- Email: [email protected]
The ubiquitin pathway for protein degradation
Biochemistry in Israel
TIBS 16 - JULY 1991
INTRACF.I.LULAR PROTEIN breakdown is important for cellular functions such as the modulation of the levels of regulatory proteins and the removal of abnormal proteins. The biochemical mechanisms involved in this process were for a long time unclear. Early observations suggested that metabolic energy was required for the degradation of cellular proteins. Studies on the mode of action of an ATP-dependent proteolytic system from reticulocytes led to the discovery of a major pathway for selective protein degradation. Resolution and reconstitution experiments showed that a heat-stable polypeptide was required for the action of this system. This polypeptide was subsequently identified as ubiquitin, which consisted of 76 amino acids and was highly conserved in evolution. It was then found that the polypeptide is covalently linked to protein substrates in an ATP-requiring process. It was proposed that proteins are committed for degradation by their ligation to ubiquitin. The discovery of the ubiquitin system was reviewed in Ref. I and subsequent biochemical work has been reviewed in Ref. 2. In this review, recent information on the mechanisms of the selectivity of ubiquitin-protein ligation and on the mode of degradation of proteins conjugated to ubiquitin is discussed.
Enzymatic reactions In the Iigation of ubiquitinto proteins Presently available information on the enzymatic reactions involved in the ligation of ubiquitin to proteins is summarized in Fig. I. The process is initiated by a specific ubiquitin-activating enzyme, E~, which catalyzes the formation of ubiquitin adenylate and the transfer of activated ubiquitin to a thiol site on the same enzyme (Ref. 3; Fig. la, step I). The activated amino acid residue of ubiquitin has been identified as the carboxy-terminal glycine. The genes of El from several eukaryotes have recently been clone6: yeast and human enzymes are 53% identical, indicating a remarkable conservation in evolution. As might be expected for an essential enzyme, the deletion of E~ in yeast is lethaP. A. Hershko is at the Unit of Biochemistry, Facultyof Medicine and the Rappaport Institute, TechnionIsrael Instituteof Technology,Haifa 31096, Israel.
The ubiquitin pathway for protein degradation Cellular proteins are marked for selective degradation by their ligation to the polypeptide ubiquitin. Recent studies have revealed information on the mechanisms involved in the selection of proteins for ligation to ubiquitin and on the mode of degradation of ubiquitinated proteins. Much remains to be learned about the high selectivity of this degradation pathway. Recent evidence that the cell-cycle regulatory proteins, cyclins, are degraded by the ubiquitin pathway points the way to future challenges in ubiquitin research.
Following activation, ubiquitin is transferred from the thiol site of El to the thiol site of one of several ubiquitin carrier proteins, known as Ezs (Ref. 5; Fig. la, step 2). The subject of E2 proteins was recently reviewed in TIBS by Jentsch and co-workers s. There are at least seven species of Ez in animal cells and yeasts, some of which transfer activated ubiquitin to amino groups of protein substrates in the presence of a third enzyme, E3 (Ref. 5, Fig. Ib). Other Ezs can transfer ubiquitin to certain proteins (such as histones) in the absence of E~ (Ref. 7, Fig. Ic). Of the seven E2 genes that have been cloned in yeast 6, three (UBCI, UBC4 and UBCb), appear to be involved in protein breakdown, as indicated by decreased rates of degradation of abnormal proteins in
(a)
mutants generated by gene disruption. Double mutants in UBC4and UBC5have greatly reduced growth rates, while deletion of all three genes is lethal8, emphasizing the essential functions of the ubiquitin proteolytic system. Other species of Ez may not be involved in protein degradation, but play other roles such as the modification of protein function by ubiquitin ligation. Thus, one of the E2 species in yeast has been identified as the product of the RAD6 gene, which is required for DNA repair, sporulation and induced mutagenesis. Another E2 protein in yeast is the product of CDC34,which plays a role in cell-cycle progression at the GI~S phase transition (for review see Ref. 6). The target proteins ubiquitinated by these Ez species remain unknown.
oII
oII
1 Ub-C-OH+ ATP+ EI-SH ~
O II 2 E~-S-C-Ub "t- E2-SH ~
EI-S-C-Ub+ AMP+ PPi
O II E2-S-C-Ub -I- E~-SH
(b) 1 E3 -t- Protein-NH 2 =
~ E3• Protein-NH2
O II 2 E3e Protein-NH 2 -!- EfS-C-Ub ~ _ -
(c)
0 II E2-S-C-Ub -I- Protein-NH 2 ,,
O II Protein-NH-C-Ub + E~.+ EfSH
0 II --- Protein-NH-C-Ub -t- E2-SH
Figure 1 Enzymaticreactions in the ligation of ubiquitinto proteins. (a) Activation of ubiquitin and transferto E2; (b) E3-dependentligation;(©) E3-indepeneientligation.Ub-COOH,ubiquitin.
