The two products of the KRAS locus, K-Ras4A and K-Ras4B, are encoded by alternative fourth exons and therefore, possess distinct membrane-targeting sequences. The common activating mutations occur in exons 1 or 2 and therefore, render both splice variants oncogenic. K-Ras4A has been understudied, because it has been considered a minor splice variant. By priming off of the splice junction, we developed a quantitative RT-PCR assay for K-Ras4A and K-Ras4B message capable of measuring absolute amounts of the two transcripts. We found that K-Ras4A was widely expressed in 30 of 30 human cancer cell lines and amounts equal to K-Ras4B in 17 human colorectal tumors. Using splice variant-specific antibodies, we detected K-Ras4A protein in several tumor cell lines at a level equal to or greater than that of K-Ras4B. In addition to the CAAX motif, the C terminus of K-Ras4A contains a site of palmitoylation as well as a bipartite polybasic region. Although both were required for maximal efficiency, each of these could independently deliver K-Ras4A to the plasma membrane. Thus, among four Ras proteins, K-Ras4A is unique in possessing a dual membrane-targeting motif. We also found that, unlike K-Ras4B, K-Ras4A does not bind to the cytosolic chaperone δ-subunit of cGMP phosphodiesterase type 6 (PDE6δ). We conclude that efforts to develop anti–K-Ras drugs that interfere with membrane trafficking will have to take into account the distinct modes of targeting of the two K-Ras splice variants.

KRAS is the most frequently mutated oncogene. The incidence of specifi c KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specifi c mutant KRAS proteins. We used a crossdisciplinary approach to compare KRAS G12D , a common mutant form, and KRAS A146T , a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRAS A146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factorinduced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRAS G12D and KRAS A146T exhibit distinct tissue-specifi c effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specifi c phenotypes result from allele-specifi c signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. SIGNIFICANCE: Although epidemiologic and clinical studies have suggested allele-specifi c behaviors for KRAS , experimental evidence for allele-specifi c biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specifi c KRAS mutants in human cancers from different tissues is due to their distinct signaling properties.

Primary hyperoxalurias (PHs) are autosomal recessive disorders caused by the overproduction of oxalate leading to calcium oxalate precipitation in the kidney and eventually to end-stage renal disease. One promising strategy to treat PHs is to reduce the hepatic production of oxalate through substrate reduction therapy by inhibiting liver-specific glycolate oxidase (GO), which controls the conversion of glycolate to glyoxylate, the proposed main precursor to oxalate. Alternatively, diminishing the amount of hepatic lactate dehydrogenase (LDH) expression, the proposed key enzyme responsible for converting glyoxylate to oxalate, should directly prevent the accumulation of oxalate in PH patients. Using RNAi, we provide the first in vivo evidence in mammals to support LDH as the key enzyme responsible for converting glyoxylate to oxalate. In addition, we demonstrate that reduction of hepatic LDH achieves efficient oxalate reduction and prevents calcium oxalate crystal deposition in genetically engineered mouse models of PH types 1 (PH1) and 2 (PH2), as well as in chemically induced PH mouse models. Repression of hepatic LDH in mice did not cause any acute elevation of circulating liver enzymes, lactate acidosis, or exertional myopathy, suggesting further evaluation of liver-specific inhibition of LDH as a potential approach for treating PH1 and PH2 is warranted.