FIFo-TYPE ATPases are found in the plasma membrane of prokaryotes, the mitochondrial inner membrane of ani- mal cells, and in both the chloroplast thylakoid ...
REVIEWS FIFo-TYPE ATPases are found in the plasma membrane of prokaryotes, the mitochondrial inner membrane of animal cells, and in both the chloroplast thylakoid membrane and the mitochondrial inner membrane of plants. This key enzyme synthesizes ATP in response to an electron-transfer-generated proton gradient and, in the reverse direction, generates an ATP-hydrolysisdri,zen proton gradient for use in ion and substrate-transport processes. As the name implies, F~Fo-typeATPases are composed of two parts: an F~ part, which is made up of five different subunits, ~, [~, 7, ~ and e, present in the stoichiometry 3:3:1:1:1, and an Fo part which, in bacteria such as Escherichia coli (the ECF~Fo complex), contains three different subunits, a, b and c, in the molar ratio 1:2:10-12 ~efs I-3). The F~Fo-type ATPase from chloroplast (CF~Fo) is very similar to that of bacteri.'.. The mitochondrial enzyme (MFfo) has (x, 15and 7 subunits that are highly homoiogous to their bacterial ~nd chloroplast counterparts; the 5subunit is the homolog of the E-subunit in bacteria and chloroplasts, while the e-subunit is unique to the mitochondrial enzyme4. F~Fo from mitochondria has a more complicated Fo part, with as many as 10-12 different subunits 4,5. The F~Fo complex functions in a highly cooperative manner. The rate of ATP hydrolysis at any of three catalytic sites in the F, part (on ~-subunits; see later) is very slow but can be speeded up 103-106-foldby substrate binding at a second site (reviewed in Refs 1, 3). Careful kinetic measurements have established that the reaction ATP ADP.P~ is close to equilibrium in catalytic sites, so that the energy derived from proton translocation is used duriug ATP synthesis to release tightly bound product/(rP, rather than being used directly in ATP formation (reviewed in Refs 5, 6). How the proton gradient drives product ATP release and how ATP hydrolysis is coupled to proton translocation is only now beginning to be studied in any detail. In this review, recent structural data on Frotype ATPases are summarized, with particular focus on the y- and E-subunits and the role that these so )units appear R. A. Capald|, R. Aggeler, P. Tudna and S. Wilkens are at the Instituteof Molecular Biology,Universityof Oregon,Eugene, OR97403, USA.
284
TIBS 19 - JULY 1994
Coupling between catalytic sites and the proton channel in FiFo.typeATPases Roderick A. Capaldi, Robert Aggeler, Paola Turina and Stephan Wilkens FIFo-type ATPases catalyse both ATP-driven proton translocation and proton-gradient-driven ATP synthesis. Recent cryoelectronmicroscopy and low-resolution X-ray studies provide a first glimpse at the structure of this complicated membrane-bound enzyme. The F1 part is roughly globular and linked to the membrane-intercalated Fo part by a narrow stalk domain, which contains the 7-, & and E-subunits along with domains of the />subun!t of the Fo part. Here, we review evidence that conformational and positional changes in the 7- and ~-subunits provide the coupling between c~.alytic sites and proton translocation within the Fro complex. protein (OSCP) in MF~ and the b-subunit of the Fo portion. Figure lc shows the MF~ structure as viewed from below (from the stalk side). Six major masses Structure of the F,Fo complex Figure 1 presents a summary of of density can be seen in a pseudorecent structural studies of F~Fo-type hexagonal arrangement surrounding a ATPases. Figure I a shows a side view of central cavity. Inside this central cavity, the intact ECF~Fo, as observed by cryo- extending from the stalk, are two electronmicroscopy 7, a technique in (x-helices running perpendicular to the which specimens of the enzyme are pre- plane of this view, one of which extends served in their native structure in a thin high into the F~ molecule. Figure ld layer of amorphous ice. The average of shows the hexagonal projection of ECF~ many images shows the F~ part as a viewed by cryoelectronmicroscopy 9. roughly globular structure of around For identification of subunits, the ECF~ 100A in diameter, oriented with the molecule has been reacted with Fab' major masses, the (x- and [5-subunits, fragments derived from monoclonal running perpendicular to the plane of antibodies to the (x-subunit. It can be the membrane. The Fo part of the com- seen that these Fab' fragments superplex is seen to be mostly buried in the impose on alternating masses at the lipid bilayer but it extends out a small periphery, indicating that the (x- and way on the F~ side of the membrane. ~-subunits must alternate in their The Fl and Fo parts of ECF~Foare joined hexagonal arrangement. The projection by a stalk, 45A long. Figure lb presents image in Fig. ld clearly shows the cavity the recently obtained 6.5A.