posttranslational processing of the ras superfamily of small gtp-binding proteins

Biochimica et Biophysica Acta, 1155 (1993) 79-96 79 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-419X/93/$06.00

BBACAN 87261

Posttranslational processing of the ras superfamily of small GTP-binding proteins

Christopher M.H. Newman and Anthony I. Magee Laboratory of Eukaryotic Molecular Genetics, National Institute for Medical Research, Mill Hill, London (UK)

(Received 3 August 1992)

Contents

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

II.

III.

C-Terminal processing of ras proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 A. Elucidation of the processing pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 B. Sequence of processing events and importance for membrane binding . . . . . . . . . . . . . . . 83 C. Functional importance of membrane binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 D. Mechanism and specificity of membrane attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

C-Terminal processing of ras-related proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 A. Proteins with CAAX motif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 B. Proteins with a CC or CXC C-terminal motif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

IV.

V.

Enzymology of C-terminal modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 A. Prenylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 B. Proteolytic processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 C. Carboxyl-methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 D. Palmitoylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Other posttranslational modifications of ras-related proteins . . . . . . . . . . . . . . . . . . . . . . . . . 90 A. Myristoylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 B. ADP-ribosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 C. Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

I. Introduct ion

A c t i v a t e d ras g e n e s h a v e b e e n i d e n t i f i e d in up to

3 0 % o f all h u m a n t u m o u r s , i n c l u d i n g 9 0 % of p a n c r e -

a t ic a n d 5 0 % of co lon ic c a r c i n o m a s [1]. M a n y o f t h e s e

t u m o u r s a r e pa r t i cu l a r ly r e s i s t an t to c o n v e n t i o n a l t he r -

a p e u t i c r eg imes , a n d h e n c e the d e v e l o p m e n t o f a g e n t s

Correspondence to: A.I. Magee, Laboratory of Eukaryotic Molecular Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK.

w h i c h i n t e r f e r e spec i f ica l ly wi th ras f u n c t i o n has be-

c o m e a m a j o r goa l o f o n c o g e n e r e sea rch .

I t has b e e n k n o w n for s o m e t i m e t h a t t he o n c o g e n i c

p o t e n t i a l o f a c t i v a t e d ras p r o t e i n s is cr i t ica l ly d e p e n -

d e n t u p o n c o r r e c t l oca l i za t i on to t h e i n n e r su r f ace o f

t he p l a s m a m e m b r a n e [2-4] , a l t h o u g h ras p r o t e i n s do no t c o n t a i n any pa r t i cu l a r l y h y d r o p h o b i c s e q u e n c e s

w h i c h cou ld m e d i a t e this i n t e r a c t i o n [5]. I t is n o w c l ea r

t ha t m e m b r a n e a s soc i a t i on is a c h i e v e d ind i rec t ly

t h r o u g h a c o m p l e x se r ies o f p o s t t r a n s l a t i o n a l m o d i f i c a -

t ions . T h e i d e n t i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f t h e

e n z y m e s tha t ca ta lyze t h e s e r eac t ions , as a s t a r t ing

80

point for the development of potentially therapeutic inhibitors, has become the focus of intense research interest [6].

The ras proteins form part of a much larger group of related proteins, the ras superfamily, which currently comprises more than 50 small (molecular mass 20-29 kDa) monomeric GTP-binding proteins, all of which show clear sequence homology to the products of the mammalian ras genes [7,8]. Members of the superfamily are involved in many cellular functions, including growth regulation and differentiation (ras), intracellular trafficking (rab and arf) and cytoskeletal control (rho and rac) [7,9]. Five different subfamilies have been defined on the basis of structural rather than functional similarity [8], with the three largest of these (the ras, rab and rho subfamilies) being particularly closely related. In general, members of different subfamilies share about 30% sequence identity, whereas the figure for two members of the same subfamily is at least 40% and is often much higher.

All members of the superfamily function via a switching cycle of GTP-binding and hydrolysis [10,11]. Thus, an 'on' or 'active' GTP-bound form is generated from an 'off' or 'inactive' GDP-bound form by guanine-nucleotide exchange. A number of proteins which regulate this cycle have now been identified, of which some are highly specific for individual members of the superfamily whereas others show a more relaxed specificity, at least in vitro [12-14]. Such proteins fall into two main groups, namely the GTPase-activating proteins (GAPs) which enhance the intrinsic GTPase activity of ras superfamily members and therefore inacti- vate the protein, and GTP-exchange proteins (GEPs), which modulate the rate of guanine-nucleotide binding. This latter group is further divided into GDP dissociation inhibitors (GDI), which inhibit the release of GDP and thereby trap the protein in the inactive conformation, and G D P / G T P dissociation stimulators (GDS) which stimulate the release of both GDP and GTP. Since the concentration of GTP within the cell is approximately an order of magnitude higher than that of GDP, GDS action increases the proportion in the GTP-bound active conformation. A number of these regulatory proteins may also form upstream or downstream elements within the functional pathways which involve ras superfamily members [9,14-17].

Although the roles of the ras superfamily members are diverse, most of them must be associated with the cytoplasmic face of specific cellular membranes in order to function. As will be described in detail below, targeting to these specialized sites is achieved by coop- eration between at least two distinct regions of the proteins. The first is usually at the extreme C-terminus where a series of lipid modifications increases the hydrophobicity of the proteins and thus their membrane binding affinity. Secondly, a region of protein

sequence just upstream of the C-terminus (the hypervariable domain) apparently encodes the determinants of targeting to the correct destination, presumably through interaction with membrane lipid head groups a n d / o r membrane proteins.

II. C-Terminal processing of ras proteins

Ras proteins were first identified as the transforming principles of the Harvey (HaSV) and Kirsten (KiSV) rat sarcoma viruses, which were generated by passage of Harvey and Kirsten murine leukaemia viruses through rats. Three cellular ras genes have been identified in mammals, called N-, Harvey (H-) and Kirsten (K-) which code for highly homologous proteins of approx. 21 kDa (collectively known as p21 ras) that are thought to play a central role in growth control [5,9]. The K-ras gene can code for two products differing only in their C-terminal regions by virtue of splicing to two alternative fourth exons (4A and 4B). Homologues of the mammalian ras genes have been identified in all species so far tested, including the RAS1 and R A S 2

genes of Saccharomyces cerevisiae [18]. The mammalian ras genes yield proteins which are

85% homologous to one another, particularly within four domains which are involved in guanine-nucleotide binding and hydrolysis [5,7,9]. Appreciable divergence between the three sequences is restricted to a 'hypervariable' domain near the C-terminus [5]. Residues 32-40 are conserved in all mammalian p21 ras proteins and form part of an exposed loop on the surface of the protein [19,20]. This region has been termed the 'effector domain' since mutagenic studies suggested that this domain represents the site of interaction with downstream elements of the ras signalling pathway [21,22]. Subsequent experiments have shown that the same residues are necessary for interaction with ras-GAP [23-25] and probably NF1 [26], though whether one or both of these molecules represent downstream targets of p21 ras remains a subject of much debate [9,15,16].

ras genes containing mutations at codon 12, 13 or 61 are frequently detected in human tumours [1]. These activating mutations yield p21TM proteins which are constitutively locked in the active GTP-bound state due to a reduction in intrinsic GTPase activity and their failure to respond to binding of ras-GAP a n d / o r other proteins with GAP activity, such as the product (neurofibromin) of the neurofibromatosis gene, NF1.

Mutations at sites of interaction with the guanine base (residues 116, 117, 119 and 146) decrease the affinity of the mutant p21 r~s for nucleotide and hence favour the exchange of bound GDP for cytosolic GTP. Such 'exchange' mutants cause morphological transformation in vitro. Mutations at position 146 have been detected in several human tumours.

81

II-A. Elucidation of the processing pathway

As mentioned above, the primary structure of p21 ras is not suggestive of a direct membrane binding, and yet an early study by Willingham et al. showed that 95% of p21 v-Hras expressed in MDCK cells was localized to the inner surface of the plasma membrane [2]. The same group then noticed that newly synthesized p21 vHra~ was found in the cytosolic supernatant fraction (S100) following subcellular fractionation and that subsequent association with the membrane pellet fraction (P100), was accompanied by an increase in electrophoretic mobility on SDS-polyacrylamide gels [27]. This observation raised the possibility that the ability to bind to membranes is not an inherent property of p21 ras but is achieved only following posttranslational modification. Furthermore, the results of formic acid and V8 proteinase cleavage studies suggested that the modification(s) responsible for the change in migration rate occurred within the C-terminal half of the protein [27].

