- Email: [email protected]
Protein prenylation: a modification that sticks
SCOTT POWERS
POST-TRANSLATIONALMODIFICATION
Protein prenylation: a modification that sticks A seemingly obscure post-translational modification is turning out to be not only widespread, but crucial to protein localization and function. In 1979, Kamitya e t aL reported that a secreted mating peptide from the basidiomycetous yeast R h o d o s p o r i d i u m t o r u l o i d e s contained a carboxy-terminal S-farnesyl cysteine [1]. What seemed at the time an anomalous post-translational modification is today known to be a relatively widespread modification to a large group of eukaryotic proteins that terminate with special cysteinecontaining sequences. Modification of eukaryotic proteins by the covalent attachment of isoprenoid lipid (prenylation) is now thought to to be of critical importance for a variety of proteins involved in oncogenesis, secretion, nuclear structure, and signal transduction. Protein prenylation, unlike other post-translational modifications such as phosphorylation, is a stable modification that in many cases is absolutely essential for the protein's correct subcellular localization and function. Farnesyl, a 15-carbon lipid that is derived from the cholesterol precursor mevalonate, is not the only iso prenoid lipid that can be attached to prenylated proteins: geranylgeranyl, famesyl's 20-carbon relative, also serves as a lipid donor in protein prenylation, and in fact may be the predominant choice of donor [2]. In either case, the isoprenoid lipid is linked to the protein's carboxyterminal cysteine residue via a stable thioether bond; and so far it seems that only one isoprenoid is linked to any particular protein. On the basis of investigations of individual proteins discussed below, the stable modification of proteins by
Protein Ras2(yeast) RaplA (human) Transducin, y-subunit (bovine) Go,Gi,Gs, y-subunit (bovine brain) Nuclear lamin A (human) cGMP phosphodiesterase, 0~'-subunit Yptl (yeast) Rab2 (human) Sec4 (yeast)
114
prenylation is very important for their function and is a necessary step in their targeting to specific subcellular membranes. Proteins that are known or likely to be prenylated include nuclear lamins, r a s and ras-like proteins, G-proteins, cGMP phosphodiesterase and certain secreted peptides (Table 1). Much of what we know about the effects of prenylation is based on studies of the proto-oncogenic ras proteins and the nuclear lamins A and B, which are targeted to the plasma membrane and to the nuclear envelope, respectively. In both cases, these proteins are modified by farnesylation of the cysteine contained within the carboxyterminal sequence CAAX [3-5], where the cysteine (C) is followed by two aliphatic (A) amino acids (valine, leucine or isoleucine) and then any amino acid (X) at the carboxy terminus (Fig. 1). Preventing the famesylation of r a s proteins by any of several different methods results in the accumulation of cytoplasmic precursor forms of the protein that are severely dampened in their ability to influence cell growth, presumably because they now fail to interact with target proteins in the plasma membrane [3,4]. Furthermore, prenylation is probably necessary for the two other carboxy-terminal processing events that occur with r a s proteins: removal of the three amino acids following the carboxy-terminal cysteine, and its carboxymethylation [6]. These two events seem to occur with all proteins that contain the CAAX sequence, but their functional significance remains unclear. In the case of nuclear lamins, blocking prenylation by mutagenesis
Carboxy-terminal sequence G S GG CC PK K K SC E L K G GC E K K F FC Q S P Q NC K K SK T C N TGG G C Q AGGG C S S K SN C
I I S LLL V I S A I L SI M LM L C C C
Prenylation Farnesyl Geranylgeranyl Farnesyl Geranylgeranyl Farnesyl ? ? ? ?