-resolution inside the (x3[~3 barrel structure, and structure of MF~ (Ref. 8). it shows a inside this cavity an asymmetrically globular protein with a 40A-long exten- placed seventh mass that has been sion or stalk, indicating that this feature shown, by difference maps and a gold in the FIFocomplex must include Fl sub- labeling technique, to includ~ the 7- and units and almost certainly the single- a-subunits 9-n. A comparison of Figs lc copy 7- and e-subunits (the subunit and ld indicates that the central mass nomenclature for ECF~Fo will be used seen in cryoelectronmicroscopy is the throughout this review). In addition, the projection through both the perpendicustalk may contain segments of the larly running (x-helices within the %[53 &subunit of the F~ part (or the equiv- barrel and the associated stalk strucalent oligomycin-sensitivity-conferring ture seen in the X-ray determination. to play in energy coupling within the enzyme complex.
© 1994,ElsevierScienceLtd 0968-0004/94/$07.00
REVIEWS
TIBS 19 - JULY 1 9 9 4
Nucleotide-bindingsites While not revealed at the level of structural resolution obtained so far, there are a total of six nucleotidebinding sites in the F] part =-s. Three of these sites exchange nucleotides rapidly during ATP hydrolysis and are thought to be catalytic sites. There are three additional nucleotide-binding sites that exchange ATP or ADP much more slowly than the rate of enzyme turnover, and these are called noncatalytic sites ~2. These noncatalytic sites have a structural role and may also play a part in regulating ATPase and ATP synthesis activitya. The catalytic sites have been located on [l-subunits, while the noncatalytic sites appear to be at the interface between (t- and ~subunits L2. Predictions of the secondary structure of [I-subunits 13, combined with sitedirected mutagenesis studies (see, for example, Refs 14, 15), provide strong evidence of the similarity between the structure of the catalytic site in F~ and that in other nucleotide trisphosphatases, including p21 ~' (Ref. 16) and elongation factor Tu (EF-Tu)l~, whose three-dimensional structures have been determined to high resolution. The characteristic nucleotide-binding fold in these two proteins and, by analogy, in F~ is a ~sheet comprising five parallel strands and one antiparallel strand, surrounded on both sides by (t-helices, with several loops connecting the elements of secondary structure.
direct proton channeling is inconsistent with results obtained for F~Fo of Propionigenium modestum, which is structurally very similar to the E. coil enzyme, but can interchangeably pump Na+ or H÷ (Ref. 20). The most likely alternative is that the F] and Fo sectors communicate via conformational changes which, from the structural data above, would be expected to involve the stalk-iorming subunits. Recent structural studies support this idea and identify the 7- and e-subunits as being involved in this conformational activity.
Arrangement of the y- and E-subunitsin the F,Fo complex The 6.5.~ structure of MF~ (Ref. 8) provides important information on the arrangement of the 7-subunit. The two long (t-helices that extend from within
Catalytic sites and the proton channel are coupled via conformatlonalchanges Figure 2 presents a model of the disposition of subunits in the ECF~Fo complex based on the available structural data. The catalytic sites in the three subunits of ECF~Fo (as well as in CF~Fo and MFtFo) are separated from the proton channel by at least the distance of the stalk, i.e. 45A. Energy transfer measurements in CF~Fo indicate that the catalytic sites are approximately halfway up the F~ part TM and, as seen in Fig. 2, this gives a distance between these sites and the proton channel of 100 ~ or more.