Around the same time, several proteins, in particular the human transferrin receptor [28] and the membrane glycoproteins of VSV and Sindbis viruses [29], were shown to contain covalently bound fatty acid, a substituent which could convert an otherwise soluble protein into one which will bind more readily to membranes. Sefton et al. incubated HaSV-transformed human A431 cells with [3H]palmitic acid and demonstrated incorporation of label into the membrane-associated but not the cytosolic form of p21 v-n . . . . [30]. This incorporated label was sensitive to treatment with mild alkali or hydroxylamine, compatible with a thioester linkage to the protein rather than incorporation of catabolized fatty acid into the protein backbone. It was therefore suggested that the posttranslational addition of fatty acid (acylation) could be the means by which p21 r~ acquires an affinity for membranes. Willumsen et al. then demonstrated that the C-terminus, and in particular the highly conserved Cys-186 residue (Table I), is essential for the acylation, membrane-association and transforming potential of p21 v-H-ro~ [3,4]. Indepen- dent confirmation that palmitic acid is attached to p21 r~ proteins was provided by Buss and Sefton [31]. As a result of these studies, the hypothesis that the attachment of palmitic acid to Cys-186 of p21 ras proteins mediated their attachment to the plasma membrane gained widespread support.

The next two years saw the publication of a number of reports whose results suggested that the post-trans- lational processing of ras proteins may be more complex than bad previously been imagined. Tamanoi and his colleagues reported that the primary translation products of both the yeast RAS1 and RAS2 genes and the mammalian H-ras gene over-expressed in S. cerevisiae were first detected in the cytosolic S100 fraction

TABLE I

C-Terminal sequences o f some members o f the ras superfamily

All sequences are human unless otherwise indicated. Underl ined cysteine residues represent confirmed a n d / o r potential palmitoylation sites.

H-ras D E S G P G C M S C K C V L S N-ras D D G T Q G C M G L P C V V M K(A)-ras E K T P G C V K I K K C I I M K(B)-ras G K K K K K K S K T K C V I M R A S l ( S . cereeisiae) A N A R K E S S G G C C I I S RAS2(S. cerecisiae) S E A S K S G S G G C C I I S

r ap lA a P V E K K K P K K K S C L L L rap lB b P V P G K A R K K S S C Q L L rap2A Q P D K D D P C C S A C N I Q rap2B Q S N G D E G C C S A C V I L

rhoA L Q A R R G K K K S G C L V L rhoB R Y G S Q N G C I N C C K V L rhoC L Q V R K N K R R R G C P I L

racl C P P P V K K R K R K C L L L rac2 C P Q P T R Q Q K R A C S L L

ralA R K S L A K R I R E R C C I L ralB S S K N K K S F K E R C C L L

G25K(placenta) L E P P E P K K S R R C V L L G25K(brain) L E P P E T Q P K R K C C I F

r ab lA K I Q S T P V K Q S G G G C C rab lB( ra t ) K I D S T P V K S A S G G C C r ab2( ra t / can ine ) H G G N Q G G Q Q A G G G C C rab9(canine) T V S L H R K P K P S S S C C rab l0(can ine) I S S G G G V T G W K S K C C

Y P T l ( S . cerevisiae) K G Q S L T N V N T G G G C C SEC4(S. cerevisiae) S I N S G S G N S S K S N C C

Y P T l ( S . pombe) V G Q G T N V S Q S S S N C C YPT3(S. pombe) T M N D L N K K K S S S Q C C

rab3AC Q L S D Q Q V P P H Q D C A C rab3BC R L S D T P P L L Q Q N C S C rab4 S P R R T Q A P N A Q E C G C rab6 E K P Q E Q P V S E G G C S C rab7(canine) D K N D R A K T S A E S 12 S C ram (rat) T D Q L S E E K E K G L C G 12

YPT5(S. pombe) S E A R P A A Q P S G S C S C

a Also known as smg b Also known as smg c Also known as smg

p21A/K-rev l . p21B. p 2 5 A / s m g p25B.

but were then converted to a form that migrated slightly faster on SDS-polyacrylamide gels whilst still remain- ing in the S100 fraction [32,33]. These partially processed forms were then further modified by fatty acid acylation, as assessed by incorporation of label from [3H]palmitic acid, which was associated with binding to cellular membranes but little or no further shift in electrophoretic mobility. Similar results were obtained when studying the posttranslational processing of normal p21TM .. . . overexpressed in a mouse fibroblast cell

35 line. In pulse-chase experiments with [ S]methionine,

82

newly synthesized p21Nras chased rapidly into two faster migrating forms [34]. Only the fastest migrating of the three forms was labelled by [3H]palmitic acid and was localized to the membrane P100 fraction. Furthermore, hydroxylamine treatment of the P100 fraction released bound p21N . . . . into the supernatant and caused a slight decrease in the mobility of the protein [34]. Whilst all these data still supported the hypothesis that acylation of ras proteins is important for membrane-binding, they also gave the first indica- tion that acylation may be preceded by a modification step which affects electrophoretic mobility but is not sufficient for stable membrane association.

In 1986, Powers et al. isolated a S. cerevisiae mutant which suppressed the action of a transforming ras gene [35]. It was found that ras protein in these cells was not acylated and failed to associate with membranes. Inter- estingly, the same mutant ceils failed to secrete the a- mating factor. The gene responsible for this phenotype was named RAM (for RAS and a-factor maturation function). The common element between the a-factor and ras proteins appeared to be the presence of a conserved cysteine residue (Cys-186 in mammalian p21 ras proteins) within the C-terminal sequence Cys-A- A-X (A, aliphatic; X, any amino acid), a motif which has subsequently become known as the 'CAAX box' [36] (Table I). Whilst these data suggested that the RAM gene product might encode a fatty acyltrans- ferase responsible for palmitoylation of ras and a-factor, Powers et al. did not exclude the possibility that RAM encodes a protein required to 'prepare' ras and other proteins for acylation. Tamanoi's group also isolated a S. cerevisiae mutant in which ras processing was almost completely abolished, which they named dprl (defective in the processing of ras) [37]. Both yeast RAS and mammalian p21H-ras expressed in dprl cells remained as precursors and accumulated in the cytoplasm, showing no evidence of the increased electrophoretic mobility seen in wild-type cells. Genetic analyses as well as cloning of the gene indicated that DPR1 is allelic with RAM [33,38], suggesting that the D P R 1 / R A M gene in fact codes for a protein involved in a modification step which precedes fatty acylation.

Possible candidates for the processing step(s) which precede fatty acylation of ras proteins were suggested by the known biology of other proteins and peptides which terminate with a CAAX motif. For example, the cDNA sequence of the S. cerevisiae a-factor terminates in the sequence Cys-Val-Ile-Ala [39], but amino-acid sequencing of mature secreted a-factor indicated a cysteine residue at the extreme C-terminus [40], suggesting that the last three amino acids had been removed post-translationally. Furthermore this cysteine residue was shown to be S-alkylated by the fifteen carbon isoprenoid group, farnesol, an intermediate in the biosynthetic pathway for cholesterol [41,42], and

also carboxyl-methylated [40]. Identical modifications of the C-terminal cysteine residue of the mating factors of basidiomycetous fungi had been documented almost a decade earlier [43-45], but the presence of a CAAX motif in their primary sequence was only revealed after cloning of the genes involved [46].