©
1991 C u r r e n t B i o l o g y
of the CAAX sequence precludes the mutant lamin from associating with the membranous nuclear envelope [5]. Thus, for both ras proteins and lamins, carboxy-terminal prenylation is a necessary step for their localization to target membranes. Somewhat surprisingly, the mechanistically simple notion that the lipid moiety is solely responsible for targeting prenylated proteins to membranes does not seem to be true, as other portions of both ras proteins and lamins are absolutely required in conjunction with the prenylated carboxy-termini for membrane targeting. For ras proteins, sequences immediately preceding the CAAX sequence are required for plasma membrane association; in their absence, the mutant prenylated ras proteins remain largely soluble and dispersed throughout the cytoplasm [3]. For nuclear lamins, alteration of the coiled-coil motif results in a mutant prenylated protein that is dispersed throughout the nucleoplasm [5]. The coiled-coil motif of lamins, also found in intermediate filaments, is very likely to be involved in protein-protein interactions. The exact function of the membrane targeting sequences preceding CAAX in ras proteins is not clear; but for some ras proteins these sites contain cysteines that can be palmitoylated [3,4]. Nuclear lamins also contain a nuclear localization sequence that directs the protein into the nucleus independently of the CAAX sequence and coiledcoil motif. Thus, the precise membrane localization of prenylated proteins can be dependent on numerous targeting sequences. CAAX may not be the only carboxy-terminal sequence that designates proteins for prenylation. Recent information suggests that the requirement for two aliphatic (A) amino acids within CAAX is not absolute and that the terminal amino acid X plays an important part in designating which isoprene donor (famesyl or geranylgeranyl) becomes attached [7]. It is suspected but not yet proven that CC is also a carboxy-terminal prenylation designation sequence. CC is found at the carboxy-termini of the large subfamily of ras-like proteins that are essential components of the endoplasmic reticulum/Golgi secretory apparatus, such as the yeast proteins Yptl and Sec4. The list of proteins that contain CAAX or CC carboxy-terminal sequences now numbers well over 50 and is rapidly expanding as more and more protein sequences are determined (Table 1).The most compelling evidence that these sequences actually designate proteins for prenylation comes from experiments where CAAX was added onto the carboxy-terminus of a normally cytoplasmic protein, resulting in a prenylated protein with increased hydrophobic properties [3]. Little is yet known of the enzymes responsible for prenylating proteins. How many protein prenyltransferases are there and what is their specificity in terms of lipid donor and peptide recognition sequence? When and where do they act to prenylate proteins? Recently, a protein prenyltransferase that can transfer farnesyl onto ras proteins was purified to near homogeneity [8]. Amazingly, tetramefic CAAX peptides of various compositions can competitively inhibit the farnesylation of ras proteins by this soluble enzyme, demonstrat-
Volume 1
Number 2
1991
Protein with CAAX sequence
:
FarnesylX~added
MethylatiOn of carb0xy- ferminus
Farnesyl 8roup anchors protein to plasma membrane
Fig. 1. Protein farnesylation leads to membrane localization.
115
ing that the CAAX s e q u e n c e itself is the m a i n d e t e r m i n a n t in r e c o g n i t i o n b y the e n z y m e [8]. Similar activity has b e e n d e t e c t e d in o t h e r eukaryotic ceils a n d two yeast g e n e s that g o v e r n the e n z y m e activity res p o n s i b l e for f a m e s y l a t i o n o f r a s p r o t e i n s have b e e n identified, R A M 1 / D P R 1 a n d R A M 2 [9]. O n the basis o f the ability o f bacterially p r o d u c e d R A M I / D P R 1 p r o t e i n to reconstitute famesyltransferase activity in r a m 1 yeast extracts, it is likely that this g e n e p r o d u c t is a structural s u b u n i t o f the e n z y m e [10]. Yeast p r o t e i n s e q u e n c e s with h o m o l o g y to R A M 1 / D P R 1 have b e e n f o u n d a n d m a y e n c o d e structural c o m p o n e n t s o f o t h e r p r o t e i n prenyltransferases [11]. T h e n u m b e r , structural c o m p o s i t i o n , a n d specificity o f p r o t e i n prenyltransferases is b e i n g actively p u r s u e d b y several laboratories a n d w e c a n e x p e c t solid a n s w e r s to s o m e basic q u e s t i o n s c o n c e r n i n g these e n z y m e s i n the n e a r future.
References 1. KAMIYAY, SAKUP4dA, TAMURAS, TAKAHASHIN, TSUCHIYAT, ABE K, FUKUI S: Structure of Rhodotorucine A, a peptidal factor inducing mating tube formation in R h o d o s p o r i d i u m toruloides. Agric Biol Chem 1979, 43:363-369. 2. FARNSWORTHCC, GELB MH, GLOMSETJ r Identification of geranylgeranyl-modified proteins in HeLa cells. Science 1990, 247:320-322. 3. HANCOCKJF, MAGEEAI, CHILDSJE, MARSHALLCJ: All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 1989, 57:1167-1177. 4. CASEYPJ, SOKSKIPA, DER CJ, Buss JE: p21 ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci USA 1989, 86:8323-8327.