Earlier proposals of direct proton transfer between catalytic sites and the proton channel (see, for example, Ref. 19) seem unlikely, in part because of the long distance involved and the difficulty in insulating the proton channel within the narrow stalk region. Moreover,
•
~
the (t3~13barrel into the stalk region are almost certainly the conserved aminoand carboxy-terminal parts of the 7subunit, which are predicted by sequence analysis to be (t-helical ,.'n,structure. Based on sequence analysis, the middle part of this subunit is arranged mostly as ~turns and ~sheet structures. Protease digestion, biotin-avidin labeling and monoclonal-antibody-binding experiments all indicate that this middle region is outside the (t3~3 barrel and involved in binding the ~-subunit2L Structure-prediction algorithms indicate that the amino-terminal half of the E-subunit comprises mostly ~-turn and ~sheet structures, while the carboxyterminal 50 residues are in an (thelix-turn--~-helix arrangement 22. Crosslinking shows that the E-subunit is bound to the (t- and ~-subunits via the
,. i~ ~ "~
. .
Rgure 1 Structural information on the F1Fo ATPase. (a) Side view of the intact E. co/i F1Fo, obtained by cryoelectronmicroscopy~, showing the roughly globular I:1 part linked to the membraneintercalated Fo part by a 45 A stalk. (b) Side view and (©) view from the bottom (nearest the Fo part) of I:1 from beef-heart mitochondria (reproduced, with permission, from Ref. 8). These views show the globular nature of F1 and the contribution of this part of the complex to the stalk. (d) E. co/i I:1 in the hexagonal projection, immunodecorated with Fab' fragments of a monoclonal antibody against the wsubunits. These Fab' fragments superimpose their density on alternating masses around the periphery. Note the asymmetry of ECF1 as revealed by the ~subunits. The centrally located mass is the projection through both the stalk and the mass of smaller subunits within the (7.3[33 barrel.
285
REVIEWS carboxy-terminal domain 23'24, while the amino-terminal domain interacts with .~he 7-subunit 23. The most conserved segment of the E-subunit in different bacterial species and in the chloroplast enzyme is the region between residues 30 and 40, which contains four invariant residues: Gly30, Glu31, His38 and Pro40 (Ref. 25). This region appears to make contact with the Fo part, as judged by the results of three recent studies. LaRoe and Vik26 have shown that mutations of Glu31 and His38 alter the coupling between F~ and Fo without significantly affecting ATPase activity. We have found that a Cys at position 38 in the mutant EH38C is shielded from reaction with ~4C-labeled N-ethylmaieimide LNEM) in the intact ECF~Fo, while this site is rapidly modified by the reagent in isolated ECF~ (R. Aggeler and R. A. Capaldi, unpublished). Reaction of ECF~ EH38C with NEM did not prevent
TIBS 19 - JULY 1994
binding of the isolated F~ part to ECFo, but coupling of ATPase activity to proton channeling was disrupted, as measured by the altered sensitivity to dicyclohexyl carbodiimide of the ATPase activity. Finally, Fillingame and colleagues have shown that this interaction of the E-subunit with the Fo part appears to involve the c-subunit. They find that mutations of Glu31 in the E-subunit suppress the mutation Q42E in the c-subunit 27. Thus, changing Gin42 to Glu in the polar loop of the c-subunit uncoupled ATPase activity from proton translocation in ECF~Fo, while a second mutation, changing Glu31 in the Esubunit to Gly, Val or Lys, recoupled ATP-driven proton translocation.