The first evidence that mammalian CAAX proteins may also be modified by isoprenoid groups in vivo came from the demonstration that HeLa and CHO cells incorporated label from the isoprenoid precursor [3H]mevalonic acid into a number of proteins, including lamin B, which terminates with the sequence Cys- Ala-Ile-Met [47,48]. A number of mutually complemen- tary papers then reported that mammalian ras proteins are also modified in an analogous fashion to yeast and fungal mating factors. Clarke et al. demonstrated that activated human p21Hras expressed in transformed rat embryo fibroblasts is carboxyl-methylated [49]. Gutier- rez et al. showed that p21TM . . . . over-expressed in mouse fibroblasts was rapidly converted from a precursor form (pro-p21) into a faster migrating form (c-p21) which, although still largely cytosolic, partitioned into the detergent-rich phase of Triton X-114, indicating the acquisition of increased hydrophobic properties compared to pro-p21 [50]. This form did not, however, incorporate label from [3H]palmitic acid. After a one- hour chase the c-p21 form had largely chased into the membrane P100 fraction (m-p21), where it co-migrated with [3H]palmitic-acid-labelled protein. Isoelectric fo- cusing of the same samples indicated charge differences between all three forms, again suggesting that the protein is modified in some way in at least two distinct steps. Replacement of Cys-186 by serine in p21 n-ras abolished both the initial mobility shift and increase in hydrophobicity as well as palmitoylation, an effect identical to that of the d p r l / r a m mutation in S. cerevisiae. Gutierrez et al. also showed that the conver- sion of pro-p21 to c-p21 is accompanied by cleavage of the AAX sequence and by carboxyl-methylation. Han- cock et al. extended these observations by showing that the c-p21 and m-p21 forms of p21H .... could be labelled with [3H]mevalonic acid, and that the Ser-186 mutation blocked both mevalonic acid-labelling and carboxyl-methylation as well as palmitoylation [51]. Casey et al. also demonstrated labelling of p21H-ras with [3H]mevalonic acid in vivo and were able to con- firm the identity of the attached group as all trans- farnesol by HPLC analysis of the cleaved label [52].

All these data strongly suggested that the Ser-186 mutation blocks palmitoylation of p21 ra~ indirectly by interfering with a processing step which mandatorily precedes acylation, most likely farnesylation of Cys-186. This hypothesis was further strengthened by the observation that p21K . . . . (B), whose only cysteine adjacent to the C-terminus is contained within the CAAX box (Table I), was not labelled by [3H]palmitic acid and yet

exhibited a comparable shift in mobility on SDS-polyacrylamide gels, chased into the detergent-rich phase of Triton X-114, could be labelled with [3H]mevalonic acid and was carboxyl-methylated [51]. All of these events could be blocked by the Ser-186 mutation [51]. Further mutagenic studies by Hancock et al. mapped the true palmitoylation sites within p21 nras to two adjacent cysteine residues (Cys-181 and Cys-184) within the C-terminal hypervariable domain [51].

It therefore appears that ras proteins are subject to a highly complex series of posttranslational modifications, beginning with a triplet of modifications (farnesylation, -AAX proteolysis and carboxyl-methylation) focused on the C-terminal CAAX motif, followed in some cases by palmitoylation of one or more adjacent cysteine residues within the C-terminal hypervariable domain. Recent data suggest that the RAS2 protein of S. cerevisiae is modified in an identical fashion [53,54].

II-B. Sequence of processing events and importance for membrane-binding

The abolition of p21 ras processing by the Ser-186 mutation indicates that a cysteine residue at this position is necessary to trigger the triplet of modifications of the CAAX motif but does not identify the order in which these events take place in vivo, in particular whether farnesylation precedes AAX proteolysis or vice-versa. Inhibition of endogenous mevalonate synthesis using the HMG-CoA reductase inhibitors com- pactin or mevinolin depletes mammalian cells of poly- isoprenoids and completely abolishes processing of ras proteins in vivo [41,42,51]. Furthermore, mevalonate starvation of S. cerevisiae mutants blocked in sterol biosynthesis due to mutations in HMG-CoA synthase or HMG-CoA reductase leads to the accumulation of unprocessed ras proteins in the cytosol [55]. These data strongly suggest that farnesylation of the cysteine residue within the CAAX sequence is the first modification to occur on mammalian and yeast ras proteins. As mentioned above, CAAX modification appears mandatorily to precede acylation.

The triplet of CAAX modifications appear to be very closely coupled. HMG CoA-reductase inhibitors block all ras modifications and ras-specific inhibitors of proteolysis and methylation are not yet available. It has therefore been difficult to assess the contribution of each CAAX modification to the membrane-binding of p21 ra~ proteins in vivo. By progressive reconstitution of CAAX processing in vitro, however, Hancock et al. demonstrated an approximately equivalent contribution of farnesylation, AAX proteolysis and carboxyl- methylation to membrane-binding of wild-type p21K-~as(B) [56]. Furthermore, RAS2 protein expressed in a S. cerevisiae ste14 null mutant, which lacks func-

83

tional ras methyltransferase activity, still chases from a precursor into a faster migrating form which, unlike RAS2 protein expressed in wild-type yeast, is not methylated and binds relatively poorly to membranes [57]. These latter data suggest that methylation con- tributes to the membrane-binding of processed RAS2 in wild-type S. cerevisiae, either directly and/or by an effect upon the efficiency of palmitoylation. Although both proteolysis and carboxyl-methylation contribute to in-vitro and in-vivo membrane-binding, the critical modification for function seems to be farnesylation since mutants defective in proteolytic processing and carboxyl-methylation are still partly membrane-bound and transforming [58].

Hancock et al. showed that p21Hras proteins which are fully CAAX-modified but mutationally blocked for palmitoylation (equivalent to the c-p21 form) associate very weakly with cellular membranes [51]. This finding raised an apparent paradox, since wild-type p21K . . . . (B) is also not palmitoylated and yet binds with high affinity to membranes. The p21Kras(B) protein contains a string of six consecutive lysine residues within the C-terminal hypervariable domain. Hancock et al. pos- tulated that interaction of these positively charged residues with polar lipid head groups could act as a functional alternative to palmitoylation as a 'second signal' for membrane localization. Progressive substitution of these lysines with uncharged glutamine residues resulted in the mutant p21Kras(B) proteins expressed in COS cells becoming increasingly localized to the S100 fraction despite normal CAAX-processing [59]. Replac- ing the same residues with another positively charged amino acid, arginine, yielded a mutant protein which was localized to the plasma membrane, indicating that the critical feature of the lysine-rich domain in wild- type p21K . . . . (B) (the 'polybasic domain') is its positive charge [60].

II-C. Functional importance of membrane-binding

Many studies have shown that activated p21 ras proteins which remain unprocessed due to additional mutations within the CAAX box are completely unable to induce morphological transformation of NIH3T3 fibroblasts in vitro and are found exclusively in the cytoplasm [3,4,51]. Furthermore, maturation of Xeno- pus laevis oocytes induced by an oncogenic human p21 n ... . protein can be inhibited by mevinolin or com- pactin [61]. In S. cerevisiae, strains containing RAS2 mutations analogous to the mammalian transforming alleles (e.g., Gly-19 to Val-19) are sensitive to heat and cold shock and lose viability upon starvation [62]. The isolation of mutants able to suppress this phenotype led to the discovery of the DPR1/RAM gene [35], which has subsequently been shown to code for one

84

subunit of the yeast farnesyl-transferase [63] (see below). This abnormal phenotype can also be suppressed by mevalonate limitation of strains auxotrophic for mevalonate [55] or by further mutation of the activated RAS2 protein to disrupt the normal CAAX sequence [64]. In all these studies RAS proteins were confined to the S100 cytoplasmic fraction.

There does not appear to be a simple correlation between the degree of membrane-binding of activated p21 ~os proteins and their transforming potential. Thus activated p21K .... (B) and p21H . . . . proteins which are further mutated to remove the polybasic domain or upstream palmitoylation sites, respectively, are both subject to normal CAAX-processing, associate weakly (< 10%) with membranes and yet have a significant transforming activity that is only 2-10-fold lower than that of the parent activated protein [51,59,60]. The most likely explanation for this relatively weak correlation is that cellular transformation by activated ras proteins is triggered in an all-or-none fashion once the concentration of activated ras in a particular membrane compartment, most likely the plasma membrane, reaches a critical but low level. This is in contrast to the situation with transformation induced by the over- expression of normal ras proteins, where the degree of morphological transformation is directly related to the level of p21TM expression [65]. The mechanism of membrane-attachment appears to be relatively unimportant, since the membrane-association and -transforming activity of mutant oncogenic ras proteins that are defective in CAAX-processing can be restored by the cova- lent addition of another fatty acid, myristate, to the N-terminus [66,67]. In addition, activated p21K .... (B) or p21H .... which are modified by C20 geranylgeranyl rather than C~5 farnesyl (see Section III) are fully transforming [60,69].