© 1991,ElsevierSciencePublishers,(UK) 0376-5067/91/$02.00
265
Biochemistry in Israel has a strong influence. Ciechanover et al. showed that the tRNAmediated transfer of arginine to the amino termini of proteins with acidic residues at this position converts them to good substrates for the ubiquitin system (for review see Ref. 14). Varshavsky and co-workers, using sitedirected mutagenesis, changed the amino-terminal residue of some test proteins expressed in Ub site (s) yeast and found dramatic acceleration of degradation rates caused by cerFigure 2 tain amino acid residues Schematic representation of the different binding at the amino terminus ~5. sites of E3o~.Ub, ubiquitin. Reproduced from Ref. 17. The basis of this 'aminoend rule' is due to the The participation of E3 is generally, specificities of the protein-binding sites although not always 9, required for pro- of the ubiquitin-protein ligase, E3a. By tein degradation via the ubiquitin path- the use of simple amino acid derivaway. in this process, many ubiquitin tives (such as dipeptides), three types molecules are attached to the substrate of protein substrates could be distinprotein via isopeptide linkages between guished ~6. Type ! protein substrates the carboxy-terminai glycine residue of have a basic amino acid residue at their ubiquitin and the ~-NH2groups of lysine amino termini, and their binding to the residues of the protein, in addition, sub- ligase is inhibited by peptides that have strate-bound polyubiquitin chains are Arg, Lys or His at the amino-terminal usually built up TM, with each additional position. Type ll protein substrates ubiquitin unit bound to Lys48 (Ref. 11) have bulky, hydrophobic amino-termiof the previous one. Ubiquitin-protein nal amino acid residues, and their bindligation takes place in two main ing to ligase is specifically inhibited by stagesl2: first, protein substrates with derivatives of Phe, Tyr, Trp or Leu. suitable structures are bound to spe- Type IP protein substrates have amino cific sites of E:~ (Fig. lb, step 1), and termini that are neither basic nor then activated ubiquitin is transferred hydrophobic, and their binding to the from E., to the protein (Fig. l b, step 2). iigase is not inhibited by any dipeptide. Due to the high specificity of its pro- it was suggested that Type ! and Type ll tein-substrate binding sites, E~ appears protein substrates bind to sites of the to have a centrally important role in the ligase specific for basic or hydrophobic selection of proteins suitable for degra- amino-terminal residues, respectively ~6. dation. Some of the structural signals in The mode of interaction of Type III proproteins recognized by certain species teins with E:~aremained unknown. of E~are described below. E~a was purified by affinity chromatography on immobilized Type ! or Mode of action of the ubiquitin-protein Type I! protein substrates ~7. The eniigase, E3 zyme binds strongly to such protein Two species of E~, termed E~a and substrate affinity columns and cannot E~[~,have been isolated from extracts of be eluted with salt at high concenreticulocytes and of these, E~a has been trations. However, the enzyme can be more thoroughly studied. One signal specifically eluted with dipeptides that recognized by E3a is the amino-terminal have amino-terminal residues similar to residue of the protein. It was first found that of matrix-bound protein substrate. that a free amino-terminal a-amino Preparations of iigase purified on either group is required for the degradation of Type ! or Type ll protein substrate many proteins by the ubiquitin affinity columns acted on both types of system ~'~. Subsequent work by several protein substrates, indicating that the investigators showed that the nature of enzyme contains separate binding sites the amino-terminal amino acid residue for both basic and hydrophobic amino Type I 'head' site
[
l
266
i
E2e
TIBS 16 - JULY 1991
acid residues ~7. Currently available information on the different binding sites of E:~a is illustrated schematically in Fig. 2. There are separate 'head' sites (defined as those binding specific amino-terminal residues), for Type I and Type ll amino-termini. In addition, the existence of a 'body' site that interacts with other regions of the protein substrate is indicated by observations that proteins with blocked a-amino groups bind to E:~a, provided that their methionine residues are oxidized ]2, or tha~ they are otherwise denatured. The observation that E3~ acts on some Type Ill proteins ~7 may be explained by the interaction of the 'body site' with some as yet undefined structures in such proteins. The existence of an E2-binding site in E30~(Fig. 2) is indicated due to the formation of a tight complex between these two enzymes TM. The formation of an E2"E3complex might facilitate the transfer of activated ubiquitin from Ez to the protein substrate bound to E3. The presence of ubiquitin-binding site(s) on E30~ (Fig. 2) is indicated by observations that the enzyme binds to ubiquitin-Sepharose S and that protein conjugated to ubiquitin binds to E3 more strongly than the free protein TM. The tight binding of ubiquitin-conjugated proteins to E:~ might be responsible for the sequential addition of several ubiquitin units to the substrate protein. While the amino-terminal signal for protein degradation is strongly conserved in evolution and presumably has some important functions, it does not seem to be involved in the degradation of most cellular proteins. For the majority of intracellular proteins, the specificity of the enzyme that removes the initiator Met residue following protein synthesis ensures that the initiator Met is usually not removed if the adjacent amino acid is of the labilizing class ~9. The conclusion that the amino-terminal signal is not involved in general protein turnover is supported by the recent findings of Varshavsky and co-workers that the disruption of the gene of the yeast homologue of E3(zdoes not impair viability or growth under a variety of conditions 2". Since mutations in other enzymes of the ubiquitin proteolytic system are lethal, it may be assumed that other ubiquitin-protein iigases, recognizing other degradation signals, are involved in the degradation of cellular proteins. One possible candidate is E:~, a recently discovered species of ligase that is specific for Type ill proteins2L E~[~ was partially purified
TIBS 16 - JULY 1991
Biochemistry in Israel