Conformationalchanges in the 7-subunit
There is strong genetic evidence for a role of the 7-subunit in coupling catalytic-site events and proton channeling. Futai and colleagues 28 have shown that an M23K mutation in the amino-terminal part of the 7-subunit causes the uncoupling of ATP hydroly~is from proton pumping. This mutation can be suppressed by mutations at any of several sites in the carboxy-terminal part of the 100A subunit between residues 269 and 280. More recently, these workers have found that mutations in the carboxy-terminal part of the 7-subunit can be suppressed by several mutations in the amino terminus between residues 18 and 35 (Ref. 29). In terms of the sLructure in 45 A Fig. 2. it can be envisaged that movements of the amino- and carboxy-terminal T (z-helices of the 7-subunit relative to one another are a 50 A critical part of energy coupling within the F,Fo complex. Our recent studies have provided direct evidence of structural changes in the 7subunit during energy coupling. We have found that the Figure2 Schematic model for the coupling of catalytic sites position of the central mass (three ovoids in the F1 part) with the proton channel in (seen in hexagonal view, as the Fo part through the y- and E-subunits. The ~ and Ein Fig. 1), which includes subunits are shown as part of the stalk, the moveboth the 7- and E-subunits, is ments of which alter the linkage of Fz to Fo different whether Mg2+-ATP during ATP-driven proton translocation and in the opposite direction during ATP synthesis. or Mg~+-ADP are bound to
286
the catalytic sites a°. By introducing Cys residues into the 7-subunit by sitedirected mutagenesis, we have been able to incorporate fluorescence and crosslinking reagents as reporter groups of conformational change. Figure 3 shows data for an experiment using the mutant 7T106C, in which the introduced Cys was selectively modified by the fluorophore N-[4-[7(diethylamino)-4-methylcoumarin-3-yl]maleimide (CM), and the effect of nucleotide binding on the fluorescence of this probe was monitored 31. On binding ATP under conditions in which only one mole of substrate can bind per mole of F~ (unisite ATPase conditions), there was an initial enhancement of fluorescence as a result of substrate binding, fop lowed by a quenching of the fluorescence in a time course that followed exactly the rate of ATP hydrolysis. Similar data were obtained with CM bound at position 8 in the 7-subunit On the mutant yS8C), and with CM bound at position 108 in the ~-subunit (in the mutant ESI08C). The same fluorescence changes were seen, not only in unisite catalysis as described above, but also under multisite conditions where there is catalytic cooperativity. A nucleotide-dependent structural change in the 7-subunit was also resolved in crosslinking studies 32,33. Figure 4b shows the crosslinking of Cys8 of the 7-subunit to the J3-subunit, generated by a novel crosslinking reagent, N-maleimido-N'-(a,-azido-2,3,5,6 tetrafluorobenzamido) cystamine (TFPAM. SS1; Fig. 4a). In these experiments, the reagent is bound to the 7-subunit Cys by reaction of the maleimide moiety and then covalently crosslinked by UV irradiation, which activates the tetrafluorophenylazide moiety. With this class of reagents, crosslinking yields of 30-50% have been obtailled 23. With uncleaved Mg2÷-ATPin the catalytic sites (obtained, for example, by using excess unlabeled ATP to slow ATP hydrolysis, or by adding the nonhydrolysable ATP analog AMP-PNP), crosslinked products with apparent molecular masses of 102 kDa and 84 kDa were produced. By contrast, with Mg2÷-ADP in the catalytic sites, the predominant crosslinked product had an apparent molecular mass of 108kDa, while there was very little 102 kDa product and no 84 kDa species present.