The role of membrane-binding in the physiological function of normal ras proteins is poorly understood. For example, a RAS2 protein which is blocked for CAAX-processing (RAS2 Ser319) and remains in the cytoplasm can nevertheless complement a rasl ras2

strain of S. cerevisiae when expressed at high levels [64]. This suggests that stable membrane-association is not a prerequisite for normal ras function, and that sufficient association with membrane components may be achieved by increasing the concentration of the protein throughout the cell. Indeed, reversible membrane-binding may play an important role in the regulation of normal ras activity. This idea stems from the observation that the palmitate attached to p21N-ras

exhibits a high rate of turnover ( t , = 20 min) compared to the protein itself (t~= 24 h)~[34]. It is therefore conceivable that palmitoylated ras proteins undergo repeated cycles of acylation/deacylation in vivo. In- deed, recent data from our laboratory suggest that the rate of turnover of palmitate attached to p21N .... is

modulated by the action of a dialysable factor present in fetal calf serum (J. de Bony, J. Childs and A.I. Magee, unpublished observations).

In contrast to activated p21 ras proteins, the cellular effects of normal p21TM proteins are critically dependent upon the mechanism of membrane-attachment. Thus, normal p21N .... becomes transforming when modified by myristic acid at its N-terminus [67]. In S. cereuisiae, a raplA protein that had been mutated to give it RAS-like properties could only complement a ras2 strain lacking RAS function when it was modified by C~5 farnesyl and not C20 geranylgeranyl [68]. Fur- thermore, normal p21H . . . . that is prenylated with C20 geranylgeranyl rather than the usual C15 farnesyl (see below) exhibits a dominant-negative phenotype when expressed at moderate levels in NIH3T3 cells [69]. It is tempting to speculate that these aberrant phenotypes are the result of stable rather than reversible membrane-binding. Whatever the mechanism, it is clear that differences between the behaviour of normal and oncogenic ras proteins could be of potential importance in the development of agents which specifically interfere with the membrane-targeting of these proteins.

II-D. Mechanism and specificity o f membrane attachment

Farnesylation, carboxyl-methylation and palmitoylation all increase the overall hydrophobicity of p21 ras proteins [70] and favour association with hydrophobic structures such as the lipid bilayers of cellular membranes. The positively charged polybasic region of p21K . . . . (B) also favours membrane-binding, possibly through interaction with negatively charged head groups. Nevertheless, p21 ras proteins are not equally distributed between the various endomembrane compartments in vivo, but are highly targeted to the cytoplasmic aspect of the plasma membrane. This observation suggests that the plasma membrane may contain specific receptors which recognize one or more of these modifications and/or the polybasic domain, possibly in conjunction with adjacent protein sequences. A number of candidate protein receptors for lamin B have been identified, though it is not yet clear whether the modified C-terminus is directly involved in the lamin B/receptor interaction [71]. Similarly, a 32-kDa plasma-membrane protein has been identified which recognizes the myristoylated N-terminus of the src oncogene product [72]. Interestingly, normal p21H .... which is modified by myristate at its N-terminus but is not CAAX modified is still localized to the plasma membrane [67].

Hancock et al. showed that chimeric proteins consisting of the C-terminal 17 amino acids of p21K . . . . (B) or the last 10 amino acids of p21H .... fused to a soluble

protein, protein A, were posttranslationally processed and localized to the P100 fraction when expressed in COS cells [51,60]. Immunofluorescence studies revealed that these chimeric proteins were localized to the plasma membrane when transiently expressed in MDCK cells. Furthermore, a p21Kra~B) protein lacking the polybasic domain and a non-palmitoylated p21 n . . . . protein were both shown to bind to membranes when the overall hydrophobicity was enhanced by CAAX modification with C20 geranylgeranyl rather than C15 farnesyl [60]. In contrast to the protein A chimeras, however, these proteins were not specifically targeted to the plasma membrane and were poorly transforming. Modification of p21Kras<B) with an intact polybasic domain by C20 geranylgeranyl did not disrupt targeting to the plasma membrane, suggesting that palmitoylation and the polybasic domain, but not the isoprenoid group, have effects upon the specificity as well as the avidity of membrane-binding.

It therefore appears that the CAAX box and the C-terminal hypervariable domain contain all the necessary signals for posttranslational modification and targeting of p21 ra~ proteins to the plasma membrane. Whether membrane proteins are involved in the recog- nition of these modifications and/or the polybasic sequence remains unclear, but the existence of specific receptors involved in the function of normal but not activated p21 ras could explain why the mechanism of membrane-attachment has a major influence on the cellular response to these proteins.

III. C-Terminal processing of ras-related proteins

Eukaryotic cells incorporate label from [3H]mevalonic acid into a number of proteins [73-75] other than p21 r~s. Indeed, quantitation of prenylated cysteines in mouse tissues suggests that 0.5-2% of all proteins are isoprenylated in vivo [76]. Recent studies, however, have demonstrated that 80-90% of these proteins are not modified by C15 farnesyl, as is the case for ras proteins, but by a Cz0 geranylgeranyl isoprenoid [77,78]. Nevertheless, a large number of [3H]mevalonic- acid-labelled proteins migrate with an apparent molecular weight within the range 20-30 kDa on SDS-polyacrylamide gels and have subsequently been shown to be members of the ras superfamily. Whilst these proteins contain cysteine residues at or near their carboxyl-termini, not all possess a typical CAAX box. In particular, most members of the rab subfamily terminate in a CC or CXC motif (Table I) and yet many have been shown to be modified by C20 geranylgeranyl in vitro and/or in vivo.

I l iA . Proteins with a CAAX motif

In 1989, Noda's group reported the isolation of a cDNA clone from a human fibroblast cDNA expres-

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sion library on the basis of its ability to suppress the transformed phenotype of p21V-Kras-transformed mouse NIH3T3 cells (hence the name Krev-1) [79]. Two other laboratories coincidentally isolated the same gene, Tavitian's group by screening a human cDNA library with the Drosophila Dras3 gene (they named the gene rap for ras-proximate) [80], and Takai's group by screening a bovine brain cDNA library with an oligo- nucleotide probe generated from a partial amino-acid sequence of an unidentified 21-kDa GTP-binding protein purified from bovine brain membranes (they called the gene smgp21 for small g protein) [81]. In fact, four r a p / s m g p 2 1 p r o t e i n s ( r a p l A / s m g p 2 1 A , raplB/smgp21B, rap2A and rap2B) have now been identified, all with a molecular mass of approx. 21 kDa, with Krevl being equivalent to raplA/smgp21A [79- 83]. The rap proteins share 50% amino-acid identity with mammalian p21 ras, including consensus sequences for guanine-nucleotide binding and GTPase activity, and terminate with an identifiable C A A sequence (Table I). Furthermore, the putative effector domain of the ras proteins is strictly conserved in the rapl proteins and rap2B, whereas that of the rap2A protein differs by one amino acid.

Kawata et al. demonstrated incorporation of label from [3H]mevalonolactone into smgp21B in human platelets [84]. By using GC/MS analysis of Raney nickel-treated smgp21B the attached isoprenoid was identified as all-trans-geranylgeranyl. Further analysis indicated that the C-terminal AAX sequence had been cleaved posttranslationally and suggested that the re- sultant C-terminal cysteine residue was at least partially carboxyl-methylated. Buss et al. then reported that raplA when overexpressed in insect cells is also modified by C20 geranylgeranyl, albeit inefficiently, and carboxyl-methylated [85]. Rap2A overexpressed in rat and human cell lines also incorporated label from [3H]mevalonic acid land exhibited shifts in mobility and hydrophobicity that were very similar to those observed with p21 ras proteins [86], though the chain length of the attached isoprenoid is not yet known. Rap2A also incorporated label from [3H]palmitic acid [86], and inspection of the cDNA sequence (Table I) reveals two potential acylation sites (Cys-176 and Cys-177) just upstream of the the CAAX box. Less is known about the posttranslational processing of rap2B, but the cDNA sequence contains both a CAAX box and potential acylation sites adjacent to the C-terminus and platelets incorporate label from [3H]mevalonic acid into a 21-kDa protein which is specifically immuno- precipitated by an antiserum raised against rap2B expressed in E. coli [83].