to contain three different protease activities. It thus seems that CF-3conMgATP CF-1 -t- C F - 2 + 20S Protease =, 26S Complex tains the 'catalytic core' (CF-3) of the 26S protease complex. indeed, a mutation in a subunit of the 20S (b) ATP ADP + P, ~ Ks,,2+ P protease in yeast caused (Ub),-Protein " ~ ' / - _ nUb -F Peptides the accumulation of 26S complex ubiquitin-protein conjuisopeptidase (?) gates 27, while deletion of the genes of such subRgure 3 units is lethaF 7.28. Breakdown of proteins ligated to ubiquitin by the 26S The functions of CF-I complex. (a) Formation of active 26S complex; and CF-2 and the roles of (b) degradation of protein ligated to ubiquitin. CF-1, ATP in the assembly and CF-2 and CF-3, conjugate-degradingfactors 1-3; Ub, continued action of ubiquitin. the complex remain unknown. It might be that from extracts of reticulocytes and was an ATP-dependent modification reacshown to be required for the ubiquitin- tion is involved in complex assembly. In dependent degradation of some Type Ill the subsequent action of the 26S comproteins. Apart from its different speci- plex ATP hydrolysis appears to take ficity, it resembles E3a in several physi- place. None of the three factors has cal and enzymatic characteristics u. The appreciable ATPase activity, but signals in proteins recognized by E315 ATPase activity is detectable following complex assembly29. A possible explaremain to be elucidated. Though what we have learned about nation is that the energy from ATP E30~ and E315will presumably help our hydrolysis is required for the translocaunderstanding of the mode of action of tion of ubiquitin-protein conjugates to other ubiquitin-protein ligases, it seems the site of degradation, and that the that many physiologically important E3s operation of the translocation machinhave yet to be discovered. Ciechanover ery is possible only in the complete 26S et al. have shown that some N-a-acety- structure. There is obviously much more lated proteins can be degraded by the to learn about the mode of action of the ubiquitin system~2. Such proteins are 26S complex. In addition, the identity not good substrates for either E3a or and mode of action of isopeptidases E315.it seems reasonable to assume that that allow ubiquitin to be recycled from many different ubiquitin-protein ligases end products of the protein breakdown exist, some of which are highly specific pathway remain to be elucidated. for certain target proteins; this might be the case in the cell.cycle-regulated Future challenges:selectivity and regulation degradation of cyclins (see below). The mechanisms and selectivity of Degradation of proteinsIlgated to ubiquitin the ubiquitin pathway still require furLittle is known about the mechanism ther investigation. The complexities of of degradation of proteins ligated to this pathway, containing a multitude of ubiquitin. Rechsteiner and co-workers enzymes and at least three sites of ATP have identified and purified a large, 26S action, are understandable in terms of a ATP-dependent protease complex that machinery that ensures the high selecdegrades proteins ligated to ubiquitin 23. tivity of protein degradation and its We found that the 26S complex is com- regulation. A striking example of highly posed of three distinct components 24, selective and regulated protein degracalled conjugate-degrading factors (CF-I, dation is that of cyclins in the cell cycle. CF-2 and CF-3). Assembly of these three Cyclins are proteins that are synthefactors to form the active 26S multi- sized and then rapidly degraded at difenzyme complex requires MgATP (Ref. ferent phases of the cell cycle, and thus 24, Fig. 3, step a). ATP is also required serve as oscillators that determine cellfor the action of the 26S complex in the cycle progression 3°. In preliminary degradation of ubiquitin-conjugated experiments, we have found evidence proteins (Fig. 3, step b). One of the fac- indicating that cyclin degradation is tors, CF-3, has been subsequently carried out by the ubiquitin system, as identified as the 20S 'multicatalytic' shown by the specific inhibition of protease complex 25.26, a particle known cyclin degradation in a cell-free system
from clam embryos by a derivative of ubiquitin that cannot form polyubiquitin chains3L While our studies were in progress, Kirschner and coworkers reported a similar conclusion, based on a different experimental approach 32.These investigators showed that bacterially expressed derivatives of cyclin are ligated to ubiquitin in extracts of Xenopus eggs arrested in mitosis, but not in interphase extracts. Based on partial mutational analysis, it was proposed that a 'destruction box' of the sequence RAALGNISNat residues 42-50 of sea urchin cyclin B is required for its degradation 32. The cumulative evidence from these studies opens up the way for the analysis, by direct biochemical methods, of the mechanisms of regulation of cyclin degradation. For example, a cyclin-specific E3 may be converted to an active form at a specific stage of the cell cycle. The cyclin story is one example that promises that the future of the ubiquitin field will be at least as exciting as the past.
(a)
Acknowledgements [ thank A. Ciechanover for helpful comments on the manuscript. Work in my laboratory was supported by NIH grant DK-25614 and by grants from the United States-lsrael Binational Science Foundation. Work with I. Rose was supported by American Cancer Society Grant BC-596.
References 1 Hershko, A. and Ciechanover, A. (1982) Annu. Rev. Biochem. 51, 335-364 2 Hershko, A. (1988) J. Biol. Chem. 263, 15237-15240 3 Ciechanover, A., Heller, H., Katz-Etzion, R. and Hershko, A. (1981) Proc. Natl Acad. Sci. USA 78, 761-765 4 McGrath, J. P., Jentsch, S. and Varshavsky, A. (1991) EMBOJ. 10, 227-236 5 Hershko, A., Heller, H., Elias, S. and Ciechanover, A. (1983) J. Biol. Chem. 258, 8206-8214 6 Jentsch, S., Seufert, W., Sommer, T. and Reins, H. A. (1990) Trends Biochem. Sci. 15, 195-198 7 Pickart, C. M. and Rose, I. A. (1985) .I. Biol. Chem. 260, 1573-1581 8 Seufert, W., McGrath, J. P. and Jentsch, S. (1990) EMBOJ. 9, 4535-4541 9 Haas, A., Reback, P. M., Pratt, G. and Rechsteiner, M. (1990) J. Biol. Chem. 265, 21664-21669 10 Hershko, A. and Heller, H. (1985) Biochem. Biophys. Res. Commun. 128,1079-1086 11 Chau, V. et aL (1989) Science 243,1576-1583 12 Hershko, A., Heller, H., Eytan, E. and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999 13 Hershko, A. et aL (1984) Proc. Natl Acad. Sci. USA 81, 7021-7025 14 Ciechanover, A. and Schwartz, A. L. (1989) Trends Biochem. Sci. 14, 483-488 15 Bachmair, A., Finley. D. and Varshavsky, A. (1986) Science 234, 179-186 16 Reiss, Y., ~;,ah., D. and Hershko, A. (1988)
