Detecting Changes in the Relative Expression of KRAS2 Splice Variants Using Polymerase Colonies

Butz, James A.; Roberts, Kim G.; Edwards, Jeremy S.

doi:10.1021/bp0343054

Cited by 21 publications

(15 citation statements)

References 11 publications

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“…More compelling was the analysis of fresh colorectal tumors, where expression levels of the two transcripts were equal over a panel of 17 samples. It is likely that our results differ from those previously reported (10,15,16), because we used splice variant-specific RT-qPCR primers and measured absolute mRNA levels against a standard curve generated with each experiment. Our results analyzing mRNA were supported by studies of protein expression.…”

Section: Discussioncontrasting

confidence: 84%

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K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif

Tsai

Lopes

Zhou

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

195

256

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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.

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Section: Discussioncontrasting

confidence: 84%

“…1B), with particular prominence in cell lines derived from colon carcinoma and melanoma. K-Ras4A accounted for 10-50% of total KRAS transcripts, levels significantly higher than previously reported (10,15,16). The relative abundance of the two transcripts did not seem to correlate with K-Ras mutations.…”

Section: Significancecontrasting

confidence: 62%

K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif

Tsai

Lopes

Zhou

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

195

256

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“…When the two transcripts were amplified in the same PCR (Fig. 3), the B isoform was predominant in both tissues, as has been reported previously (34,41,44,45). There are some discrepancies between reported mRNA ratios, which may reflect tissue sampling and/or species differences.…”

Section: Discussionsupporting

confidence: 70%

“…There are some discrepancies between reported mRNA ratios, which may reflect tissue sampling and/or species differences. Human (44) and mouse (41,45) colon have been reported to express both K-ras transcripts at similar levels. The higher B:A ratio found in the present study may be due to the use of distal, rather than total, colon.…”

Section: Discussionmentioning

confidence: 98%

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Mammalian K-ras2 Is a Corticosteroid-Induced Gene in Vivo

Brennan

Fuller

2006

Endocrinology

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Aldosterone acts via the mineralocorticoid receptor to regulate gene expression. A number of aldosterone-induced genes have been characterized in the distal colon and/or the distal nephron. Using the Xenopus kidney-derived A6 cell line, the K-ras transcript of the K-ras gene was identified as aldosterone induced, with a role in epithelial sodium transport. This study sought to establish whether K-ras expression is also increased in mammalian epithelia in vivo in response to aldosterone. RNA was extracted from the kidney and distal colon of rats treated with aldosterone or dexamethasone. Northern blot analysis and real-time RT-PCR were performed using probes and primers specific for the K-rasA isoform and for total K-ras. The expression of both total K-ras and of the A isoform is induced in the distal colon by aldosterone and by dexamethasone. Given the relative abundances of the two isoforms, this would appear to indicate induction of both isoforms. The time course of the response is consistent with a primary transcriptional response. In contrast to the documented up-regulation in the amphibian kidney, we did not observe regulation by corticosteroids in the kidney. However, regulation in a subpopulation of cells cannot be excluded.

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