Characterization of the hydrophobic binding site of isoprenylcysteine carboxyl methyltransferase

Kalub J Hahne, Purdue University

Abstract

CaaX proteins, including the three predominant Ras isoforms, have a C-terminal tetrapepide motif where "C" is a cysteine, "a" is an aliphatic amino acid and "X" is one of several residues. Proteins terminating in a CaaX box require three post-translational modifications; the final step being the α-carboxyl methylesterification of the terminal cysteine. This methylation reaction is completed by a unique class of methyltransferases known as isoprenylcysteine carboxyl methyltransferase (Icmt). Oncogenic K-Ras mutations are responsible for 30% of all cancers and 90% of pancreatic cancers and methyl esterification is required for K-Ras plasma membrane localization where it needs to be in order to function properly. Icmt is the only protein that methylates K-Ras, driving the pursuit of Icmt as a target for anti-cancer therapeutics. Human Icmt and the yeast homolog, Ste14p, (33- and 26-kDa, respectively) are integral membrane proteins localized to the endoplasmic reticulum (ER). The Icmt family of proteins is believed to bind isoprenylated substrates and the co-substrate via an ordered bi-bi reaction mechanism. These proteins use S-adenosyl-L-methionine (SAM) as a methyl source and have a SAM binding site distinctly different from all other known methyltransferases. Our research has focused on the characterization of the hydrophobic binding site of human Icmt and Ste14p. Here, we report the cellular characterization of a novel human Icmt inhibitor, STAB-F3-Diol. Our findings determine that STAB-F3-Diol deters the growth of pancreatic cancer cell lines, as well as inhibiting Ras activation and Erk 1/2 phosphorylation. In addition, this inhibitor reversed both the K-Ras driven cellular transformation of the PaTu 8902 pancreatic cancer cell line and plasma membrane localization of EGFP-K-Ras. We also investigated the oligomeric state of metal affinity purified His10myc3N-Ste14p. Furthermore, we determine the ability of Ste14p and human Icmt to recognize and photo-crosslink with a variety of N-acetyl-S-farnesyl-L-cysteine (AFC) and a-factor benzophone-labeled substrates. We found that tagged Ste14p functions as a homodimer or higher order oligomer. The oligomerization state of Icmt is essential to the characterization of the protein and development of substrate based inhibitors. From these studies, we concluded that both the yeast and human proteins were able to recognize and photo-crosslink with each of the substrates to varied degrees. We also found that Ste14p was able to methylate both the AFC and a-factor substrates whereas human Icmt was only able to methylate the labeled AFC substrates.

Degree

Ph.D.

Advisors

Hrycyna, Purdue University.

Subject Area

Biochemistry

COinS