267
Biochemistry in Israel
TIBS 16 - JULY 1991
J. BioL Chem. 263, 2693-2698 17 Reiss, Y. and Hershko, A. (1990) J. Biol. Chem.
265, 3685-3690 18 Reiss, Y., Heller, H. and Hershko, A. (1989) J. BioL Chem. 264, 10378-10383 19 Sherman, F., Stewart, J. W. and Tsunusawa, F. (1985) BioEssays 3, 27-31 20 Bartel, B., Wunning, I. and Varshavsky, A. (1990) EMBO J. 9, 3179-3189 21 Heller, H. and Hershko, A. (1990) J. Biol. Chem. 265, 6532-6535 22 Mayer, A., Siegel, N. R., Schwartz, A. L. and
FIBROBLAST GROWTH FACTORS are a family of seven structurally related polypeptides characterized by high affinity to heparin. They are highly mitogenic for cells derived from mesoderm and neuroectoderm and are among the most potent inducers of the formation of mesenchyme and new blood vessels ~-~. This gene family includes the prototypes acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) which, unlike most other polypeptide growth factors, are primarily cell-associated and lack a conventional signal sequence for secretion ~-:~. Studies with neutralizin?, anti-bFGF antibodies demonstrate, however, the occurrence of an extracellular autocrine activity of bFGF:~-5,suggesting that FGF may be released from cells via an unusual secretory route that does not require a signal peptide. A family of high affinity ceil-surface FGF receptors with intrinsic tyrosine kinase activities has been identified, as have low affinity receptors that consist of heparan sulfate proteoglycans (HSPGs) I-:~. Binding of FGF to the high affinity receptors leads to stimulation of protein kinase activity and activation of early gene transcription. However, the relationship of these events to FGFstimulated cell proliferation remains unclear ~. Several observations indicate that intracellular target sites for the growth factor may be required to complete the mitogenic response 6. This idea is supported by the demonstration of nuclear localization of FGFTM, although the role of nuclear translocation in the mechanI. Vlodavsky, R. Bar-Shavit, R. Ishai-Michaeli and P, Bashkin are at the Department of Oncology, Hadassah University Hospital, Jerusalem 91120, Israel. Z. Fuks is at the Department of Radiation Oncology, SloanKettering Institute for Cancer Research, 1275 York Ave, New York, NY 10021, U3A.
268
23 24 25 26
Ciechanover, A. (1989) Science 244, 1480-1483 Hough, R., Pratt, G. and Rechsteiner, M. (1987) J. Biol. Chem. 262, 8303-8313 Ganoth. D., Leshinsky, E., Eytan, E. and Hershko, A. (1988) J. Biol. Chem. 263, 12412-12419 Eytan, E., Ganoth, D., Armon, T. and Hershko, A. (1989) Prec. Natl Acad. Sci. USA 86, 7751-7755 DriscoU, J. and Goldberg, A. L. (1990) J. Biol. Chem. 265, 4789-4792
27 Heinemeyer, W. et aL (1991) EMBO J. 10,
555-562 28 Fujiwara, T. et al. (1990) J. Biol. Chem. 265, 16604-16613 29 Armon, T., Ganoth, D. and Hershko, A. (1990) J. Biol. Chem. 265, 20723-20726 30 Minshull, J., Golsteyn, R., Hill, C. S. and Hunt, T. (1990) EMBO J. 9, 2865-2875 31 Hershko, A. et aL J. Biol. Chem. (in press) 32 Glotzer, M., Murray, A. W. and Kirschner, M. W. (1991) Nature 349, 132-138
Extracellular sequestration and release of fibroblast growth factor a regulatory mechanism
Basic fibroblast growth factor, (bFGF), promotes the formation of new blood capillaries and is sequestered and protected by binding to heparan sulfate (HS), both on the cell surface and in the extracellular matrix. Release of HS-bound bFGF by heparin-like molecules and HS-degrading enzymes (i.e., heparanase) provides a novel mechanism for regulation of the growth of capillary blood vessels in normal and pathological situations. The extracellular matrix also serves as a storage depot for other growth factors and enzymes. ism of action of FGF remains unclear 6. While aFGF has been found mainly in neural tissues, bFGF was identified in most of the solid tissues and cultured cells examined ~-3. Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell (EC) proliferation in these tissues is usually very low, with turnover time measured in years. How then are these EC growth factors prevented from acting continuously on the vascular endothelium and in response to what signals do they become available for stimulation of capillary EC proliferation? One possibility is that they are sequestered from their site of action by binding to heparan sulfate 0-1S) on cell surfaces and in the extraceilular matrix (ECM) and saved for emergencies such as wound repair and new blood vessel formation. This article summarizes our results on the sequestration of bFGF by HS in the ECM and cell surface and its release by heparin-like molecules, enzymes and intact cells.
