 200 nm (tetramer) in length sl,2:' sT, presumably formed by head-to-head association of pairs of heterodimers. Their actin binding sites are at the tail ends of the dimers 2',2:'. The brain protein tetramers appear to be more rigid and straighter than spectrin2~.~".sL (7) Like spectrin, the brain protein is associated with membranes 17 21.~7, from which it can

TABLE I. Concentrations of brain spectrin-like calmodulin-binding protein in rat brain subcellular fractions 2°.

Concentration of brain Fraction spectrin-like protein

% of the total protein in given fractions

Crude mitochondrial fraction . . . . . . . . . . . . . . 3. I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

P~ a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 ~B ~ P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2

c - _ZZ_____________________ ~.2 Microsomal fraction . . . . . . . . . . . . . . . . . . . . . . . . 3.9

A (myelin fragments), B (synaptosomes plus mitochondria) and C (mitochondria) are subfractions of the crude mitochondrial fraction. Px 2, Pa 4 and Ps s, consisting of mainly myelin fragments, synaptic membranes, and synaptosomal mitochondria, respec- tively, were derived from B after hypo-osmotic break- age of synaptosomes.

be solubilized with 6 M urea 1:' or with a low ionic strength medium 2°.2~.27 but not with Triton X-1001". However, about 30% of the total amount of the brain spectrin analogue is present in the cytosolic frac- tion ~°. (8) The brain protein was shown to contain a binding site for erythrocyte anky- rin although its affinity was slightly lower than that of spectrin 27. It is attractive to assume that these spectrin analogues associate with the plasma membrane through their corresponding ankyrin analogues since a wide distribution of immunoreactive forms oferythrocyte anky- rin in mammalian cells has been shown recently (Bennett). (9) The brain protein was immunoprecipitated by antibody against spectrin 27. However, the cross- reactivity was only partial as the brain pro- tein could displace binding of ~2q-labeled spectrin to the antibody by a maximum of 20% (Ref. 27). (1~0 The peptide maps of the brain protein were significantly differ- ent from those of spectrin ~7. ( 11 ) Like spec- trin, the brain protein was shown to be phosphorylated by endogenous protein kin- ases present in both the soluble and mem- brane fractions aU. Interestingly, these kin- ase activities were dependent upon the pre- sence of both Ca ~ and calmodulin :~°.

As for the physiological significance of the spectrin-like proteins, they may per- form functions analogous to those of ery- throcyte spectrin, i.e. they may work as membrane anchorage of actin fibers, and spectrin analogues-actin fibers complex attached to the membrane may confer the structural integrity on the plasma mem- brane and control the mobility of trans- membrane proteins (including surface receptors) in the plane of the membrane. In addition, the brain spectrin analogue may be implicated in the mechanism of axo-

T I B S - F e b r u a r y 1983

plasmic transport s4 and in the synapse func- tion possibly CaS-induced release of transmitter substances at the nerve termi- nals. The latter view is supported by the fact that the brain protein is concentrated in synaptic membranes (Table I) TM 20.

References 1 Kakiuchi, S. and Yamazaki, R. (1970) Proc.

Japan Acad. 46, 387-392 2 Kakiuchi, S., Yamazaki, R. and Nakajima, H.

(1970) Proc. Japan Acad. 46, 587-592 3 Cheung, W. Y. (1970) Biochem. Biophys. Res.

(- ommun. 38,533-538 4 Teo, T. S. and Wang, J. H. ( 1973)J. Biol. (hem.

248, 595(r-5955 5 Hartshorne, D. J. and Siemankowski, R. F. ( 1981 )

Ann. Rev. Physiol. 43, 19-30 6 Sobue, K., Muramoto, Y., Fujita, M. and

Kakiuchi, S. ( 1981 ) Proc. Natl Acad. Sci. U.S.A. 78, 5652-5655

7 Sobue, K., Morimoto, K., Inui, M., Kanda. K. and KakiuchL S. (1982) Biomed. Res. 3, 188-196

8 Timasheff, S. N. and Grisham, L. M. (1980)Ann. Rev. Bioehem. 49, 56.5-59 I

9 Sakai, H. (1980) Biomed. Res. I, 359-375 10 Sobue, K., Fujita, M., Muramoto, Y. and

Kakiuchi, S. (1981) FEBS Lett. 132, 137-1~} I1 Kakiuchi, S. and Sobue, K. 11981)FEBS Lett.

132, 141-143 12 Sobue, K., Morimoto, K., Kanda, K., Fukunaga,

K , Miyamoto, E. and Kakiuchi, S. (1982) Biochem. Int. 5,503-510

13 Branton, D., Cohen, C. M. and Tyler, J. (1981) (ell 24, 24-32

14 Sobue, K., Muramoto. Y., Fujita, M. and Kakiuchi, S. (1981) Biochem. Biophys. Res. Commun. I~.~J, 1063-1070

15 Jarrett, H. W. and Kyte, J. (1979)J. Biol. (hem. 254, 8237-8244

16 Raess, B. U. and Vincenzi, F. F. (1980) Mol. Pharmacol. 18,253-258

17 Teshima, Y. and Kakiuchi, S. 11978)J. Cyclic Nucl. Res. 4, 219-231

18 Sobue, K., Muramoto, Y., Yamazaki, R. and Kakiuchi, S. (1979) FEBS Lett. 105, 105-109

19 Kakiuchi, S., Sobue, K. and Fujita, M. (1981) FEBS Lett. 132, 144-148

20 Kakiuchi, S., Sobue, K., Morimoto, K. and Kanda, K. Biochem. Int. (in press)

21 Kakiuchi, S., Sobue, K., Kanda, K., Morimoto, K., Tsukita, S., Tsukita, S., Ishikawa, H. and Kurokawa, M. (1982) Biomed. Res. 3, 41~r-410

22 Davies, P. J. A. and Klee, C. B. (1981) Biochem. Int. 3,203-212

23 Shimo-oka, T. and Watanabe, Y. (1981) J. Biochem. (70kyo) 90, 1297-1307

24 Levine, J. and Willard, M. ( 1981 )J. Cell Biol. 90, 631~343

25 Glenney, J. R. Jr, Glenney, P. and Weber, K. ( 1982 ) J. Biol. (_hem. 257. 9781-9787

26 Glenney, J. R. Jr, Glenney, P., Osborn, M. and Weber, K. (1982)Cell 28,843-854

27 Bennett, V., Davis, J. and Fowler, W. E. (1982) Nature (London) 299, 126-131

28 Palfrey, H. C., Schiebler, W. and Greengard, P. (1982) Proc. Natl Aead. Sci. U.S.A. 79, 378tr-3784

29 Sobue, K., Kanda, K., Inui, M., Morimoto, K. and Kakiuchi, S. FEBS Lett. (in press)

30 Sobue, K., Kanda, K., Yamagami, K. and Kakiuchi, S. 11982)Biomed. Res. 3,561-570