REVIEWS
TIBS 19 - JULY 1994
As might be expected if confor- may take place in the Emational changes are a necessary part subunit during the coupling of ATP hydrolysis and coupled proton process, as observed by cryo) translocation, covalent crosslinking of electronmicroscopy. In this the y-subunit to a ~subunit inhibited experiment, the e-subunit enzyme activity in the presence of any was labeled with a 14A nucleotide. Cleavage of the disulfide gold particle by reaction of $ bond in the crosslink led to regener- a monomaleimido-gold comsT ation of full activity for enzyme pound with a Cys introduced crosslinked in the presence of ATP, indi- at position 38 (in the mutant cating that it is the crosslink per se, ~-138C). With Mg2÷-ATP in rather than the chemical modification the catalytic sites, the by the tetrafluorophenylazide moiety, E-subunit-bound gold particle Time that caused the inhibition. Disulfide- is localized predominantly bond breakage regenerated some, but near a [5-subunit, while with Rgure 3 not all, activity when the crosslinking Mg2~ADP bound, it is localStructural changes of the y-subunit during AIP hydrolywas done in the presence of Mg2+-ADP, ized predominantly near an sis followed by fluorescence changes of CM bound at residue 106 (see text). The presence of ATP in cataindicating that the formation of the a-subunit, a shift of 20.~ lyric sites, obtained by, for example, using an ATP 108 kDa species involves modification by (Ref. 11). regenerating system (a) or by adding Mg2*--AMP-PNP, A shifting of the ~-subunit tetrafluorophenylazide of a site on the gives a fluorescence enhancement that is not seen in lS-subunit that is important for activity. between different sites is the presence of Mg2+-ADP(b). When ATP is added in This site of insertion was located to the indicated by an altered bindsubstoichiometric amounts (0.3 mole per mole of ECF1, so-called unisite catalysis) there is a fluorsegment of the ~subunit between ing affinity of this subunit escence enhancement followed by a quenching of this residues 145 and 155 by peptide m a p to the core ECF~ complex fluorescence (c). The time course of the fluorescence ping and sequencing, with most of (%P37) in the presence of change can be simulated using kinetic constants that the modification at Phe148 (Ref. 33). different nucleotides. Thus, describe unisite catalysis (straight-line piot). Note that Significantly, this is the glycine-rich loop with Mg2÷-ADP in the catathe scale of the fluorescence change in (c) is enlarged threefold with respect to traces (a) and (b). part of the catalytic site, placing the lytic sites, a binding conamino terminus of the 7-subunit within stant of less than 3riM has been measured, while in the presence involving the E-subunit have been ob17 A of the catalytic sites. The conformational changes in the of Mg~*-AMP-PNP or ATP plus EDTA served in trypsin digestion studies 34,36, T-subunit resolved by cryoelectron- the binding constant is close to 3OhM as well as in crosslinking studies 23,32. microscopy, fluorescence experiments (C. Tan and R. A. Capaldi, unpublished). Additionally, there is evidence of shifts in and crosslinking studies were not Nucleotide-dependent structural changes the E-subunit as a part of ATP synthesis. observed in enzyme from which the e-subunit had been removed. This is an import(a) (b) eelleC108000 ant finding because ECF= is a 02000 highly active ATPase in the == 84000 absence of the e-subunit 34,3s ie, eu, and, therefore, the structural changes studied are not solely 0 F F a part of catalytic-site coop erativity. Rather, removing ~roCHZCHzS-SCHzCHzNHCO " - ~ N 3 the e-subunit appears to Y ~ uncouple the cooperativity F F from movements of the T-subb unit that are part of energy transmission within the protein complex.
~
Conformational changes in the e-subunit The altered behavior of the y-subunit when the e-subunit is removed implicates the E-subunit in c o u p ling within the F=Fo complex, and suggests that the 7- and E-subunits work in unison. Figure 5 shows the shift that
1234 Rgure 4 (a) The cleavable hetero-bifunctional photoactivated crosslinker N-maleimido-N'-(4-azido-2,3,5,6 tetrafluorobenzamido) cystamine (TFPAM-SS1).(b) Crosslinking of the mutant ECF1 ~$8C with TFPAMSSI. In lanes 3 and 4, the photolysis to activate the azide to a nitrene was conducted in the presence of ATP (5 raM) and EDTA(0.5 mM) or ADP (5 mM) + Mg2* (5.5 raM), respectively. Lanes I and 2 are the control experiments for lanes 3 and 4, respectively, in which the crosslinking reagent was added but not activated. The crosslinking patterns in the two nucleotide conditions are clearly different. The crosslinked products obtained in ATP+ EDTAwere also observed for enzyme reacted with TFPAM-SS1 in the presence of the nonhydrolysable ATP analog AMP-PNP+ Mg2+, and when ATP+ Mg2* was added and the enzymewas kept in the cold to prevent complete hydrolysis of the substrate.