Basic FGF is stored within basement membranesin vitro and in vivo Our studies on the control of cell proliferation and tumor progression by its local environment focus on the interaction of cells with the ECM that is produced by cultured corneal and vascular EC9. This ECM closely resembles the subendothelium in vivo in its morphologica! appearance and molecular composition 9. We have demonstrated that EC and other cell types plated in contact with the subendothelial ECM produced in vitro, no longer require the addition of soluble bFGF in order to proliferate and express their differentiated functions. They do, however, deposit bFGF into their ECM~". Likewise, cultured cardiac myocytes were shown to deposit aFGF into their ECM but not conditioned medium~L In subsequent studies, bFGF was extracted from the subendothelial ECM produced in vitro ~j and from basement membranes of the cornea r', suggesting that ECM may serve as a reservoir for
© 1991, Elsevier Science Publishers, (UK) 037~-5067/91/$02.00
TIBS 16 - JULY 1991
INTRACF.I.LULAR PROTEIN breakdown is important for cellular functions such as the modulation of the levels of regulatory proteins and the removal of abnormal proteins. The biochemical mechanisms involved in this process were for a long time unclear. Early observations suggested that metabolic energy was required for the degradation of cellular proteins. Studies on the mode of action of an ATP-dependent proteolytic system from reticulocytes led to the discovery of a major pathway for selective protein degradation. Resolution and reconstitution experiments showed that a heat-stable polypeptide was required for the action of this system. This polypeptide was subsequently identified as ubiquitin, which consisted of 76 amino acids and was highly conserved in evolution. It was then found that the polypeptide is covalently linked to protein substrates in an ATP-requiring process. It was proposed that proteins are committed for degradation by their ligation to ubiquitin. The discovery of the ubiquitin system was reviewed in Ref. I and subsequent biochemical work has been reviewed in Ref. 2. In this review, recent information on the mechanisms of the selectivity of ubiquitin-protein ligation and on the mode of degradation of proteins conjugated to ubiquitin is discussed.
Enzymatic reactions In the Iigation of ubiquitinto proteins Presently available information on the enzymatic reactions involved in the ligation of ubiquitin to proteins is summarized in Fig. I. The process is initiated by a specific ubiquitin-activating enzyme, E~, which catalyzes the formation of ubiquitin adenylate and the transfer of activated ubiquitin to a thiol site on the same enzyme (Ref. 3; Fig. la, step I). The activated amino acid residue of ubiquitin has been identified as the carboxy-terminal glycine. The genes of El from several eukaryotes have recently been clone6: yeast and human enzymes are 53% identical, indicating a remarkable conservation in evolution. As might be expected for an essential enzyme, the deletion of E~ in yeast is lethaP. A. Hershko is at the Unit of Biochemistry, Facultyof Medicine and the Rappaport Institute, TechnionIsrael Instituteof Technology,Haifa 31096, Israel.
The ubiquitin pathway for protein degradation Cellular proteins are marked for selective degradation by their ligation to the polypeptide ubiquitin. Recent studies have revealed information on the mechanisms involved in the selection of proteins for ligation to ubiquitin and on the mode of degradation of ubiquitinated proteins. Much remains to be learned about the high selectivity of this degradation pathway. Recent evidence that the cell-cycle regulatory proteins, cyclins, are degraded by the ubiquitin pathway points the way to future challenges in ubiquitin research.
Following activation, ubiquitin is transferred from the thiol site of El to the thiol site of one of several ubiquitin carrier proteins, known as Ezs (Ref. 5; Fig. la, step 2). The subject of E2 proteins was recently reviewed in TIBS by Jentsch and co-workers s. There are at least seven species of Ez in animal cells and yeasts, some of which transfer activated ubiquitin to amino groups of protein substrates in the presence of a third enzyme, E3 (Ref. 5, Fig. Ib). Other Ezs can transfer ubiquitin to certain proteins (such as histones) in the absence of E~ (Ref. 7, Fig. Ic). Of the seven E2 genes that have been cloned in yeast 6, three (UBCI, UBC4 and UBCb), appear to be involved in protein breakdown, as indicated by decreased rates of degradation of abnormal proteins in
(a)
mutants generated by gene disruption. Double mutants in UBC4and UBC5have greatly reduced growth rates, while deletion of all three genes is lethal8, emphasizing the essential functions of the ubiquitin proteolytic system. Other species of Ez may not be involved in protein degradation, but play other roles such as the modification of protein function by ubiquitin ligation. Thus, one of the E2 species in yeast has been identified as the product of the RAD6 gene, which is required for DNA repair, sporulation and induced mutagenesis. Another E2 protein in yeast is the product of CDC34,which plays a role in cell-cycle progression at the GI~S phase transition (for review see Ref. 6). The target proteins ubiquitinated by these Ez species remain unknown.
oII
oII
1 Ub-C-OH+ ATP+ EI-SH ~
O II 2 E~-S-C-Ub "t- E2-SH ~
EI-S-C-Ub+ AMP+ PPi
O II E2-S-C-Ub -I- E~-SH
(b) 1 E3 -t- Protein-NH 2 =
~ E3• Protein-NH2
O II 2 E3e Protein-NH 2 -!- EfS-C-Ub ~ _ -
(c)
0 II E2-S-C-Ub -I- Protein-NH 2 ,,
O II Protein-NH-C-Ub + E~.+ EfSH
0 II --- Protein-NH-C-Ub -t- E2-SH
Figure 1 Enzymaticreactions in the ligation of ubiquitinto proteins. (a) Activation of ubiquitin and transferto E2; (b) E3-dependentligation;(©) E3-indepeneientligation.Ub-COOH,ubiquitin.
