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dependent on FTase, while the other isoforms, mutations of which are more common in human cancers, can also undergo geranylation by geranyl-geranyl-transferase (GGTase) if FTase activity is inhibited.

FTase inhibitors (FTIs) are currently of clinical interest in human cancers, with pre-clinical and phase-1 clinical results demonstrating acceptable toxicities, despite farnesylation of many normal human proteins other than p21-Ras. Initial focus has been in common human cancers which harbor activating p21-Ras mutations, such as lung, colon, and pancreas. However, interest also exists in the potential use of FTIs in tumors where secondary activation of p21-Ras has been demonstrated such as human malignant gliomas, with encouraging pre-clinical data. However, FTIs have not been fully evaluated in clinical trials of patients with malignant gliomas, perhaps as a result of the disappointing initial results in the more common systemic cancers. These tempered results likely represent the ability of the commonly mutated p21-Ras isoforms to shuttle their processing to geranylation and lack of clear understanding of what is actually being targeted by FTIs. Nevertheless, targeting p21-Ras in gliomas still remains to be fully explored, with its likely role if any, to be in combinatorial therapy with other conventional and biological therapies.

UNDERSTANDING p21-RAS STRUCTURE AND FUNCTION

p21-Ras Proto-oncogene and p21-Ras Protein

After ligand-receptor interaction, receptor activation leads to a large variety of biochemical events in which small G-proteins such as p21-Ras are very important. Small G-proteins are a superfamily of regulatory GTP hydrolases that cycle between two conformational states, induced by the binding of either guanosine diphosphate (GDP: inactive) or guanosine triphosphate (GTP: active) [1,2]. Three p21-Ras proto-oncogenes have been identified: the Ha-Ras gene (homologous to the Harvey murine sarcoma virus oncogenes), the K-Ras gene (homologous to Kristen murine sarcoma virus oncogenes), and the N-Ras gene (which was first isolated from a neuroblastoma cell line and does not have a retroviral homologue) [3-7]. The p21-Ras oncogenes encode four 21-Kd proteins between 188 and 189 amino acids long, called Ha-Ras, N-Ras, K-Ras4A, and K-Ras4B, the latter two resulting from two alternatively spliced

K-Ras gene products. There is high sequence homology between these four p21-Ras proteins, with the first 86 amino acids being identical, the next 78 having 79 per cent homology, and the remaining 25 amino acids being highly variable.

Post-translational Modification of p21-Ras p21-Ras proteins are produced as cytoplasmic precursor pro-Ras proteins and require several post-translational modifications to become fully biologically active (see Fig. 12.1). Pro-Ras is sequentially modified by:

1. prenylation of the cysteine residue,

2. proteolytic cleavage of the AAX peptide by proteases,

3. carboxymethylation of the new C-terminal carboxylate by carboxy-methyl transferase, and

4. palmitoylation [8-10].

Three different enzymes catalyze prenylation of protein intermediates: (A) farnesyltransferase (FTase), (B) geranyl-geranyltransferase type-I (GGTase-I), and (C) geranyl-geranyltransferase type-II (GGTase-II) [8-10]. FTase transfers a farnesyl diphosphate (FDP), while GGTase-I transfers a geranyl-geranyl diphosphate (GGDP) to the cysteine residue of the CAAX motif. GGTase-II transfers geranyl-geranyl groups from GGPPs to both cysteine residues of CC or CXC motifs.

Farnesyltransferase (FTase)

FTase is a heterodimeric enzyme, which catalyzes farnesylation, resulting in prenylation of the cysteine in the C-terminal CAAX motif of p21-Ras. [11-13]. Farnesylation involves attachment of a 15-carbon farnesyl moiety in a thioether covalent linkage to the cysteine. FTase is composed of 48Kd-a and 45Kd-p subunits [14-16], with Zn+2 and Mg+2 required for function. The terminal residue of the C-terminal CAAX motifs in proteins directs differential affinity to FTase [17,18]. For example, if X is methionine (e.g., K-Ras4B) then there is a 10-30X increased affinity to FTase, compared to proteins where X is either serine or glycine (e.g., Ha-Ras). From the crystalline structure, FTase contains two clefts, which may represent the FDP and CAAX binding sites [19]. A Zn+2 atom lies in the junction between the two clefts, with both FDP and CAAX probably binding with the p-subunit of FTase, whereas the a-subunit may stabilize the p-subunit and catalyze transfer of the farnesyl isoprenoid moiety [20].

FIGURE 12.1 Post-translational modification of p21-Ras p21-Ras is synthesized as a pro-peptide, which undergoes a series of post-translational modifications, resulting in attachment to the inner surface of the plasma membrane, where it can be cycled from inactive GDP- to active GTP-bound state. First, FTase or GGT'ase transfers a farnesyl or a geranyl-geranyl group from FPP or GGPP to the thiol group of the cysteine residue in the CAAX motif of p21-Ras. The C-terminal tri-peptide is then removed by a CAAX-specific endoprotease (PPSEP) in the endoplasmic reticulum. A PPSMTase attaches the methyl group from S-adenosylmethionine (SAM) to the C-terminal cysteine. Finally, a prenyl-protein-specific palmitoyltranferase (PPSPTase) attaches palmitoyl groups to cysteines near the farnesylated C-terminus. See Plate 12.1 in Color Plate Section.