287
REVIEWS
TIBS 1 9 - J U L Y 1 9 9 4
part, it can be en~saged that ATP..hydrolysis-driven movements of the 7- and E-su~ units cause protonation of sidechains in the c-subunit, and consequent structural changes within Fo that facilitate unidirectional proton translocation across the membrane. In the reverse direction, redox-linked proton translocation through Fo could alter the interaction of the E-subunit with the csubunits, for instance, by changing the ionization of sidechains in the c-subunit (b) (c) on the F~ side of the membrane. The resulting shift in the interaction of the esubunits and conformational changes in the y-subunit could then alter the affinity of catal~ic sites for nucleotides. (f Additional information on the molecular mechanism by which the catalytic site reacRgure S tion ATP ~ ,~d)P + P= affects, Density maps of hexagonal projections of ECF1. (a) or is affected by, conforSummarizes various locations of the central mass (~) and of the ~-subunit (El resolved in cryoelectronmational changes involving microscopy studies, Labeling of the three ec- and the y- and ~-subunits may be subunits is based on reproducible differences in the revealed by the soon-to-be shape of the densities of the ~-subunits seen in the completed high-resolution hexagonal views (see Ref. 11). (b) and (el show the structure of MF~ by Walker, density profile of ECF~ labeled with anti-ec-subunit Fab's, as well as with a 14A gold maleimlde bound at Leslie and their colleagues in a Cys introduced into the ~-subunit (in the mutant Cambridge, UK. More difficult eH38C). The two images show extremes in the posto detail at present is the ition of the gold cluster and, therefore, the e-subunit interaction of the ~-subunit (electron dense region indicated by the arrowhead). and possibly the y-subunit The lines drawn from o~1 to ~3 and ~1 to cx2 provide a reference for the nucleotide-dependent shift of the with the Fo part of the come-subunit (see text). plex; a complete picture of this association and the McCarty and colleagues 37 have found changes involved in coupling must await that an ~-subunit-specific antibody fails high-resolution structural data on the to bind to CF~ in the dark, but reacts Fo part. However, even for the simplest with the enzyme in thylakoids upon Fo complex, that from E. coil, there is still illumination, when ATP synthesis is uncertainty about the stoichiometry of occurring. Energy-dependent changes the ¢-subunits; and while there is good in the reaction of a Lys residue in the evidence ot an cx-helical hairpin folding carbo~-terminal part of the ~-subunit of the c-subunits 39, the multimer of CF~ have also been reported 3s, arrangement of this subunit and its interactions with the a- and/>- subunits The mechanismof coupling:future studies remain to be determined (reviewed in and prospects Refs 39, 40). It is hoped that studies of The studies reviewed here suggest the Fo part of the F~Focomplex will soon that energy coupling within the FjFo show the rapid progress now being complex involves shifts of the E-subunit, made on the F~part of the complex. . . . .
probably in association with a domain of the 7-subunit. As the ~-subunit appears to interact with the protonchannel-forming c-subunits of the Fo
288
Acknowledgements Work on FnFo ATPases in this laborat o r y is supported by NIH grant HL
24526. We thank our many colleagues working on ATPases for important discussions.
References Owing to TIBS' policy of short reference lists, the number of references cited in this article has been limited. Some work has therefore unfortunately been left unacknowledged. 1 Senior,A. E. (1988) Physiol. Rev. 68, 177-231 2 Pederson, P. L. and Amzel, L. M. (1993) J. Biol. Chem. 268, 9937-9940 3 Boyer, P. D. (1993) Biochim. Biophys. Acta 1140, 215-250 4 Walker, J. E. et aL (1990) Phil. Trans. R. Soo. London Ser. B 326, 367-378 5 Ludwig, B., Prochaska, L. and Capaldi, R. A. (1980) Biochemistry 1.9, 1516-1523 6 Penefsky, H. S. and Cross, R. L. (1991) Adv. Enzymol. 64, 173-214 7 Lucken, U., Gogol, E. P. and Capaldi, R. A. (1990) Biochemistry 29, 5339-5343 8 Abrahams, J. P. et al. (1993) EMBO J. 12, 1775-1780 9 Gogol, E. P., Aggeler, R., Sagermann, M. and Capaldi, R. A. (1989) Biochemistry 28, 4717-4724 10 Wilkens, S. and Capaldi, R. A. (1992) Arch. Biochem. Biophys. 