© 1991,ElsevierSciencePublishers,(UK) 0376-5067/91/$02.00
265
Biochemistry in Israel has a strong influence. Ciechanover et al. showed that the tRNAmediated transfer of arginine to the amino termini of proteins with acidic residues at this position converts them to good substrates for the ubiquitin system (for review see Ref. 14). Varshavsky and co-workers, using sitedirected mutagenesis, changed the amino-terminal residue of some test proteins expressed in Ub site (s) yeast and found dramatic acceleration of degradation rates caused by cerFigure 2 tain amino acid residues Schematic representation of the different binding at the amino terminus ~5. sites of E3o~.Ub, ubiquitin. Reproduced from Ref. 17. The basis of this 'aminoend rule' is due to the The participation of E3 is generally, specificities of the protein-binding sites although not always 9, required for pro- of the ubiquitin-protein ligase, E3a. By tein degradation via the ubiquitin path- the use of simple amino acid derivaway. in this process, many ubiquitin tives (such as dipeptides), three types molecules are attached to the substrate of protein substrates could be distinprotein via isopeptide linkages between guished ~6. Type ! protein substrates the carboxy-terminai glycine residue of have a basic amino acid residue at their ubiquitin and the ~-NH2groups of lysine amino termini, and their binding to the residues of the protein, in addition, sub- ligase is inhibited by peptides that have strate-bound polyubiquitin chains are Arg, Lys or His at the amino-terminal usually built up TM, with each additional position. Type ll protein substrates ubiquitin unit bound to Lys48 (Ref. 11) have bulky, hydrophobic amino-termiof the previous one. Ubiquitin-protein nal amino acid residues, and their bindligation takes place in two main ing to ligase is specifically inhibited by stagesl2: first, protein substrates with derivatives of Phe, Tyr, Trp or Leu. suitable structures are bound to spe- Type IP protein substrates have amino cific sites of E:~ (Fig. lb, step 1), and termini that are neither basic nor then activated ubiquitin is transferred hydrophobic, and their binding to the from E., to the protein (Fig. l b, step 2). iigase is not inhibited by any dipeptide. Due to the high specificity of its pro- it was suggested that Type ! and Type ll tein-substrate binding sites, E~ appears protein substrates bind to sites of the to have a centrally important role in the ligase specific for basic or hydrophobic selection of proteins suitable for degra- amino-terminal residues, respectively ~6. dation. Some of the structural signals in The mode of interaction of Type III proproteins recognized by certain species teins with E:~aremained unknown. of E~are described below. E~a was purified by affinity chromatography on immobilized Type ! or Mode of action of the ubiquitin-protein Type I! protein substrates ~7. The eniigase, E3 zyme binds strongly to such protein Two species of E~, termed E~a and substrate affinity columns and cannot E~[~,have been isolated from extracts of be eluted with salt at high concenreticulocytes and of these, E~a has been trations. However, the enzyme can be more thoroughly studied. One signal specifically eluted with dipeptides that recognized by E3a is the amino-terminal have amino-terminal residues similar to residue of the protein. It was first found that of matrix-bound protein substrate. that a free amino-terminal a-amino Preparations of iigase purified on either group is required for the degradation of Type ! or Type ll protein substrate many proteins by the ubiquitin affinity columns acted on both types of system ~'~. Subsequent work by several protein substrates, indicating that the investigators showed that the nature of enzyme contains separate binding sites the amino-terminal amino acid residue for both basic and hydrophobic amino Type I 'head' site
[
l
266
i
E2e
TIBS 16 - JULY 1991
acid residues ~7. Currently available information on the different binding sites of E:~a is illustrated schematically in Fig. 2. There are separate 'head' sites (defined as those binding specific amino-terminal residues), for Type I and Type ll amino-termini. In addition, the existence of a 'body' site that interacts with other regions of the protein substrate is indicated by observations that proteins with blocked a-amino groups bind to E:~a, provided that their methionine residues are oxidized ]2, or tha~ they are otherwise denatured. The observation that E3~ acts on some Type Ill proteins ~7 may be explained by the interaction of the 'body site' with some as yet undefined structures in such proteins. The existence of an E2-binding site in E30~(Fig. 2) is indicated due to the formation of a tight complex between these two enzymes TM. The formation of an E2"E3complex might facilitate the transfer of activated ubiquitin from Ez to the protein substrate bound to E3. The presence of ubiquitin-binding site(s) on E30~ (Fig. 2) is indicated by observations that the enzyme binds to ubiquitin-Sepharose S and that protein conjugated to ubiquitin binds to E3 more strongly than the free protein TM. The tight binding of ubiquitin-conjugated proteins to E:~ might be responsible for the sequential addition of several ubiquitin units to the substrate protein. While the amino-terminal signal for protein degradation is strongly conserved in evolution and presumably has some important functions, it does not seem to be involved in the degradation of most cellular proteins. For the majority of intracellular proteins, the specificity of the enzyme that removes the initiator Met residue following protein synthesis ensures that the initiator Met is usually not removed if the adjacent amino acid is of the labilizing class ~9. The conclusion that the amino-terminal signal is not involved in general protein turnover is supported by the recent findings of Varshavsky and co-workers that the disruption of the gene of the yeast homologue of E3(zdoes not impair viability or growth under a variety of conditions 2". Since mutations in other enzymes of the ubiquitin proteolytic system are lethal, it may be assumed that other ubiquitin-protein iigases, recognizing other degradation signals, are involved in the degradation of cellular proteins. One possible candidate is E:~, a recently discovered species of ligase that is specific for Type ill proteins2L E~[~ was partially purified
TIBS 16 - JULY 1991
Biochemistry in Israel