FIGURE 12.1 Post-translational modification of p21-Ras p21-Ras is synthesized as a pro-peptide, which undergoes a series of post-translational modifications, resulting in attachment to the inner surface of the plasma membrane, where it can be cycled from inactive GDP- to active GTP-bound state. First, FTase or GGT'ase transfers a farnesyl or a geranyl-geranyl group from FPP or GGPP to the thiol group of the cysteine residue in the CAAX motif of p21-Ras. The C-terminal tri-peptide is then removed by a CAAX-specific endoprotease (PPSEP) in the endoplasmic reticulum. A PPSMTase attaches the methyl group from S-adenosylmethionine (SAM) to the C-terminal cysteine. Finally, a prenyl-protein-specific palmitoyltranferase (PPSPTase) attaches palmitoyl groups to cysteines near the farnesylated C-terminus. See Plate 12.1 in Color Plate Section.

The a-subunit undergoes phosphorylation, which regulates the activity of FTase [21]. Of importance towards potential complications of inhibiting FTase, there are over 200 normal proteins, which also undergo farnesylation in addition to p21-Ras. These include nuclear laminins A and B, the g-subunit of the retinal trimeric G-Protein Transducin, Rhodopsin Kinase, and a peroxisomal protein termed PxF [9,10].

After farnesylation, there is endoproteolytic removal of the three carboxy-terminal amino acids (AAX) by a cellular thiol-dependent zinc metallo-peptidase [22,23] and subsequent methylation of the carboxyl group of the prenylated cysteine residue by an uncharacterized methyltransferase. All the p21-Ras proteins, except K-Ras, undergo further palmitoyla-tion at 1-2 cysteines near the farnesylated carboxy-terminus [8-10,24-26]. In contrast to farnesylation and proteolysis, palmitoylation and methylation of p21-Ras are thought to be reversible and may have a regulatory role. Although each of the post-translational modifications increases the hydro-phobicity of p21-Ras and contributes to its association with the plasma membrane, the initial farnesylation step alone is sufficient to promote substantial membrane association and confer transforming potentials [26-28].

Geranyl-Geranyl Transferase (GGTase)

There are two other protein prenyltransferases, GGTase-I and GGTase-II, which can prenylate the C-terminal ends of proteins like FTase, by attaching either one or two 20-carbon geranyl-geranyl isopren-oid lipid moieties [9,26]. GGTase-I and FTase share an identical a-subunit, but have distinct p-subunits. Like FTase, GGTase-I is a Zn+2 metalloenzyme that requires Mg+2 for catalysis, however, in contrast to FTase which binds FDP with approximately 30X greater affinity than GGDP, GGTase-I binds GGDP approximately 300X greater than FDP. GGTase-I consists of three subunits, of which the catalytic unit is the p-heterodimer, however, GGTase-II requires an additional protein known as the Rab escort protein to facilitate interaction of its substrate proteins with the enzyme. GGTase-I preferentially prenylates proteins in which the X residue is leucine, but their substrate specificities are not absolute.

The cumulative results of studies indicate, that although membrane localization is critical for p21-Ras function modification, with which specific isoprenoid lipid moiety (FDP or GGDP) this is achieved, is not a critical issue. The potential for cross-prenylation of FTase and GGTase-I implies that GGTase-I might be able to restore the function of p21-Ras and other proteins after FTase inhibition, which probably has implications for the development of resistance to FTIs.

Activation and Inactivation of p21-Ras p21-Ras functions as a membrane-associated biologic switch that relays signals from ligand-stimulated receptors to cytoplasmic effector signaling cascades (see Fig. 12.2). Ligand binding to the extracellular domain of receptor tyrosine kinases (RTKs) causes receptor dimerization, stimulation of protein tyrosine kinase activity, and autophosphorylation [28-30]. Specific phosphorylated tyrosines on the cytoplasmic domains of receptors act as docking sites to recruit adapter proteins, such as Shc and Grb2 to the inner cell membrane, through interactions of specific

Activation of AP-1 gene activation

Proliferation

FIGURE 12.2 Activation and inactivation of p21-Ras. In response to growth factor receptor activation and tyrosine (Y) phosphorylation, Grb2 which is complexed to p21-Ras guanine nucleotide exchange factor Sos, is recruited in proximity to mature p21-Ras attached to the inner cell membrane. Sos catalyzes exchange of GTP for GDP to form activated p21-Ras-GTP. Activated p21-Ras-GTP interacts and activates several effector pathways, including Raf-MAPK, PI-3K, Rac and Rho, to transmit signals to other cytoplasmic regions and the nucleus. Although there is a small intrinsic GTPase activity to revert activated p21-Ras-GTP to inactive p21-Ras-GDP, this hydrolysis is catalyzed by a family of proteins termed Ras:GAPs (GTPase Activating Proteins), among which is neurofibromin, the gene product lost in the cancer pre-disposition syndrome, Neurofibromatosis-1. See Plate 12.2 in Color Plate Section.