116
5. HOLTZD, TANAKARA, HARTWIGJ, McKEONF: The CAAX motif of lamin A functions in conjunction with the nuclear localization signal to target assembly to the nuclear envelope. Cell 1989, 59:969-977. 6. GuTIERREZL, MAGEEAI, MARSHALLCJ, HANCOCKJF: Post-translational processing of p21 ras is two-step and involves carboxymethylation and carboxy-terminal proteolysis. EMBO J 1989, 8:1093-1098. 7. REISSY, STRADLEYSJ, GIERASCHLM, BROWNMS, GOLDSTEINJL: Sequence requirement for peptide recognition by rat brain p21 ras protein farnesyltransferase. Proc Natl Acad USA 1991, 88:732-736. 8. REISSY, GOLDSTEINJL, SEABRAMC, CASEYPJ, BROWNMS: Inhibition of purified p21 ras farnesyl:protein transferase by CysAAX tetrapeptides. Cell 1990, 62:81-88. 9. GOODMANLE, JUDD SR, FARNSWORTHCC, POWERS8, GELB MH, GLOMSETJA, TAMANOIF: Mutants of Saccharomyces cerevisiae defective in the farnesylation of ras proteins. Proc Natl Acad Sci USA 1990, 87:9665-9669. 10. SCHAFERWR, TRUEBLOODCE, YANGC, MAYERMP, ROSENBERGS, POULTERCD, KIMSH, RINEJ: Enzymatic coupling of cholesterol intermediates to a mating pheromone precursor and to the Ras protein. Science 1990, 249:1133-1139. 11. PETERSEN-BJORNS, HARRINGTONTR, FRIESENJD: An essential gene in Saccharomyces cerevisiae shares an upstream regulatory element with PRP4. Yeast 1990, 6:345-352.
Scott Powers, D e p a r t m e n t o f Biochemistry, University o f Dentistry a n d Medicine o f N e w Jersey, R o b e r t W o o d J o h n s o n Medical School, Piscataway, N e w Jersey 08854, USA.
© 1991 Current Biology
POST-TRANSLATIONALMODIFICATION
Protein prenylation: a modification that sticks A seemingly obscure post-translational modification is turning out to be not only widespread, but crucial to protein localization and function. In 1979, Kamitya e t aL reported that a secreted mating peptide from the basidiomycetous yeast R h o d o s p o r i d i u m t o r u l o i d e s contained a carboxy-terminal S-farnesyl cysteine [1]. What seemed at the time an anomalous post-translational modification is today known to be a relatively widespread modification to a large group of eukaryotic proteins that terminate with special cysteinecontaining sequences. Modification of eukaryotic proteins by the covalent attachment of isoprenoid lipid (prenylation) is now thought to to be of critical importance for a variety of proteins involved in oncogenesis, secretion, nuclear structure, and signal transduction. Protein prenylation, unlike other post-translational modifications such as phosphorylation, is a stable modification that in many cases is absolutely essential for the protein's correct subcellular localization and function. Farnesyl, a 15-carbon lipid that is derived from the cholesterol precursor mevalonate, is not the only iso prenoid lipid that can be attached to prenylated proteins: geranylgeranyl, famesyl's 20-carbon relative, also serves as a lipid donor in protein prenylation, and in fact may be the predominant choice of donor [2]. In either case, the isoprenoid lipid is linked to the protein's carboxyterminal cysteine residue via a stable thioether bond; and so far it seems that only one isoprenoid is linked to any particular protein. On the basis of investigations of individual proteins discussed below, the stable modification of proteins by
Protein Ras2(yeast) RaplA (human) Transducin, y-subunit (bovine) Go,Gi,Gs, y-subunit (bovine brain) Nuclear lamin A (human) cGMP phosphodiesterase, 0~'-subunit Yptl (yeast) Rab2 (human) Sec4 (yeast)
114
prenylation is very important for their function and is a necessary step in their targeting to specific subcellular membranes. Proteins that are known or likely to be prenylated include nuclear lamins, r a s and ras-like proteins, G-proteins, cGMP phosphodiesterase and certain secreted peptides (Table 1). Much of what we know about the effects of prenylation is based on studies of the proto-oncogenic ras proteins and the nuclear lamins A and B, which are targeted to the plasma membrane and to the nuclear envelope, respectively. In both cases, these proteins are modified by farnesylation of the cysteine contained within the carboxyterminal sequence CAAX [3-5], where the cysteine (C) is followed by two aliphatic (A) amino acids (valine, leucine or isoleucine) and then any amino acid (X) at the carboxy terminus (Fig. 1). Preventing the famesylation of r a s proteins by