299, 105-110 11 Wilkens, S. and Capaldi, R. A. (1994) Biol. Chem. Hoppe-Seyler375, 43-51 12 Cross, R. L. (1988) J. Bioenerg. Biomernbranes 20, 395-406 13 Duncan, T. M. and Cross, R. L. (1992) J. Bioenerg. Biomembranes 24, 453-461 14 Futai, M., Iwamoto,A., Omote, H. and Maeda, M. (1992) J. Bioenerg. Biomembranes 24, 463-468 15 Senior, A. E. (1992) J. Bioenerg. Biomembranes 24, 479-484 16 Berchtold, H. et al, (1993) Nature 365, 126-132 17 Tong, L,, de Vos, A. M., Milburn, N. V. and Kim, S.H. (1991) J. Mol, Biol. 217, 503-516 18 Richter, M. L., Snyder, B., McCarty, R. E. and Hammes, G. G. (1985) Biochemistry 24, 5755-5763 19 Mitchell, P. (1985) FEBS Lett, 181, 1-7 20 Lanbinger, W. and Dimroth, P, (1989) Biochemistry 28, 7194-7198 21 Tang, C., Wilkens, S. and Capaldi, R. A. (1994) J. BioL Chem. 269, 4467-4472 22 Skakoon, E. N. and Dunn, S. D. (1993) Arch. Biochem. Biophys. 302, 272-278 23 Aggeler, R. et al. (1992) Biochemistry 31,2956-2961 24 Dallman, H. G., Flynn,T. G. and Dunn, S. D. (1992) J. Biol. Chem. 267, 18953-18960 25 Kuki, M. et al. (1988) J. Biol. Chem. 263, 17437-17442 26 LaRoe, D. J. and Vik, S. B. (1992) J. Bacteriol. 174, 633-637 27 Zhang, Y., Oldenburg, M. and Fillingame, R. H. J. BioL Chem. (in press) 28 Nakamoto, A., Miki, J., Maeda, M. and Futai, M. (1990) J. BioL Chem. 265, 5043-5048 29 Nakamoto, A. and Futai, M. (1994) Biophys. J. 66, Al18 30 Gogol, E. P., Johnston, E., Aggeler, R. and Capaldi, R. A. (1990) Proc. Natl Acad. Sci. USA 87, 9585-9589 31 Turina, P. and Capaldi, R. A. (1994) J. Biol. Chem. 269, 13465-13471
REVIEWS
TIBS 19 - JULY 1994 32 Aggeler, R. and Capaldi, R. A. (1992) J. BioL Chem. 267, 21355-21359 33 Aggeler, R. et al. (1993) J. Biol. Chem. 268, 20831-20837 34 MendeI-Hartvig,J. and Capaldi, R. A. (1991) Biochemistry 30, 1278-1284
FUELED BY THE discovery of proteins with Src homology 2 (SH2) domains and their role as phosphotyrosine-binding sites, several models for the propagation of extracellular signals through messenger systems involving tyrosine kinases are rapidly coming into focusL Receptors with intrinsic kinase activity, such as the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors, have led the field, in part because they use the simplest pathway (Fig. I). Activation of these receptors by ligand-induced dimerization stimulates autophosphorylation of multiple tyrosyl residues, which associate directly with various SH2-containing proteins, including phosphoinositide 3-kinase (PI 3-kinase), Ras GTPaseactivating protein (Ras-GAP), phospholipase C7, Grb2, c-Fyn, c-Src and probably other SH2-containing proteins 2-6.The characteristic biological responses to PDGF and EGF result, at least in part, from the exact collection of interacting SH2-containing proteins recruited to the receptors for these growth factors in various cellular situations. Oilier related paradigms are emerging because not all receptors with intrinsic tyrosine kinase activity directly engage SH2-containing proteins. Receptors for insulin and insulin-like growth factor-1 (IGF-1) undergo tyrosine autophosphorylation during ligand stimulation. Autophosphorylation strongly activates these receptors, but it does not mediate the association of known SH2containing proteins. Instead, these receptors phosphorylate insulin-receptor substrate 1 0RS-1), which functions as a tyrosine-phosphorylated docking protein to recruit the SH2-containing proteins into a signaling complex (Fig. 1)7. The interleukin 4 (IL-4) receptor illustrates an additional complexity, since its activation leads to
35 Dunn, S. D., Zadorozny,V. D., Tozer, R. G. and Orr, L. E. (1987) Biochemistry 26, 4488-4493 36 Mendel-Hartvig, J. and Capaldi, R. A. (1991) Bioci~emistry 30, 10987-10991 37 Richter, M. L. and McCarty, R. E. (1987) J. Biol. Chem. 262, 15037-15040
The IRS.I signaling system Martin G. Myers, Jr, Xiao Jian Sun and Morris F. White Insulin-receptor substrate 1 (IRS-1) is a principal substrate of the receptor tyrosine kinase for insulin and insulin-like growth factor 1, and a substrate for a tyrosine kinase activated by interleukin 4. IRS-1 undergoes multisite tyrosine phosphorylation and mediates downstream signals by 'docking' various proteins that contain Src homology 2 domains. IRS-1 appears to be a unique molecule; however, 4PS, a protein found mainly in hemopoietic cells, may represent another member of this family.