As with the p21 ras proteins, mature rap proteins are consistently isolated in the P100 fraction of eukaryotic ceils following subcellular fractionation [83-86]. Im- munofluorescence studies have shown that raplA is

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localized to the Golgi region [87] and that rap2A colocalizes with markers of the endoplasmic reticulum [86]. These observations are perhaps somewhat surpris- ing, given that raplA contains a polybasic region (Ta- ble I) and rap2A is palmitoylated [86], since these signals appear to target ras proteins to the plasma membrane. The polybasic domain of raplA differs from that of p21Kras(B) only in the insertion of a single proline residue within the string of six lysine residues, and the two potential acylation sites of rap2A (Cys-176 and Cys-177) are consecutive, unlike those of p21 n . . . .

(Cys-181 and Cys-184) (Table I). Whether these subtle differences, either alone or in conjunction with adjacent sequences, can account for this very different subcellular localization remains unclear. It is also possible that the C20 geranylgeranyl modification of rap proteins cooperates with the polybasic region and/or palmitoylation in a different way to the Ca5 farnesyl group attached to ras proteins, leading to altered subcellular targeting. The above mentioned observation that mutant ras proteins modified by C20 geranylgeranyl are still targeted to the plasma membrane, however, does not support this hypothesis [60].

The mechanism by which raplA suppresses transformation by activated p21 ras remains unclear. Because these proteins share an identical effector domain, it has been suggested that raplA acts by competing for regulatory or target proteins of p21 r~s. The difference between the biological activity of raplA and activated ras proteins does not seem to be a function of the chain length of the attached isoprenoid, since a mutant raplA protein modified by Ca5 farnesyl rather than C20 geranylgeranyl can still suppress transformation of NIH3T3 cells by activated p21 n-r~s and does not itself cause transformation [69]. Furthermore, Buss et al. showed that a chimeric protein consisting of the first 110 amino acids of an activated p21Hras fused to the C-terminal 74 amino acids of raplA still caused transformation of NIH3T3 cells [85]. The efficiency of transformation was an order of magnitude lower than that of the parent activated p21H-~s protein, however, and it is tempting to speculate that this reflects a failure of efficient localization to the plasma membrane.

A number of other ras superfamily members which terminate in a CAAX sequence have been shown to be modified by C20 geranylgeranyl. These include racl, rac2 and rhoA p21 (all probably involved in cytoskeletal control), G25K (the mammalian homologue of the CDC42 gene product in S. cereuisiae) and ralA, to which no function has as yet been ascribed [88-90]. In common with p21K-~as(B), raplA and raplB, these proteins also contain a number of basic residues adjacent to the C-terminus. Takai's group have shown that the post-translationally modified forms of raplA, rhoA p21 and rhoB p21 all bind to a variety of purified membranes in vitro [91-93]. These proteins also bind to

boiled or trypsinized membranes and to phosphatidyl- serine-linked Affi-Gel, suggesting that specific protein receptors are not necessary for membrane-binding. The ral proteins have been localized to the plasma membrane of bovine and rat kidney cortical cells [94], and both [3H]mevalonic-acid-labelled racl and rac2 overexpressed in COS cells are isolated in the P100 fraction following subcellular fractionation [95]. Recent evidence, however, indicates that a small GTP-binding protein, p21 rac, is found in the cytosol of quiescent human neutrophils, but that activation of the superox- ide-generating system may be accompanied by translocation of racl to the plasma membrane [96,97]. Maltese and Sheridan reported that mevinolin treatment of cultured murine erythroleukaemia (MEL) cells almost abolished membrane-binding of endogenous G25K, and yet [3H]mevalonolactone-labelled G25K protein was found primarily in the cytoplasm, even after prolonged labelling to approach steady state [98]. Furthermore, the distribution of membrane-binding of modified rho proteins appears to vary markedly between different cell types, since CAAX-modified rhoA has been purified from such diverse sources as bovine brain membranes [99], aortic smooth muscle cytosol [100] and adrenal gland cytosol [101].

These data suggest that the level of membrane-binding of ras-related proteins may be determined by one or more factors in addition to CAAX-modification plus a 'second signal' (the polybasic domain or palmitoylation). Takai's group has shown that smgp21 GDS is active on the post-translationally modified forms of smgp21A and -B, p21K .... (B) and rhoA p21, but not on their unprocessed counterparts [102]. Furthermore, the interaction of smgp21 GDS with smgp21B is inhibited by geranylgeranylated but not unmodified C-terminal peptides, suggesting that smgp21 GDS binds to the modified C-terminus of intact smg p21B [103]. Both rho p21 GDS and rho p21 GDI are also active only upon the post-translationally modified form of rhoA p21 [92]. Of particular interest is the finding that binding of smgp21 GDS to smgp21B or p21Kras(B) not only stimulates guanine nucleotide exchange but also inhibits binding to purified membranes in vitro and induces dissociation of pre-bound proteins from membranes [14]. Given that the binding of rho p21 GDI to rho Bp21 has the same effect [104], GEP-binding may account for the observed variations in membrane-association of some ras superfamily members in vivo. Indeed, it is possible that GEP-induced modulation of guanine nucleotide-binding and membrane localization could play a central role in the biological function of many ras-related proteins (for further discussion, see Ref. 14).

Of all the ras superfamily members which terminate in a CAAX motif, only the mammalian and yeast ras proteins have so far been shown to be farnesylated.

Closer examination of the C-Xl-X2-X 3 sequences of isoprenylated GTP-binding proteins reveals that a leucine residue commonly occupies the X 3 position of geranylgeranylated but not farnesylated proteins. A number of in-vitro studies have now attempted to define in detail the structural features which determine whether proteins are geranylgeranylated or farnesylated [105,106]. On the basis of currently available evidence it appears that modification by a specific isoprenoid is determined by the C-terminal X 3 residue, as suggested below.

Farnesylation:

Cys-X 1 X 2 X3

V S

I M L C

O

Geranylgeranylation: S F V I

I L L N N

Proteins in which X 3 = residues with small relatively hydrophilic side chains such as S,M are the major substrates for farnesylation whilst proteins in which X 3 = residues with large hydrophobic side chains, e.g., L, are the primary candidates for geranylgeranylation although exceptions to these rules do occur. Substitu- tions in the X 1 and the X z positions appear to influence isoprenylation efficiency but do not change the specificity of the reaction. Glycine or proline in the X~ position impair effective isoprenylation. Large hydrophobic residues are preferred in the X 2 position, whereas charged residues are inhibitory. The X 2 position also affects proteolytic processing [58]. This high degree of specificity suggested the existence of more than one prenyl-transferase. Indeed purified protein and/or C-terminal peptides from proteins which are known to be either geranylgeranylated or farnesylated have been used as substrates in order to separate and purify a number of different prenyl-transferase enzymes (see Section IV).

III-B. Proteins with a CC or CXC C-terminal motif

The rab subfamily currently comprises more than 20 related proteins, the majority of which terminate in either a CC or CXC motif. The first of these to be identified were the products of the S. cerevisiae genes YPT1 and SEC4, both of which terminate with a CC motif [107,108]. The mammalian rab genes were first isolated by screening a rat brain library with oligo- nucleotides corresponding to a six amino-acid sequence which is highly conserved amongst ras and ras-related proteins [109]. Current evidence suggests

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that rab subfamily members are involved in the control of vesicular trafficking [110], and individual members have been localized to highly specific compartments of the endomembrane system [111-115].