any of several different methods results in the accumulation of cytoplasmic precursor forms of the protein that are severely dampened in their ability to influence cell growth, presumably because they now fail to interact with target proteins in the plasma membrane [3,4]. Furthermore, prenylation is probably necessary for the two other carboxy-terminal processing events that occur with r a s proteins: removal of the three amino acids following the carboxy-terminal cysteine, and its carboxymethylation [6]. These two events seem to occur with all proteins that contain the CAAX sequence, but their functional significance remains unclear. In the case of nuclear lamins, blocking prenylation by mutagenesis
Carboxy-terminal sequence G S GG CC PK K K SC E L K G GC E K K F FC Q S P Q NC K K SK T C N TGG G C Q AGGG C S S K SN C
I I S LLL V I S A I L SI M LM L C C C
Prenylation Farnesyl Geranylgeranyl Farnesyl Geranylgeranyl Farnesyl ? ? ? ?
©
1991 C u r r e n t B i o l o g y
of the CAAX sequence precludes the mutant lamin from associating with the membranous nuclear envelope [5]. Thus, for both ras proteins and lamins, carboxy-terminal prenylation is a necessary step for their localization to target membranes. Somewhat surprisingly, the mechanistically simple notion that the lipid moiety is solely responsible for targeting prenylated proteins to membranes does not seem to be true, as other portions of both ras proteins and lamins are absolutely required in conjunction with the prenylated carboxy-termini for membrane targeting. For ras proteins, sequences immediately preceding the CAAX sequence are required for plasma membrane association; in their absence, the mutant prenylated ras proteins remain largely soluble and dispersed throughout the cytoplasm [3]. For nuclear lamins, alteration of the coiled-coil motif results in a mutant prenylated protein that is dispersed throughout the nucleoplasm [5]. The coiled-coil motif of lamins, also found in intermediate filaments, is very likely to be involved in protein-protein interactions. The exact function of the membrane targeting sequences preceding CAAX in ras proteins is not clear; but for some ras proteins these sites contain cysteines that can be palmitoylated [3,4]. Nuclear lamins also contain a nuclear localization sequence that directs the protein into the nucleus independently of the CAAX sequence and coiledcoil motif. Thus, the precise membrane localization of prenylated proteins can be dependent on numerous targeting sequences. CAAX may not be the only carboxy-terminal sequence that designates proteins for prenylation. Recent information suggests that the requirement for two aliphatic (A) amino acids within CAAX is not absolute and that the terminal amino acid X plays an important part in designating which isoprene donor (famesyl or geranylgeranyl) becomes attached [7]. It is suspected but not yet proven that CC is also a carboxy-terminal prenylation designation sequence. CC is found at the carboxy-termini of the large subfamily of ras-like proteins that are essential components of the endoplasmic reticulum/Golgi secretory apparatus, such as the yeast proteins Yptl and Sec4. The list of proteins that contain CAAX or CC carboxy-terminal sequences now numbers well over 50 and is rapidly expanding as more and more protein sequences are determined (Table 1).The most compelling evidence that these sequences actually designate proteins for prenylation comes from experiments where CAAX was added onto the carboxy-terminus of a normally cytoplasmic protein, resulting in a prenylated protein with increased hydrophobic properties [3]. Little is yet known of the enzymes responsible for prenylating proteins. How many protein prenyltransferases are there and what is their specificity in terms of lipid donor and peptide recognition sequence? When and where do they act to prenylate proteins? Recently, a protein prenyltransferase that can transfer farnesyl onto ras proteins was purified to near homogeneity [8]. Amazingly, tetramefic CAAX peptides of various compositions can competitively inhibit the farnesylation of ras proteins by this soluble enzyme, demonstrat-
Volume 1
Number 2
1991
Protein with CAAX sequence
:
FarnesylX~added
MethylatiOn of carb0xy- ferminus
Farnesyl 8roup anchors protein to plasma membrane
Fig. 1. Protein farnesylation leads to membrane localization.