protein modules, such as SH2 and PTB domains in these proteins. Grb2, is constitutively complexed with the enzyme Sos, a p21-Ras guanine-exchange-factor or Ras-GEF, by its SH3 domain interacting with the proline-rich region of Sos. Bringing this Grb2-Sos complex in proximity to the post-translationally modified p21-Ras in the inner cell membrane, allows activation of p21-Ras by exchanging GDP for GTP [28-30]. In addition to RTKs, a variety of non-receptor tyrosine kinases, such as Lyn and Fes and the Janus kinase JAK2, also can bind to docking adapter proteins such as Grb2 resulting in the activation of p21-Ras to its GTP-bound state.

Inactivation of p21-Ras requires hydrolysis of GTP and binding of GDP, a reaction known as GTPase. Small G-proteins such as p21-Ras have slow intrinsic GTPase function, requiring catalysis by another family of proteins known as Ras-GAPs. The two best known mammalian Ras-GAPs are p120GAP and neurofibromin, the latter being the protein product which is lost in the cancer pre-disposition syndrome Neurofibromatosis-1 (NF-1).

p21-Ras Signal Transduction Pathways

In its GTP-bound state, p21-Ras can activate several downstream effector pathways, of which activation of the serine-threonine kinase Raf-1 and subsequently, the MAPK (mitogen-activated protein kinase) pathway to the nucleus, has been most thoroughly elucidated (see Fig. 12.2) [31-50]. Other effector pathways include those mediated by phos-phatidylinositol-3'-kinase (PI3K) and small G-proteins Rac and Rho. Two mechanisms have been proposed to explain how p21-Ras-GTP activates its downstream effectors:

(A) Recruitment model: Activated p21-Ras bound to GTP and anchored to the inner cell membrane binds to the cytoplasmic effectors, facilitating their activation by other membrane proteins;

(B) Allosteric model: Activated p21-Ras binding to the effectors induces their conformational change, resulting in their activation.

Both mechanisms are likely involved, depending on the particular effector protein being activated.

p21-Ras-Raf-MAPK Signaling Cascade

Several lines of evidence indicate that Raf serine-threonine kinases (A-Raf, B-Raf, and C-Raf) are critical effectors of p21-Ras function: (A) Dominant-negative mutants of Raf can impair p21-Ras-transforming activity [31-50]; (B) Constitutively activated forms of Raf exhibit transforming activity comparable to that of p21-Ras, and are themselves sufficient to transform some of the murine cells. The activation of Raf occurs after it is recruited to the cell membrane, however, the precise mechanism(s) by which p21-Ras activates Raf is unknown. Once activated, Raf phosphorylates two MAP kinase kinases (MAPKK), MEK1 and MEK2, which are also serine-threonine kinases that phosphorylate the mitogen-activated protein kinases (MAPK), p44MAPK and p42MAPK (also known as extracellular signal-regulated kinases, ERK-1 and ERK-2). The MAPK pathways are well-conserved signaling pathways, with at least six mammalian MAPK cascades, of which those mediated by ERKs are best characterized. ERK-1 and ERK-2 are proline-directed protein kinases that phosphorylate Ser-Thr-Pro motifs of several cyto-plasmic and nuclear proteins, such as phospholipase A2 (PLA2), ribosomal protein S6 kinases (RSKs), and most importantly the transcription factor Elk-1 to alter gene transcription. Activation of the ERK pathway is responsive for many mitogenic signals from protein tyrosine kinases, hematopoietic- and G-protein-coupled growth factor receptors.

Activation of PI-3K Signaling Cascade p21-Ras-GTP binds to and activates the catalytic p110 subunit of PI-3K, a member of a family of lipid kinases that phosphorylate phosphoinositides [51-55], which activate the phosphoinositide-dependent kinases PDK-1 and PDK-2, leading to the activation of Akt and non-conventional isoforms of protein kinase C (ncPKC). PI-3K has been implicated in many distinct cellular functions, including mito-genic signaling, inhibition of apoptosis, regulation of cell size, intracellular vesicle trafficking and secretion, and regulation of actin and integrin functions. In addition to p21-Ras-dependent activation, PI3K can also directly be activated by several growth receptors, by binding the SH2 domains of the p85 regulatory subunit of PI3K directly to specific phos-phorylated tyrosine residues on activated receptors.

Activation of Rho and Rac

Activated p21-Ras regulates several small G-proteins, including Rho, Rac, and CDC42, involved in regulating cell morphology [56-60]. Like p21-Ras, these proteins cycle between GDP- and GTP-bound states, activated by GEFs similar to Sos, and inactivated by GAPs similar to Ras-GAPs. Rho modulates the actin cytoskeleton, involved in regulating membrane ruffling, formation of stress fibers, focal adhesions, and filopodia, integral to maintaining cell shape, invasion, and transformation. CDC42 and Rac are two GTPases involved in regulation of the actin cytoskeleton, the SAPK/JNK pathway and the p38 pathway.

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