phosphorylation of IRS-1 even though the IL-4 receptor does not have intrinsic tyrosine kinase activity. The IL-4 receptor, like other receptors in the cytokinereceptor superfamily, such as those for erythropoietin, growth hormone and interferon, must recruit a cytoplasmic tyrosine kinase, possibly similar to JAK1, TYK2 or Fyn, into a multimeric signaling complex to phosphorylate IRS-1. Hemopoietic cells appear to co_qtain a distinct protein substrate called 4PS, which co-migrates with IRS-1 during sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)a. The 4PS protein is functionally related to IRS-1, as it becomes phosphorylated on tyrosine during insulin, IGF-~ and IL-4 stimulation, and associates with PI 3kinasea. However, 4PS in myeloid progenitor cells (FDC-P1 and FDC-P2) is immunologically distinct from IRS-1, since it binds weakly, if at all, to antibodies against IRS-1. Moreover, polymerase chain reaction analysis and low-stringency screen;ng of an FDC-P2 cDNA library did not reveal a gene encoding IRS-1 (M. E White et ai., unpublished). Thus, 4PS may be a M. G. Myers, Jr, X. J. Sun and M. F. White are unique IRS-l-like substrate for the IL-4, at the ResearchDivision, Joslin Diabetes insulin and IGF-1 receptors in these Centerand Programin Cell and cells, but further work is necessary to DevelopmentalBiology,HarvardMedical fully characterize this protein a'9. School, Boston, MA02215, USA.
© 1994, Elsevier Science Ltd 0968-0004/94/$07.00
38 Komatsu-Takaki,M. (1992) J. Biol. Chem. 267, 2360-2363 39 Rllingame, R. H. (1990) in The Bacteria (Vol. 12) (Krulwich, T. A., ed.), pp. 345-391, Academic Press 40 Schneider, E. and Altendorf, K. (1987) Microbiol. Rev. 51, 477-497
The structure and function of IRS-1
IRS-1 was initally detected as a 185kDa phosphoprotein in antiphosphotyrosine immunoprecipitates from insulin-stimulated Fao hepatoma cells ~°. IRS-1 was purified from insulinstimulated rat liver, and its cDNA was isolated from a rat liver library IIJ2. The open reading frame predicts a molecular weight of 131 kDa, but ll~S-1 migrates to a position corresponding to 165180kDa on SDS-PAGE,owing to its high serine phosphorylation state12'In.Although IRS-I is highly conserved between species, it has little extended homology to other known proteins 7. There is a potential nucleotide-binding site near the amino terminus, which is conserved between the rat and human proteins; however, no other sequences characteristic of protein kinases are present (Fig. 2) and this region is not required for any known IRS-1 signaling functions (M. G. Myers, Jr et aL, unpublished). IRS-1 contains a pleckstrin homology (PIT) domain between amino acid residues 7 and 120 .(Fig. 2). This is the ..~ T--J most highly ,=onser,eu region of L ~ I from rat, mouse and humans ~2'~4't5.The PH domain was first recognized as an internal repeat in pleckstrin, a major substrate of protein kinase C (PKC) in platelets, and appears to be required for effective recognition and
289
