Structural and Signaling Mechanisms of ICMT-Mediated KRAS4B Methylation

Ariana L Cardillo, Purdue University

Abstract

Mutated Ras proteins are implicated in ~20% of all human cancer cases. Of these cancercausing Ras mutations, 80% of missense mutations are in the isoform KRas4B. In the decades since the discovery of KRas4B, extensive research of its signaling pathways, from protein translation to cellular outputs, have helped characterize the routes of oncogenesis. Until 2021, KRas4B had been thought to be “undruggable,” as no specific inhibitors had been approved by the Food and Drug Administration (FDA) as effective treatments. This fueled researchers to design treatments that targeted the signaling pathways up- and downstream of KRas4B association to the plasma membrane; of which multiple downstream targeting drugs are currently in various stages of clinical trials. However, these downstream effectors are implicated in multiple cellular pathways and, thus, are nonspecific. Some researchers have since focused their studies on targeting the specific upstream modifications necessary for proper KRas4B cellular localization and function. KRas4B is classified as a CAAX protein, where its four C-terminal residues consist of a cysteine (“C”), two aliphatic residues (“AA”) and a residue of various identities (“X”). CAAX proteins undergo three post-translational modification (PTMs). First, dependent on the identity of the “X” residue, an isoprenoid group of either 15 or 20 carbons is added to the C-terminal cysteine by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase) respectively. KRas4B is farnesylated since its sequence terminates with a leucine. After prenylation, the three C-terminal residues (“AAX”) are removed by the protease Ras converting enzyme-1 (Rce1). Finally, the free carboxylate of the terminal cysteine is methylated by isoprenylcysteine carboxyl methyltransferase (Icmt). Following methylation, KRas4B is translocated to the plasma membrane where it associates in a functional protein complex. Interestingly, of these three required PTMs, methylation is the only reversible step, suggesting a possible point of regulation of many CAAX proteins and their signaling pathways, including that of KRas4B. The regulatory function of methylation has long been speculated, but never fully characterized. The research described herein worked to characterize the mechanism of methylation by Icmt and to better understand KRas4B signaling as a function of methylation, with the ultimate goal of elucidating the large implications methylation may have on the regulation of KRas4B. To understand the mechanism of methylation, we first sought to identify the substrate binding site of Icmt. Icmt is an integral membrane protein localized within the outer membrane of the endoplasmic reticulum (ER) and is currently the sole methyltransferase known to act on CAAX proteins, thus providing a specific target for future chemotherapeutics. Using biochemical tools, we interrogated the ability of a model Icmt from Saccharomyces cerevisiae, Ste14, to accommodate both a hydrophilic cofactor, S-adenosyl-L-methionine (SAM), and a lipophilic isoprenylated substrate, which can have one of two different isoprenoid groups. Through alaninescanning mutagenesis in combination with enzymatic activity assays using substrate mimetics of farnesylated or geranylgeranylated peptides, we identified several residues in the N-terminal half of Ste14 that appear to decrease recognition of N-acetyl-S-farnesyl-L-cysteine (AFC) but not Nacetyl-S-geranylgeranyl-L-cysteine (AGGC). When mutated to alanine, residue Leu56, which sits in the middle of transmembrane helix 2 (TM2), preferentially methylated AGGC over AFC by a factor of over 100.

Degree

Ph.D.

Advisors

Hrycyna, Purdue University.

Subject Area

Cellular biology

COinS