CAAX sequence

The Ras proteins are synthesized as biologically inactive, cytosolic precursor proteins. They are then modified by several post-translational processing steps at the carboxyl terminal end and thereby converted into biologically active proteins localized at the plasma membrane. The cysteine of the C-terminal CAAX sequence (C is cysteine, A is generally an aliphatic amino acid, and X is methionine, serine, alanine, or glutamine) is first enzymatically S-farnesylated the AAX part is then cleaved off by a specific protease, and the free C-terminal cysteine is finally converted into a methyl ester (Scheme 1). 

In normal cells, the GDP/GTP-binding proteins, after protein synthesis, move to the cell membrane to which they become hooked by a hydrophobic farnesyl group. The y-subunit is anchored in the membrane by a post-translational modification of the C-terminal CAAX sequence (C - cystein, AA - aliphatic amino acids, X - methionine). This protein is first enzymatically farnesylated by a specific farnesyltransferase, then the AAX part is cleaved by specific proteases and finally the cystein residue is converted to a methyl ester. 

Fig. 3.14. Farnesylation at the C-terminus. The signal sequence for farnesylation is the C-termi-nal sequence CAAX. In the first step a farnesyl moiety is transferred to the cystein in the CAAX sequence. The farnesyl donor is farnesyl pyrophosphate and the responsible enzyme is farnesyl transferase. Subsequently, the three C-terminal amino acids are cleaved (A alanine, X any amino acid) and the carboxyl group of the N-terminal Cys-residue becomes methylated.

Famesylation of the Ras protein occurs at the C-terminal CAAX sequence (A aliphatic amino acid, X Ser or Thr). The famesyl residue is attached, with the help of a farnesyl protein transferase, via a thioether bond to the Cys residue of the CAAX sequence. Next, the last three amino acids are cleaved off by proteases and the carboxyl group of the C-terminal cysteine residue undergoes a methylesterification (Fig. 9.6). In addition, the Ras proteins have a palmitinic acid anchor at different Cys residues in the vicinity of the C terminus. The membrane localization of the Ki-Ras protein is also supported by a polybasic sequence close to the C terminus (see 3.7 and Fig. 3.12). 

On the other hand, a small molecule that binds to the carboxy terminus of a particular RAS might prevent FTase from acting upon this RAS without inhibiting the activity of the transferase on other substrates. Indeed, the precise CaaX-sequences and the two amino acid residues preceding them distinguish the mem-... 

To confirm further that inhibition of farnesylation of GFP-CaaX-proteins is because of the binding of the molecular forceps to their CaaX-sequence, and not because of inhibition of FTase, we performed in-vitro binding assays with FTase. Whereas MF3 clearly interacts with H-RAS at a concentration of 250 gM, two and a half times the observed IC50, we did not observe binding to Ftase (Figure 3.1.4b). Because the forceps bind to RAS and fail to bind FTase, we concluded that MF3 could not act as an enzyme inhibitor, but that its activity is caused by its interaction with the substrate [26]. 

Fig. 1.1. Kinetic scheme for the reaction catalyzed by FTase. The reaction is functionally ordered since formation of the E FPP binary complex leads to product formation. The prenylated CAAX sequence remains bound in the active site following formation of the thioether product, making product release the rate-limiting step in steady-state catalysis. Binding of an additional FPP substrate molecule promotes release of the prenylated product. Adapted from Ref. [18]. (See color plate section in the back of the book.)...

FTase and GGTase I act specifically on proteins such as Ras and Rho GTPases that harbor a C-terminal CAAX box (C=Cys, A=aliphatic, X = any amino acid) [9], which represents a recognition sequence for these enzymes. Therefore, FTase and GGTase I are also classified as CAAX transferases. Geranylgeranylation occurs when the CAAX sequence ends in leucine or phenylalanine, whereas farnesylation takes place in all other cases [10]. 

This technique has been successfully applied in those biological targets where the key structural amino acids of the native peptide for peptide recognition are known. Miscellaneous examples are found in glycoprotein Gbllb/IIIa inhibitors (33)that mimic the RGD sequence (64) or in Ras-farnesyltransferase inhibitors (34) that mimic the CAAX sequence (Fig. 15.15) (65). 

The carboxyl terminal four amino acids constitute a CAAX sequence (where A is any aliphatic amino acid and X is any amino acid) that is highly conserved among all ras and ras-related genes. This sequence is present not only in ras and related proteins but also in the carboxyl terminus of other proteins, including the o and Y-subunits of several heterotrimeric G-proteins, nuclear lamins and unprocessed yeast alpha mating factor. This motif has been shown to be responsible for carboxyl terminal modification via isoprenylation for attachment of ras (and other proteins) to the irmer leaflet of the plasma membrane (Bimbaumer, Bimbaumer, 1995 Khosravi-Far, Der, 1994). 

CaaX sequence and is famesylated, proteolyzed, and methylated, but this protein lacks upstream cysteines and is thus not palmitoylated. Instead, K-ras has a run of several lysine residues that have been shown to increase the affinity of the protein for the membrane, possibly by electrostatic interactions with the negatively charged phospholipid bilayer (Hancock et al., 1990). 

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