to contain three different protease activities. It thus seems that CF-3conMgATP CF-1 -t- C F - 2 + 20S Protease =, 26S Complex tains the 'catalytic core' (CF-3) of the 26S protease complex. indeed, a mutation in a subunit of the 20S (b) ATP ADP + P, ~ Ks,,2+ P protease in yeast caused (Ub),-Protein " ~ ' / - _ nUb -F Peptides the accumulation of 26S complex ubiquitin-protein conjuisopeptidase (?) gates 27, while deletion of the genes of such subRgure 3 units is lethaF 7.28. Breakdown of proteins ligated to ubiquitin by the 26S The functions of CF-I complex. (a) Formation of active 26S complex; and CF-2 and the roles of (b) degradation of protein ligated to ubiquitin. CF-1, ATP in the assembly and CF-2 and CF-3, conjugate-degradingfactors 1-3; Ub, continued action of ubiquitin. the complex remain unknown. It might be that from extracts of reticulocytes and was an ATP-dependent modification reacshown to be required for the ubiquitin- tion is involved in complex assembly. In dependent degradation of some Type Ill the subsequent action of the 26S comproteins. Apart from its different speci- plex ATP hydrolysis appears to take ficity, it resembles E3a in several physi- place. None of the three factors has cal and enzymatic characteristics u. The appreciable ATPase activity, but signals in proteins recognized by E315 ATPase activity is detectable following complex assembly29. A possible explaremain to be elucidated. Though what we have learned about nation is that the energy from ATP E30~ and E315will presumably help our hydrolysis is required for the translocaunderstanding of the mode of action of tion of ubiquitin-protein conjugates to other ubiquitin-protein ligases, it seems the site of degradation, and that the that many physiologically important E3s operation of the translocation machinhave yet to be discovered. Ciechanover ery is possible only in the complete 26S et al. have shown that some N-a-acety- structure. There is obviously much more lated proteins can be degraded by the to learn about the mode of action of the ubiquitin system~2. Such proteins are 26S complex. In addition, the identity not good substrates for either E3a or and mode of action of isopeptidases E315.it seems reasonable to assume that that allow ubiquitin to be recycled from many different ubiquitin-protein ligases end products of the protein breakdown exist, some of which are highly specific pathway remain to be elucidated. for certain target proteins; this might be the case in the cell.cycle-regulated Future challenges:selectivity and regulation degradation of cyclins (see below). The mechanisms and selectivity of Degradation of proteinsIlgated to ubiquitin the ubiquitin pathway still require furLittle is known about the mechanism ther investigation. The complexities of of degradation of proteins ligated to this pathway, containing a multitude of ubiquitin. Rechsteiner and co-workers enzymes and at least three sites of ATP have identified and purified a large, 26S action, are understandable in terms of a ATP-dependent protease complex that machinery that ensures the high selecdegrades proteins ligated to ubiquitin 23. tivity of protein degradation and its We found that the 26S complex is com- regulation. A striking example of highly posed of three distinct components 24, selective and regulated protein degracalled conjugate-degrading factors (CF-I, dation is that of cyclins in the cell cycle. CF-2 and CF-3). Assembly of these three Cyclins are proteins that are synthefactors to form the active 26S multi- sized and then rapidly degraded at difenzyme complex requires MgATP (Ref. ferent phases of the cell cycle, and thus 24, Fig. 3, step a). ATP is also required serve as oscillators that determine cellfor the action of the 26S complex in the cycle progression 3°. In preliminary degradation of ubiquitin-conjugated experiments, we have found evidence proteins (Fig. 3, step b). One of the fac- indicating that cyclin degradation is tors, CF-3, has been subsequently carried out by the ubiquitin system, as identified as the 20S 'multicatalytic' shown by the specific inhibition of protease complex 25.26, a particle known cyclin degradation in a cell-free system
from clam embryos by a derivative of ubiquitin that cannot form polyubiquitin chains3L While our studies were in progress, Kirschner and coworkers reported a similar conclusion, based on a different experimental approach 32.These investigators showed that bacterially expressed derivatives of cyclin are ligated to ubiquitin in extracts of Xenopus eggs arrested in mitosis, but not in interphase extracts. Based on partial mutational analysis, it was proposed that a 'destruction box' of the sequence RAALGNISNat residues 42-50 of sea urchin cyclin B is required for its degradation 32. The cumulative evidence from these studies opens up the way for the analysis, by direct biochemical methods, of the mechanisms of regulation of cyclin degradation. For example, a cyclin-specific E3 may be converted to an active form at a specific stage of the cell cycle. The cyclin story is one example that promises that the future of the ubiquitin field will be at least as exciting as the past.
(a)
Acknowledgements [ thank A. Ciechanover for helpful comments on the manuscript. Work in my laboratory was supported by NIH grant DK-25614 and by grants from the United States-lsrael Binational Science Foundation. Work with I. Rose was supported by American Cancer Society Grant BC-596.
References 1 Hershko, A. and Ciechanover, A. (1982) Annu. Rev. Biochem. 51, 335-364 2 Hershko, A. (1988) J. Biol. Chem. 263, 15237-15240 3 Ciechanover, A., Heller, H., Katz-Etzion, R. and Hershko, A. (1981) Proc. Natl Acad. Sci. USA 78, 761-765 4 McGrath, J. P., Jentsch, S. and Varshavsky, A. (1991) EMBOJ. 10, 227-236 5 Hershko, A., Heller, H., Elias, S. and Ciechanover, A. (1983) J. Biol. Chem. 258, 8206-8214 6 Jentsch, S., Seufert, W., Sommer, T. and Reins, H. A. (1990) Trends Biochem. Sci. 15, 195-198 7 Pickart, C. M. and Rose, I. A. (1985) .I. Biol. Chem. 260, 1573-1581 8 Seufert, W., McGrath, J. P. and Jentsch, S. (1990) EMBOJ. 9, 4535-4541 9 Haas, A., Reback, P. M., Pratt, G. and Rechsteiner, M. (1990) J. Biol. Chem. 265, 21664-21669 10 Hershko, A. and Heller, H. (1985) Biochem. Biophys. Res. Commun. 128,1079-1086 11 Chau, V. et aL (1989) Science 243,1576-1583 12 Hershko, A., Heller, H., Eytan, E. and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999 13 Hershko, A. et aL (1984) Proc. Natl Acad. Sci. USA 81, 7021-7025 14 Ciechanover, A. and Schwartz, A. L. (1989) Trends Biochem. Sci. 14, 483-488 15 Bachmair, A., Finley. D. and Varshavsky, A. (1986) Science 234, 179-186 16 Reiss, Y., ~;,ah., D. and Hershko, A. (1988)