The first evidence that rab family proteins may be post-translationally processed came from the observations by Molenaar et al., who showed that YPTlp was evenly distributed between membrane and cytosol fractions following subcellular fractionation of S. cerevisiae and that the membrane-bound form incorporated label from [3H]palmitic acid [116]. Furthermore, palmitate labelling, membrane binding and biological function was abolished by deleting both C-terminal cysteine residues resulting in loss of viability. Interestingly, replacement of only a single cysteine residue did not cause lethality. Goud et al. then showed that Sec4p is synthesized as a hydrophilic soluble protein which rapidly chases into a membrane-bound form which partitions into the detergent-rich phase of Triton X-114, suggesting the acquisition of a hydrophobic modification [117]. Unlike YPTlp and some of the ras proteins, however, the membrane-bound form of Sec4p could not be labelled with [3H]palmitic acid. More recently, a number of studies have demonstrated that mammalian rab proteins terminating in a CC motif are modified by C20 geranylgeranyl, and that prenylation requires an intact CC motif [118-121].

rab subfamily members which terminate in a CXC motif also undergo posttranslational processing. Burstein and Macara reported that the mammalian rab3A/smg p25A protein was detectable in both cytosolic and membrane fractions of rat brain [122]. Fischer von Mollard et al. reported that both the soluble and membrane-bound forms of rab3A/smg p25A partitioned into the detergent phase of Triton X-114, in contrast to recombinant protein expressed in E. coli, which lacks all the enzymes responsible for processing of ras proteins [112]. A number of CXC proteins have now been shown to be geranylgeranylated in vitro and/or in vivo [119,120], and at least three groups have detected geranylgeranyl modification of purified rab3A [119,123,124]. Indeed, Farns- worth et al. used a combination of HPLC and mass spectrometry to show that the C-terminal region of rab3A contains two geranylgeranyl groups linked via thioether bonds to both cysteine residues of the CXC motif [124]. Whether all rab subfamily members are doubly prenylated remains unclear.

Whilst all the rab subfamily members so far studied appear to be modified by C20 geranylgeranyl, not all aspects of their posttranslational processing are identical. We have recently shown that three rab subfamily members of the fission yeast Schizosaccharomyces pombe, YPT1 (CC), YPT3 (CC) and YPT5 (CXC) are all modified by C20 geranylgeranyl in vitro and incorporate label from [3H]mevalonic acid in vivo [125]. The

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CXC protein YPT5, but neither of the two CC proteins, was also carboxyl-methylated. These data are in agreement with those of Farnsworth et al., who showed that the C-terminus of purified rab3A (a CXC protein) contains a methyl ester [124], and with those of Wei et al., who could not detect carboxyl-methylation of endogenous rab2p (a CC protein) in vivo [121]. One of the two S. pombe CC proteins, YPT3, also exhibited hydroxylamine-sensitive labelling with [3H]palmitic acid [125], as previously observed for the S. cerevisiae CC protein, YPT1 [116].

As for the p21 ras proteins, prenylation appears to be essential for membrane-binding of rab subfamily members. For example, a number of studies have shown that blockade of isoprenoid synthesis using an HMG- CoA reductase inhibitor results in the accumulation of non-isoprenylated rab proteins in the cytosol of tissue culture cells in vivo [120,121,125,126]. Furthermore, Araki et al. showed that modified rab3A purified from bovine brain but not unprocessed rab3A purified from E. coli bound to a variety of purified membranes in vitro [123]. Deletion of the CXC motif of rab3A abolished membrane-binding of the mutant protein expressed in CHO cells [126], and we have recently shown that serine substitution of both cysteine residues within the CXC motif of YPT5 completely abolishes both prenylation and membrane-association when the mutant protein is expressed in COS cells (C. Newman, T. Giannakouros, M. Craighead, J. Armstrong and A. Magee, unpublished observations). The functions of carboxyl-methylation and/or palmitoylation of rab subfamily members remain unclear, though Musha et al. have recently shown that methylation is not essential for membrane-binding of rab3A in vitro [127].

Whereas membrane-binding of p21 ras proteins is determined by posttranslational modification, plus in some cases an adjacent polybasic region, additional sequences further from the C-terminus are required for both overall membrane-association and subcellular targeting of rab subfamily members. Thus, Araki et al. showed that intact rab3A purified from bovine brain membranes, but not a C-terminal fragment, would bind to membranes in vitro [123]. Chavrier et al. showed that a hybrid protein containing the N-terminal 181 residues of rab5 fused to the C-terminal 34 residues of rab7 was detected in the late endosome fraction (rab7 is restricted to late endosomes while rab5 is associated with the plasma membrane and early endosomes) [128]. Furthermore, they demonstrated that a r a b protein from the exocytic pathway (rab2) could be targeted to an endocytic compartment by exchanging its C-terminal domain. It seems therefore that rab proteins interact through their C-terminal hypervariable domain with specific receptors on the target membrane or with cytosolic proteins (such as GEPs) mediating this targeting event.

IV. Enzymology of C-terminal modifications

IV-A. Prenyl transferases

Several lines of evidence suggest that CXIX2X 3 sequences are processed first by prenylation, followed by proteolysis and finally carboxyl-methylation. Preny- lation is performed by a single farnesyl transferase (FT) recognizing sequences where X 3 = S, M, A, C, Q and a geranylgeranyl transferase (GGTI) recognizing sequences where X 3 = L, F, I, N. These enzymes uti- lize as co-substrates the diphosphate derivatives of farnesol and geranylgeraniol respectively, which are soluble intermediates in the isoprenoid biosynthetic pathway leading to steroids, dolichols, vitamin A, and many other important cellular metabolites [41]. Other residues within the CXIX2X 3 sequence also affect activity. For example, large hydrophobic residues are preferred in the X 2 position while charged X 2 residues prevent modification. Similarly, glycine or proline in the X 1 position are inhibitory [105].

Farnesyl transferase has been purified from mammalian sources and characterized [129-132]. It is a zinc metalloenzyme [133] active on simple CX1X2X 3 peptide substrates and this has been used in its affinity purification. The enzyme is a heterodimer of a and /3 subunits of 49 and 46 kDa, respectively. The/3 subunit probably binds the protein substrate and is 37% identical to the product of the S. cerevisiae DPR1/RAM1 gene [35,38,63,134]. The role of the a subunit is less clear, but it is thought to be involved in binding the farnesyl diphosphate. A cDNA encoding this subunit from rat brain has been shown to encode a protein of 377 amino acids which appears to be the mammalian homologue of the S. cerevisiae RAM2 gene, known to be important in yeast RAS processing [135-138]. Both a and /3 subunits must be co-expressed in order to be stabilized and obtain activity [63,135,138].

Geranylgeranyl transferase I has been purified from bovine brain [139-141] and is also an a/3 heterodimer and a Zn 2+- and MgZ+-dependent metalloenzyme. The a subunit is identical to that of farnesyl transferase which may explain why the homologous RAM2 gene of S. cerevisiae is essential while the farnesyl transferase specific /3-subunit homologue RAM1 is not [137,140]. The /3-subunit of GGTI is probably the mammalian homologue of the CDC43/CAL1 gene, which is required for GGTI activity in S. cereuisiae. This gene encodes a protein homologous to the RAM1 gene [142] suggesting that there is a family of /3-subunit genes which confer protein substrate specificity on prenyl transferases.

rab C-terminal sequences (CC or CXC) are prenylated by at least one different geranylgeranyl transferase (GGTII). This enzyme does not work on simple peptide substrates, suggesting that extended upstream

sequences are required. It has been partially purified both from S. cerevisiae [105] and bovine brain cytosol [143]. The enzyme may be capable of geranylgeranylat- ing both cysteines in the CXC motif of rab3A/smg25A and also appears to be active on the CC motif of YPT1, although it is not clear whether both cysteines of this motif are prenylated in vivo or in vitro. The BET2 gene in S. cerevisiae is known to be involved in the processing of the YPT1 and SEC4 proteins (both CC motifs) and is homologous to /3-subunit genes, suggesting that it may encode the/3-subunit of GGTII [144]. This is supported by the observation that S. cerevisiae carrying a mutation in the BET2 gene are deficient in GGTII activity [137]. Recent reports [145,146] describe the purification of a mammalian rab-GGT activity, working on both rab3A (CXC) and rablA (CC) motifs, which contains three subunits. Two of these, of 60 and 38 kDa, may correspond to a and/3 subunits and form a dimer. The third subunit of 95 kD dissociates during purification and is required for enzyme activity. Peptide sequencing of this subunit reveals homology to the product of the human choroider- aemia gene which is itself homologous to rab3A-GDI [147]. rab3A-GDI binds to the prenylated tail of rab3A and forms a soluble complex, thus allowing its removal from membranes. The authors speculate that the 95- kDa subunit may perform an analogous role in strip- ping the hydrophobically modified product from the enzyme active site, thus allowing further rounds of catalysis. Interestingly, this GGT is inhibited by Zn 2÷, in contrast to FT and GGTI [145].