115
ing that the CAAX s e q u e n c e itself is the m a i n d e t e r m i n a n t in r e c o g n i t i o n b y the e n z y m e [8]. Similar activity has b e e n d e t e c t e d in o t h e r eukaryotic ceils a n d two yeast g e n e s that g o v e r n the e n z y m e activity res p o n s i b l e for f a m e s y l a t i o n o f r a s p r o t e i n s have b e e n identified, R A M 1 / D P R 1 a n d R A M 2 [9]. O n the basis o f the ability o f bacterially p r o d u c e d R A M I / D P R 1 p r o t e i n to reconstitute famesyltransferase activity in r a m 1 yeast extracts, it is likely that this g e n e p r o d u c t is a structural s u b u n i t o f the e n z y m e [10]. Yeast p r o t e i n s e q u e n c e s with h o m o l o g y to R A M 1 / D P R 1 have b e e n f o u n d a n d m a y e n c o d e structural c o m p o n e n t s o f o t h e r p r o t e i n prenyltransferases [11]. T h e n u m b e r , structural c o m p o s i t i o n , a n d specificity o f p r o t e i n prenyltransferases is b e i n g actively p u r s u e d b y several laboratories a n d w e c a n e x p e c t solid a n s w e r s to s o m e basic q u e s t i o n s c o n c e r n i n g these e n z y m e s i n the n e a r future.
References 1. KAMIYAY, SAKUP4dA, TAMURAS, TAKAHASHIN, TSUCHIYAT, ABE K, FUKUI S: Structure of Rhodotorucine A, a peptidal factor inducing mating tube formation in R h o d o s p o r i d i u m toruloides. Agric Biol Chem 1979, 43:363-369. 2. FARNSWORTHCC, GELB MH, GLOMSETJ r Identification of geranylgeranyl-modified proteins in HeLa cells. Science 1990, 247:320-322. 3. HANCOCKJF, MAGEEAI, CHILDSJE, MARSHALLCJ: All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 1989, 57:1167-1177. 4. CASEYPJ, SOKSKIPA, DER CJ, Buss JE: p21 ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci USA 1989, 86:8323-8327.
116
5. HOLTZD, TANAKARA, HARTWIGJ, McKEONF: The CAAX motif of lamin A functions in conjunction with the nuclear localization signal to target assembly to the nuclear envelope. Cell 1989, 59:969-977. 6. GuTIERREZL, MAGEEAI, MARSHALLCJ, HANCOCKJF: Post-translational processing of p21 ras is two-step and involves carboxymethylation and carboxy-terminal proteolysis. EMBO J 1989, 8:1093-1098. 7. REISSY, STRADLEYSJ, GIERASCHLM, BROWNMS, GOLDSTEINJL: Sequence requirement for peptide recognition by rat brain p21 ras protein farnesyltransferase. Proc Natl Acad USA 1991, 88:732-736. 8. REISSY, GOLDSTEINJL, SEABRAMC, CASEYPJ, BROWNMS: Inhibition of purified p21 ras farnesyl:protein transferase by CysAAX tetrapeptides. Cell 1990, 62:81-88. 9. GOODMANLE, JUDD SR, FARNSWORTHCC, POWERS8, GELB MH, GLOMSETJA, TAMANOIF: Mutants of Saccharomyces cerevisiae defective in the farnesylation of ras proteins. Proc Natl Acad Sci USA 1990, 87:9665-9669. 10. SCHAFERWR, TRUEBLOODCE, YANGC, MAYERMP, ROSENBERGS, POULTERCD, KIMSH, RINEJ: Enzymatic coupling of cholesterol intermediates to a mating pheromone precursor and to the Ras protein. Science 1990, 249:1133-1139. 11. PETERSEN-BJORNS, HARRINGTONTR, FRIESENJD: An essential gene in Saccharomyces cerevisiae shares an upstream regulatory element with PRP4. Yeast 1990, 6:345-352.
Scott Powers, D e p a r t m e n t o f Biochemistry, University o f Dentistry a n d Medicine o f N e w Jersey, R o b e r t W o o d J o h n s o n Medical School, Piscataway, N e w Jersey 08854, USA.
© 1991 Current Biology