Farnesylation motif

The use of fluorescent protein that is fused with a peptide motif for targeting proteins to plasma membrane such as the palmitoylation motif of GAP-43 [27] and the farnesylation motif (CAAX box) of K-ras [28] facilitates visualization of axons. However, it should be careful not to extremely overexpress the membrane-targeted version of fluorescent protein, because we have noticed that it sometimes causes morphological abnormality of axons. 

Farnesyl transferase from rat cells is a heterodimer consisting of a 48 kD u-snbnnit and a 46 kD /3-snbnnit. In the structure shown here, helices 2 to 15 of the u-snbnnit are folded into seven short coiled coils that together form a crescent-shaped envelope partially surrounding the /3-snbnnit. Twelve helices of the /3-snl> nnit form a novel barrel motif that creates the active site of the enzyme. Farnesyl transferase inhibitors, one of which is shown here, are potent suppressors of tumor growth in mice, but their value in humans has not been established. 

The inositol polyphosphate 5-phosphatases belong to a family of enzymes that terminate the signals generated by inositol lipid kinases and PLC. To date, two major types of 5-phosphatase have been identified, both of which share a common 5-phosphatase domain of approximately 300 amino acids, with several highly conserved motifs. Type-I enzymes are 43-65 kDa and preferentially hydrolyze 1(1,4,5)P3 and 1(1,3,4,5)P4, with the attendant formation of I(1,4)P2 and 1(1,3,4)P3, but have little or no activity towards membrane-bound phosphoinositides. The pro-totypic form of a type-15-phosphatase is a 43 kDa protein that is post-translationally modified by farnesylation of the carboxyl terminus CAAX motif this modification juxtaposes the enzyme with the membrane. Type-II enzymes are larger (75-160 kDa) and will hydrolyze both water-soluble inositol phosphates and lipids that... 

The molecular elements of that pathway were mapped with photoaffinity labeling by different investigators. Farnesyltransferase contains a and heterodimer subunits, and binds to both protein and farnesyl diphosphate. The main recognition elements for the protein is the C-terminal CAAX motif. Coleman et al. attached two benzophenones to the recognition sequence and the resulting photoprobe (38, Fig. 14) specifically labeled both subunits [125]. 

Studying the sequences of farnesylated proteins indicated that all lipidated proteins bear a cysteine residue near the C-terminus revealing the CAAX-motif, where C is a cysteine, A stands for an aliphatic amino acid, and X can be any amino acid. Database searches resulted in more prenylated proteins, all bearing the CAAX-motif, in systems from lower eukaryotes to mammals. A closer look at the mature proteins revealed that prenylation was only the first step of processing of the CAAX-motif-encoded proteins. After transfer of the isoprene unit, the last three amino acids are cleaved proteolytically by an endoprotease and the C-terminal cysteine is carboxymethylated by a methyltransferase. ... 

FTase catalyzes the covalent attachment of a farnesyl moiety via a thioether Unkage to the proteins bearing a C-terminal amino acid sequence known as the CAAX motif (Fig. 2) [12,21]. The farnesyl moiety is derived from farnesyl pyrophosphate (FPP), a 15-carbon isoprenyl intermediate in the mevalonate pathway of cholesterol biosynthesis. The binding of FPP to the enzyme has relatively high affinity (K = 1-lOnM), and FPP binding must precede the binding of the peptide substrate for successful catalysis [22,23]. 

Table 18. Bisubstrate FTIs Incorporating Both Farnesyl and Peptide Binding Motifs...

Farnesylation is an absolute requirement for a-factor biogenesis. In a rami or ram2 mutant, no subsequent steps of a-factor processing occur [43,44]. Likewise, a-factor processing does not occur when the CAAX motif Cys is mutated to a Ser residue (C33S) or when the AAX residues are deleted (AVIA) [43]. The unmodified a-factor precursor does not associate with membranes, but remains soluble, and appears to undergo degradation [43] (S. Michaelis, unpublished). 

---