Regulating the regulator: post-translational modification of RAS

Abstract

RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on RAS is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which regulate the activation state of RAS without covalently modifying it. By contrast, post-translational modifications (PTMs) of RAS proteins direct them to various cellular membranes and, in some cases, modulate GTP-GDP exchange. Important RAS PTMs include the constitutive and irreversible remodelling of its carboxy-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications, including phosphorylation, peptidyl-prolyl isomerisation, monoubiquitylation, diubiquitylation, nitrosylation, ADP ribosylation and glucosylation.

Figure 1. Ras Signaling

(A) The GDP/GTP cycle of Ras is shown. Inactive, GDP-bound Ras is activated by a GEF that induces the release of GDP and thereby permits GTP to bind. GTP binding induces a marked conformational change in Ras that allows it to bind effectors via their Ras binding domains (RBD). The “on” state of Ras is limited by its slow intrinsic GTPase activity, which is accelerated up to 10⁵ fold by the binding of a GAP, and allows Ras to return to its inactive, GDP-bound state. (B) The Ras/Raf-1/Erk pathway. This pathway is engaged by receptor tyrosine kinases (RTKs), which are activated upon growth factor binding. The adaptor protein GRB2 binds to activated (that is, phosphorylated) RTKs. GRB2 also binds the GEF son of sevenless (SOS) and brings it to the membrane where it can activate Ras. Ras initiates downstream signaling by bringing RAF1 to the membrane and activating its kinase activity. This is the best characterized Ras regulated pathway and it is frequently dysregulated in cancer. (C) Multiplex regulation of, and signaling from, Ras. The various families of GEFs, GAPs and effectors that have been reported to regulate Ras or transmit signals from Ras-GTP, are shown.

Figure 2. Post-translational modification of the C-terminal membrane-targeting region of Ras

(A) The hypervariable regions (HVR) of the four Ras isoforms are shown. These sequences contain all of the information required to target the different Ras proteins to various subcellular membrane compartments as demonstrated by the fact that they can be used in isolation to target unrelated proteins in the same way. The CAAX motif is often considered the “first signal” because a “second signal” immediately upstream of that sequence is also required for plasma membrane targeting. For N-Ras, H-Ras and K-Ras4A this consists of cysteines that are palmitoylated. For K-Ras4B (shown separately) the second signal consists of a polybasic region with a net charge of +8. The cysteines of the CAAX motifs that are farnesylated are shown in yellow. Cysteines in the second signal region that are modified by palmitate are shown in green. The lysines of the polybasic region of K-Ras4B are shown in red and serine 181, which is the principal site of phosphorylation, is show in blue. (B) CAAX processing is catalyzed by three enzymes that work sequentially: farnesyltransferase (FTase), Ras converting enzyme 1 (Rce1) and isoprenylcysteine carboxyl methyltransferase (Icmt). FTase is a cytosolic enzyme that catalyzes the first and rate-limiting reaction in the sequence. Rce1 is an ER-localized endoprotease that removes the AAX amino acids rendering the farnesylcysteine the new C-terminus. Icmt, also localized in ER membranes, methylesterifies the α-carboxyl group of the farnesylcysteine. S-adenosylmethionine (AdoMet) is used as the methyl donor in this reaction generating S-adenosylhomocysteine (AdoHcy) as a product. The end result of these modifications is to convert the C-terminus of Ras proteins from a hydrophilic to a hydrophobic domain: lapidated peptide with the charge of the C-terminal carboxylate negated by methylation. Whereas the reacions catalyzed by FTase and Rce1 are irreversible, prenylcysteine carboxyl methylation catalyzed by Icmt is readily reversible at physiologic pH, although a specific esterase that catalyzes the reverse reaction has not been identified.

Figure 3. Ras trafficking

Ras is synthesized on cytosolic free polysomes as a globular hydrophilic protein. Nascent Ras encounters farnesyltransferase in the cytosol (1) and, after farnesylation, it gains affinity for, and is transported to, membranes of the endoplasmic reticulum (ER) (2) where it encounters the subsequent CAAX processing enzymes Rce1 (3) and Icmt (4). Following CAAX processing, K-Ras4B deviates from the path of the palmitoylated Ras isoforms and proceeds directly to the plasma membrane (5) via a poorly understood pathway that may involve cytosolic chaperones. N-Ras and H-Ras proceed to the cytosolic face of the Golgi apparatus where they are palmitoylated by DHHC9–GCP16 and thereby trapped in that membrane compartment (6). From the Golgi they traffic via vesicles to the plasma membrane (7). Upon phosphorylation of serine 181, K-Ras4B can be discharged from the plasma membrane and travel back to the endomembrane system (5). N-Ras and H-Ras are discharged from the membrane by depalmitoylation, and move by retrograde transport back to the Golgi for another round of palmitoylation (8).

Figure 4. The acylation/deacylation cycle of H-Ras

During acylation, the acyl chain of a fatty acid is covalently attached to a cysteine residue of a protein via a labile thioester linkage. The most common fatty acid utilized for this purpose is palmitate, which contributes its 16 carbon saturated acyl chain to the protein. The enzymes that catalyze this lipidation reaction are known as palmitoyl-acyltransferases (PATs). H-Ras is palmitoylated on the Golgi apparatus by the PAT DHHC9-GCP16 (1) and sent to the plasma membrane via vesicular transport (2). Once on the membrane H-Ras is susceptible to depalmitoylation by a thioesterase such as acyl protein thioesterase 1 (APT1). Palmitoylated H-Ras binds to FKBP12, which catalyzes cis-trans isomerization of the peptidyl-prolyl bond immediately adjacent to the palmitoylated cysteine. This isomerization constitutes a molecular timer that promotes depalmitoylation, which allows H-Ras to leave the plasma membrane (4) and diffuse back to the Golgi (5) for another round of acylation. FK506, rapamycin, cycloheximide (CHX) and other drugs that inhibit the prolylisomerase activity of FKBP12 augment H-Ras palmitoylation by inhibiting depalmitoylation.

Figure 5. The farnesyl-electrostatic switch of K-Ras4B

The MARKS protein depicted above K-Ras4B is known to associate conditionally with the plasma membrane by virtue of a myristoylated N-terminus and a nearby polybasic region. Protein kinase C (PKC)-induced phosphorylation of serines (shown in magenta) within the polybasic region partially neutralizes its positive charge and allows MARCKS to fall of the membrane in a process known as a myristoyl-elecrostatic switch. The C-terminal farnesyl modification of K-Ras4B and the nearby polybasic region are similarly regulated by a farnesyl-electrostatic switch that is activated by PKC-mediated phosphorylation of serine 181. Serine 171 is also a phosphate acceptor that may contribute but is not required for the operation of the farnesyl-electrostatic switch, which is primarily regulated by phosphorylation of serine 181. Specifically, phosphorylation of serine 181 in K-Ras4B promotes the dissociation of Ras from membranes. The inset shows that GFP-K-Ras4B dissociates from the membrane and is internalized in live Jurkat T cells upon exposure to the PKC agonist phorbol myristate acetate (PMA).

Figure 6. Post-translational modifications of Ras

All PTMs reported for H-Ras (top) and K-Ras4B (bottom) are shown along the backbone of Ras. Sites of mono- and di-ubiquitination are indicated with blue spheres (Ub). Glucosylation and ADP-ribosylation only occur in cells intoxicated with bacterial virulence factors. The other PTMs are intrinsic to all eukaryotic cells. All of these PTMs have consequences both for Ras trafficking and signaling.