STRUCTURAL AND SIGNALING MECHANISMS OF ICMT-MEDIATED KRAS4B METHYLATION

Mutated Ras proteins are implicated in ~20% of all human cancer cases.  Of these cancer-causing 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 alanine-scanning 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 N-acetyl-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.  In agreement with a recently resolved eukaryotic crystal structure of Icmt, we hypothesized that this N-terminal region facilitates substrate binding to Icmt.  To further localize and confirm TM2 as an important region for substrate binding, we performed a multistep workflow combining photoreactive AFC-based substrate analogs, cysteine-specific cleavage reagent 2-nitro-5-thiocyanobenzoic acid (NTCB), and a library of purified Ste14 cysteine mutants.  Our data confirmed that the photoreactive substrate labels Ste14 between residues 44 and 77, which spans TM2 and part of Loop 2. 

This new information regarding substrate binding allows for more rationale design of small molecule Icmt inhibitors as chemotherapeutics.  More so, it allows for enhanced targeting of Icmt to better understand its possible role in regulating CAAX proteins such as KRas4B.  To date, there are very few comprehensive studies that examine the effects of methylation on broader cellular output levels, spanning orders of magnitude in space and time, specifically at the single-cell level.  Most research has focused on interrogating the connection of methylation and KRas4B localization and function through systems that overexpress KRas4B.  However, these results may not best represent the natural state of KRas4B and the signaling outputs being measured.  Herein, we propose to employ of more modern techniques including, single-cell studies, endogenous KRas4B monitoring within an isogenic background, and single-molecule tracking to better understand KRas4B mechanisms as a function of methylation.  With the knowledge that KRas4B mutants have different oncogenic mechanisms and the new FDA approval of sotorasib, which preferentially inhibits G12C mutated KRas4B, we also seek to explore how methylation may have differing regulatory effects on the KRas4B oncogenic variants which contributes to their divergent oncogenic mechanisms.  Herein, we describe the methodology used to test our hypotheses such as quantifying the recruitment of a Ras-GTP fluorescent sensor templated on the Ras-binding domain (RBD) and measuring signal propagation based on ERK dynamics using a kinase translocation reporter (KTR) all at the single-cell level with endogenous expression of KRas4B.  We will also present preliminary data that suggests the KRas4B oncogenic mutants have differing cell crawling rates.  Together, the structural investigation of the substrate binding site and cell-to-cell signaling of endogenous, mutant specific KRas4B will provide a structural basis for designing Icmt inhibitors not only for chemotherapy treatment, but also to better interrogate the role of methylation in regulating KRas4B and other CAAX proteins involved in various disease states.