267
Biochemistry in Israel
TIBS 16 - JULY 1991
J. BioL Chem. 263, 2693-2698 17 Reiss, Y. and Hershko, A. (1990) J. Biol. Chem.
265, 3685-3690 18 Reiss, Y., Heller, H. and Hershko, A. (1989) J. BioL Chem. 264, 10378-10383 19 Sherman, F., Stewart, J. W. and Tsunusawa, F. (1985) BioEssays 3, 27-31 20 Bartel, B., Wunning, I. and Varshavsky, A. (1990) EMBO J. 9, 3179-3189 21 Heller, H. and Hershko, A. (1990) J. Biol. Chem. 265, 6532-6535 22 Mayer, A., Siegel, N. R., Schwartz, A. L. and
FIBROBLAST GROWTH FACTORS are a family of seven structurally related polypeptides characterized by high affinity to heparin. They are highly mitogenic for cells derived from mesoderm and neuroectoderm and are among the most potent inducers of the formation of mesenchyme and new blood vessels ~-~. This gene family includes the prototypes acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) which, unlike most other polypeptide growth factors, are primarily cell-associated and lack a conventional signal sequence for secretion ~-:~. Studies with neutralizin?, anti-bFGF antibodies demonstrate, however, the occurrence of an extracellular autocrine activity of bFGF:~-5,suggesting that FGF may be released from cells via an unusual secretory route that does not require a signal peptide. A family of high affinity ceil-surface FGF receptors with intrinsic tyrosine kinase activities has been identified, as have low affinity receptors that consist of heparan sulfate proteoglycans (HSPGs) I-:~. Binding of FGF to the high affinity receptors leads to stimulation of protein kinase activity and activation of early gene transcription. However, the relationship of these events to FGFstimulated cell proliferation remains unclear ~. Several observations indicate that intracellular target sites for the growth factor may be required to complete the mitogenic response 6. This idea is supported by the demonstration of nuclear localization of FGFTM, although the role of nuclear translocation in the mechanI. Vlodavsky, R. Bar-Shavit, R. Ishai-Michaeli and P, Bashkin are at the Department of Oncology, Hadassah University Hospital, Jerusalem 91120, Israel. Z. Fuks is at the Department of Radiation Oncology, SloanKettering Institute for Cancer Research, 1275 York Ave, New York, NY 10021, U3A.
268
23 24 25 26
Ciechanover, A. (1989) Science 244, 1480-1483 Hough, R., Pratt, G. and Rechsteiner, M. (1987) J. Biol. Chem. 262, 8303-8313 Ganoth. D., Leshinsky, E., Eytan, E. and Hershko, A. (1988) J. Biol. Chem. 263, 12412-12419 Eytan, E., Ganoth, D., Armon, T. and Hershko, A. (1989) Prec. Natl Acad. Sci. USA 86, 7751-7755 DriscoU, J. and Goldberg, A. L. (1990) J. Biol. Chem. 265, 4789-4792
27 Heinemeyer, W. et aL (1991) EMBO J. 10,
555-562 28 Fujiwara, T. et al. (1990) J. Biol. Chem. 265, 16604-16613 29 Armon, T., Ganoth, D. and Hershko, A. (1990) J. Biol. Chem. 265, 20723-20726 30 Minshull, J., Golsteyn, R., Hill, C. S. and Hunt, T. (1990) EMBO J. 9, 2865-2875 31 Hershko, A. et aL J. Biol. Chem. (in press) 32 Glotzer, M., Murray, A. W. and Kirschner, M. W. (1991) Nature 349, 132-138
Extracellular sequestration and release of fibroblast growth factor a regulatory mechanism
Basic fibroblast growth factor, (bFGF), promotes the formation of new blood capillaries and is sequestered and protected by binding to heparan sulfate (HS), both on the cell surface and in the extracellular matrix. Release of HS-bound bFGF by heparin-like molecules and HS-degrading enzymes (i.e., heparanase) provides a novel mechanism for regulation of the growth of capillary blood vessels in normal and pathological situations. The extracellular matrix also serves as a storage depot for other growth factors and enzymes. ism of action of FGF remains unclear 6. While aFGF has been found mainly in neural tissues, bFGF was identified in most of the solid tissues and cultured cells examined ~-3. Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell (EC) proliferation in these tissues is usually very low, with turnover time measured in years. How then are these EC growth factors prevented from acting continuously on the vascular endothelium and in response to what signals do they become available for stimulation of capillary EC proliferation? One possibility is that they are sequestered from their site of action by binding to heparan sulfate 0-1S) on cell surfaces and in the extraceilular matrix (ECM) and saved for emergencies such as wound repair and new blood vessel formation. This article summarizes our results on the sequestration of bFGF by HS in the ECM and cell surface and its release by heparin-like molecules, enzymes and intact cells.
Basic FGF is stored within basement membranesin vitro and in vivo Our studies on the control of cell proliferation and tumor progression by its local environment focus on the interaction of cells with the ECM that is produced by cultured corneal and vascular EC9. This ECM closely resembles the subendothelium in vivo in its morphologica! appearance and molecular composition 9. We have demonstrated that EC and other cell types plated in contact with the subendothelial ECM produced in vitro, no longer require the addition of soluble bFGF in order to proliferate and express their differentiated functions. They do, however, deposit bFGF into their ECM~". Likewise, cultured cardiac myocytes were shown to deposit aFGF into their ECM but not conditioned medium~L In subsequent studies, bFGF was extracted from the subendothelial ECM produced in vitro ~j and from basement membranes of the cornea r', suggesting that ECM may serve as a reservoir for
© 1991, Elsevier Science Publishers, (UK) 037~-5067/91/$02.00