IV-B. Proteolytic processing

Following prenylation the ras-related proteins ac- quire hydrophobic properties which, depending on the specific prenyl group, result in some degree of affinity for membranes. This is much stronger in the case of geranylgeranyl than farnesyl, and is especially strong for doubly geranylgeranylated proteins such as rab3A [70]. Proteins with prenylated CXXX sequences are thus able to interact with membranes where they en- counter protease activities which remove the three X residues [50,56]. A recent report has detected three proteinase activities in S. cerevisiae, which can proteo- lytically process CXXX peptide substrates [148]. One of these is the vacuolar protease carboxypeptidase Y and is thus unlikely to be physiologically relevant due to its topological segregation from the appropriate substrates. A soluble metalloenzyme of approx. 110 kDa is a novel carboxypeptidase which can process both farnesylated and unfarnesylated CXXX substrates but is inactive on unrelated peptides. Another membrane bound protease activity is sensitive to sulphydryl reagents but not chelating agents. This is not an activity of the methyl-transferase enzyme since STE14 mu-

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tants still possess it. Either or both of these enzymes could be involved in the natural processing of CXXX- terminated prenyl proteins. Ashby et al. [149] have reported two cytosolic and one particulate protease activities from S. cerevisiae which can act on the CAAX sequence of a-factor. One of the cytosolic activities is probably carboxypeptidase Y and the second preferred the unfarnesylated substrate, therefore being unlikely to be the in-vivo proteinase. The particulate activity, and a similar activity found in rat liver membranes, endoproteolytically removed the last three amino acids as a tripeptide from farnesylated a-factor. This activity was not inhibited by unfarnesylated a-factor or a range of conventional proteinase inhibitors, but was inhibited by Zn 2÷. This unusual endoprotease is the best candidate for the authentic CAAX processing enzyme. An- other report describes an endoproteolytic activity in bovine liver microsomes which cleaves the XXX sequence from farnesylated peptide substrates [150]. The activity requires an L-cysteine and a free carboxyl group, although the number of X residues is unimportant, but does not require any sequence upstream of the cysteine residue. The relative contributions of these protease activities to processing of prenylated CXXX proteins remains to be determined.

IV-C. Carboxyl-methylation

Subsequent to proteolysis carboxyl-methylation of the C-terminal prenylated cysteine residue occurs, catalyzed by a membrane-bound carboxyl-methyl transferase utilizing S-adenosyl methionine [49,50,56]. Car- boxyl-methylation is not obligatorily coupled to proteolysis. In fact all that appears to be required is a C-terminal prenylated cysteine with a free carboxyl group. The nature of the prenyl group is not crucial although at least 15 carbons are required for efficient methylation [151,152]. The upstream sequences are also unimportant and very simple substrates, such as S-prenyl thiopropionic acid, can be methylated. A single enzyme activity seems to be active on both farnesylated and geranylgeranylated substrates [153,154] and probably acts on CXXX, CXC and CC proteins as long as their C-terminal cysteine is prenylated. The rod outer segment enzyme catalyses methyl transfer from S- adenosyl methionine using an ordered mechanism with S-adenosyl methionine binding first [155]. It is interest- ing that while all known CXC proteins appear to be doubly prenylated and methylated [124,125], at least some CC proteins are not methylated and therefore may not be prenylated on their C-terminal cysteine, but only on the upstream cysteine [121,125].

In S. cerevisiae a single gene STE14 encodes the methyl transferase responsible for modification of RAS proteins, the a-factor mating hormone and possibly all other C-terminal carboxyl-methylation [57,156]. This is

90

not an essential gene but STE14 mutants secrete bio- logically inactive a-factor and are therefore sterile. The STE14 gene product of only 140 amino acids is pre- dicted to span the membrane 4-5 times (S. Michaelis, personal communication).

Of all the C-terminal modifications only the methyl ester has the potential for turnover and therefore regulation. Turnover of carboxyl-methyl groups has been reported [152,157]. Tan and Rando [158] have identified a hydrolase activity in bovine retinal rod outer segments which is active only on prenylated methylated substrates and may be involved in catalyzing a cycle of methylation and demethylation which may modulate the activity of carboxyl-methylated proteins.

1V-D. Palmitoylation

The H, N and K(A)-ras proteins are all further processed by palmitoylation of nearby upstream cysteine residues in thioester linkage [51]. Several palmitoyl transferases utilizing palmitoyl coenzyme A as co-substrate have been reported in mammalian cells [159-162]. However, these activities have been refrac- tory to purification due to their membrane association and lability, and the lack of suitable rapid assays. One report has described a membrane-bound palmitoyltransferase active both on CXXX-modified ras protein and an unmodified protein produced in E. coli [162]. The activity was localized predominantly to membranes with a density similar to Golgi markers and may therefore correspond to the same enzyme which palmitoy- lates transmembrane proteins. Indeed, protein palmi- toyltransferases seem to be relatively unspecific for protein sequence, and may rather simply acylate cysteine residues which lie in close proximity to the cytoplasmic face of cellular membranes [163].

The palmitate groups of ras proteins and several other proteins turn over rapidly in vivo [34,164,165]. Presumably this turnover reflects the antagonistic actions of palmitoyltransferase and deacylase activities. Such a deacylase activity, active on palmitoylated N-ras protein, has been reported [162], and other deacylases have also been described [166-168]. The ras deacylase was localized to plasma-membrane fractions, suggesting that an acylation-deacylation cycle might reflect cycling of ras protein from Golgi to plasma membrane. The purification and characterization of these palmitoyl transferase and deacylase activities remains a major goal in the field.

V. Other posttranslational modifications of ras-related proteins

V-A. Myristoylation

N-terminal myristoylation is a common modification of heterotrimeric G-protein ot-subunits [169] as well as

many other cellular proteins including molecules involved in signal transduction such as pp60 src and its relatives and the regulatory subunit of cyclic AMP dependent protein kinase [170]. This involves amide linkage of the 14-carbon saturated fatty acid myristate to a glycine residue immediately following the initiator methionine. Removal of the methionine and myristoylation occur co-translationally and the acylation is catalyzed by a soluble enzyme, N-myristoyl transferase, which has been purified, cloned, and extensively studied, especially by the group of Gordon et al. [171]. The enzyme utilizes myristoyl coenzyme A and requires not only an N-terminal glycine residue, but also recognizes features of the subsequent residues, notably preferring a serine or threonine residue at position 5 after the glycine. No example of turnover of a myristoyl group has been reported, as would be expected from the stability of the amide linkage.

One naturally occurring ras protein is myristoylated, the product of the Rasheed sarcoma virus transforming gene [172]. This has arisen by fusion of the N-terminal 59 amino acids of the retroviral gag polypeptide, which contains a myristoylation signal, with the ras protein. In addition, artificially myristoylated ras proteins have been generated by attaching a myristoylation signal derived from the pp60 src or gag-ras proteins to a H-ras protein [66,67,173]. The resulting myristoylation of activated forms of the ras protein has little effect on their biological activity, even for a Ser-186 mutant which cannot be C-terminally modified by the usual route [66]. This demonstrates that the exact mode of membrane attachment is not crucial to the function of transforming ras proteins. These experiments may have worked somewhat fortuitously, since the X-ray analysis of the H-ras protein shows that the N- and C-termini are quite close together in the folded structure [174]. Thus switching from C-terminal membrane anchoring to N-terminal anchoring may have had a minimal affect on the topology of the protein at the cytoplasmic face of the membrane. An intriguing observation was reported by Buss et al. [67] when they replaced the CAAX box of a normal non-transforming ras protein with an N-terminal myristoylation site. The transforming efficiency of the resulting protein was greatly enhanced, approaching that of a mutationally activated protein. The reason for this is unclear, but one inter- pretation is that the rapid turnover of palmitate on the wild-type protein [34] is essential for regulation of its activity, perhaps by locally modulating its binding to the plasma membrane and interactions with effectors and/or detectors. Substitution of this with the irre- versible myristoylation would result in constitutively strong membrane binding and in sustained heterolo- gous interactions.

