Cadwallader, Karen Anne; (1994) Post-translational modifications and p21ras function. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The ras gene family consists of three members that encode highly similar proteins of 21Kd (p21ras/Ras). This protein is plasma membrane associated, binds guanine nucleotides and has intrinsic GTPase activity. Activating point mutations render Ras insensitive to regulation by GAP (GTPase activating protein) and it remains in the active GTP bound state. Membrane association of Ras has been shown to be essential for its biological activity. The plasma membrane targeting of Ras is accomplished by a series of post-translational modifications which occur in 2 steps. Step 1 involves the CAAX motif (C = cysteine, A = aliphatic and X = any amino acid) at the C- terminus. The cysteine is alkylated by C15 farnesyl, the -AAX amino acids are removed and the new C-terminal cysteine undergoes methylesterification. Step 2 involves palmitoylation of cysteine residues near the CAAX motif in the case of H-, N- and K-ras (A). Membrane localisation of K-ras (B) appears to involve electrostatic interaction of the polybasic region (K175-180) with the membrane. Other CAAX containing proteins (rap 1A, G25K) are prenylated with a C20 geranylgeranyl moiety rather than C15 farnesyl. Geranylgeranylation of H- and K-ras (B) also leads to membrane association of the protein but specific targeting to the plasma membrane requires the presence of the polybasic domain or the palmitoylation sites. Another family of proteins (p60src, Gag, cytochrome b5 reductase) is membrane associated by the addition of myristic acid to the N-terminus. Myristoylation can also allow Ras proteins to be membrane associated but specific plasma membrane targeting remains dependent on the presence of palmitoylation sites or a polybasic region. Upstream of the CAAX motif is the hypervariable domain - a region that shows less than 20% homology between the ras genes. The function of this domain is not known and it may simply connect the N- and C-termini. However this region could also confer specificity on the interaction of different Ras proteins with different effector and/or regulatory proteins. Deletions within this region destroy transforming ability and reduce MAP kinase activity suggesting that effector interaction is disrupted. N17 deletion mutants rescue proliferation of NIH 3T3 cells indicating that exchange factor interaction is also influenced by the hypervariable region. This thesis attempts to establish a relationship between the biological activity of Ras, its cellular location and post-translational processing events. A functional role for the hypervariable domain is also examined.

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