The ARF group of the ras superfamily consists of at least 14 members [175,176]. Many of these proteins are

involved in different aspects of intracellular membrane trafficking, probably being required for vesicle budding [176-179] and for assembly of coatomers [180]. ARFs do not contain C-terminal processing motifs, but they do have N-terminal consensus sequences for myristoylation and this modification has been found in those cases where it has been sought [181]. The amino- terminus seems to play a role in function and forms an amphipathic helix which may be stabilized by the myristate moiety [176]. Most authors have observed that myristoylation is required for function [177-179] with one exception [176]. If ARFs are indeed targeted to different membrane compartments, the myristoylation alone clearly cannot be responsible for specificity. Nevertheless, it could provide a general hydrophobic moiety which could function in conjunction with nearby protein sequences in the N-terminal variable region of the ARFs. Specificity in targeting could arise by interactions with other proteins of the targeting machinery or even with a specific receptor as has been identified for myristoylated pp60 ~rc. In the case of pp60 src a 32-kDa plasma-membrane receptor appears to recognize the myristoylated N-terminus, interacting with either or both of the myristate moiety and adjacent protein sequence [182].

V-B. ADP-ribosylation

ADP-ribosylation of the a-subunits of heterotrimeric G proteins by bacterial toxins from Borde- tella pertussis and Vibrio cholerae has been extensively used to study their functions. These toxins do not work on small GTP-binding proteins, but other bacterial ADP-ribosyl transferases have been used to study their properties. The C3 co-enzyme of Clostridium bo- tulinum has been particularly well studied. This has been shown to ADP-ribosylate small GTP-binding proteins in a number of cell types [183-186]. The modified proteins include members of the rho and rac families of ras-related proteins [187-193]. Proteins which do not appear to be modified include K-, N- and H-ras, R-ras, ral, rapl, ypt, ARF and Gp [184,185,187,193,194]. The site of ADP-ribosylation of rho has been identified as Asn-41, located near the putative effector domain of the protein [195,196]. This suggests that ADP-ribosylation will have dramatic inhibitory effects on the interactions of rho with its effectors. ADP-ribosylation has generally been found to have no effect on guanine nucleotide binding or hydrolysis, although in some cases guanine nucleotides have been reported to affect the kinetics of ADP-ribosylation [183,187,189,195, 197,198]. However, a recent report has found an increased intrinsic GTPase activity (by 50-80%) of bacte- rially expressed rhoA and rhoB proteins ADP-ribosylated with C3 [99]. This is an indirect effect, due to increased nucleotide exchange promoted by a de-

91

creased affinity of the modified protein for GDP. Whether such an effect will be seen on post-translationally modified rho proteins is not yet clear. The epidermal cell differentiation inhibitory protein of Staphylococcus aureus is an ADP-ribosyl transferase exhibiting similar substrate specificity to C3, as are the Clostridium limosum and the Bacillus cereus exoen- zymes [200-202], although the latter does not immuno- logically cross-react with C3. ras proteins themselves can be ADP-ribosylated at arginine residues by Pseu- domonas aeruginosa exoenzyme S [203] with unknown consequences.

Microinjection of C3 into living cells has dramatic consequences including the loss of actin stress fibres, as a result of the ADP-ribosylation of p21 rh° [204]. Conversely, microinjection of p21 rh° causes an increase in the number of stress fibres. Microinjecting C3 transferase into neutrophils causes loss of responsiveness to chemotactic signals. These studies have led to the hypothesis that rho proteins promote the assembly of actin stress fibres by inducing the formation of focal adhesions which act as nucleation sites. Thus ADP- ribosylation induced by bacterial toxins can be a useful tool to study the function of ras-related proteins. How- ever, there is no evidence that ADP-ribosylation is a normal physiological regulator.

V-C. Phosphorylation

Phosphorylation of ras-related proteins as a mechanism of regulation has been tested in a number of cases. Phosphorylation of K-ras(B) protein both in vitro and in vivo has been reported using both cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) [205,206]. Both kinases seem to phosphorylate the same site, probably Ser-181 in the hypervariable domain. Stoichiometric phosphorylation can be achieved in vitro but the in-vivo level may be low. Conflicting reports of phosphorylation of H-ras protein have appeared [205-207]. Saikumar et al. detected phosphorylation of p21H .... by PKA and PKC at a site different from that in p21Kras~B), probably serine 177 [206]. In no case has any effect of phosphorylation on nucleotide binding or hydrolysis, or subcellular localization been detected. S. cerevisiae RAS2 can also be phosphorylated by PKA in vitro, resulting in decreased ability to activate adenylate cyclase, but not by PKC or protein kinase P [208]. Both S. cerevisiae RAS proteins have also been shown to be phosphorylated in vivo [209]. In-vitro tyrosine phosphorylation of p21 r~s by insulin receptor kinase has been achieved, but the in-vivo relevance is unclear [210,211]. ras proteins carrying the Thr-59-activating mutation can inefficiently autophosphorylate, the bound GTP acting as phos- phate donor. Interestingly, the small fraction of protein which is phosphorylated at this site is inactive in the

92

Xenopus oocyte germinal vesicle breakdown assay [212], probably due to distortion of the effector loop. How- ever, this is unlikely to be highly relevant to human cancer since Thr-59 mutations occur mainly in ras proteins from transforming retroviruses.

Much attention has been focused on the phosphorylation of smgp21/rapl in platelets and neutrophils. This group of proteins can be phosphorylated in vitro and in vivo by PKA but not PKC [213-221]. No effects on nucleotide exchange, intrinsic GTPase or GAP- stimulated hydrolysis [222] have been noted. However, phosphorylation does cause translocation from membranes to cytosol [214,215]. This is probably due to the fact that phosphorylation, which occurs on Ser-180 near the C-terminus, greatly stimulates the binding of smgp21-GDS, resulting in increased GDS-stimulated nucleotide exchange and decreased membrane binding [222,223]. Recently it has been shown that cGMP-dependent protein kinase G can also phosphorylate smgp21B/raplB in vitro at the same site as PKA [224].

Phosphorylation also appears to have a role in con- trolling the localization and function of some members of the rab subfamily, rablA and rab4, but not rab2 and rab6, were found by Bailly et al. [225] to be phosphorylated specifically in mitotic cells by p34 cdc2 kinase: PKA did not phosphorylate any of the proteins tested. This resulted in translocation into the cytosol which may be responsible for the cessation of membrane trafficking during mitosis. Whether the translocation involves binding of the phosphorylated form to another protein such as GDS or GDI is not clear. Indeed, a role for protein phosphorylation in regulating endoplasmic reticulum to Golgi transport has recently been observed [226].

VI. Conclusions

Posttranslati0nal processing clearly plays a major role in the biological function of ras-related proteins. In particular, the attachment of lipid moieties to the C- or N-termini contribute to membrane binding and specific subcellular localization of these proteins. Regula- tion of this membrane binding is likely mediated by the family of GEP proteins (GDI and GDS), many of which interact with the modified regions of the proteins. This interaction between GEPs and ras-related proteins can itself be regulated by phosphorylation of the latter. In addition, regulated-reversible palmitoylation and/or carboxyl-methylation could also affect membrane association. Thus there are multiple means available to the cell to control the activity of this important group of proteins. The further elucidation of the processing pathways and the biological function of the modifications may lead to strategies for interfering with the activity of specific membranes of the ras

superfamily, such as the transforming ras proteins themselves, and hence to potential cancer treatments.

Acknowledgements

We thank the many colleagues who have con- tributed preprints and information toward this review, and also members of the laboratory at NIMR, particularly Thomas Giannakouros. We also thank Marilyn Brennan for preparing the manuscript. Christopher Newman is supported by an MRC Clinician Scientist Training Fellowship.

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posttranslational processing of the ras superfamily of small gtp-binding proteins

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