Post – Translational regulation of ICER

Presentation Type

Abstract

Faculty Advisor

Carlos Molina

Access Type

Event

Start Date

25-4-2025 12:00 PM

End Date

25-4-2025 1:00 PM

Description

The Inducible cAMP Early Repressor (ICER) is a group of isoforms that act as transcription factors of the cyclic AMP (cAMP) pathway, originating from the cAMP Response Element Modulator (CREM) gene. The cAMP pathway and its transcription factors are important for the development of BRAF/MEK treatment-resistant melanoma. Our laboratory has previously shown that ICER is regulated by post-translational mechanisms like phosphorylation and ubiquitination. We decided to examine whether the abnormal expression interferes with ICER’s role as a transcriptional repressor in melanoma cells. We performed a battery of expression assays to compare two cell lines – 447A expressing NHA-wtICER, a more aggressive tumour line, and 446A expressing EGFP, a less aggressive tumour line. We have performed Western blots measuring the expression levels of transgenic HA-ICER in total extract, and nuclear and cytosolic fractions of 447A cells. We further examined expression with immunocytochemistry. Both assays showed co-localization of ICER to the nucleus. Then, we used a dual luciferase assay to look for differences in PRKACA promoter activity between 446A and 447A. We quantified activity in the presence or absence of forskolin, a compound activating the cAMP pathway. Our results show that PRKACA promoter activity is lower in 447A cells compared to 446A. Additionally, PRKACA promoter activity decreases when the cAMP pathway is activated by chronic treatment with FSK. To further explore these results we have examined the relative phosphorylation level of CREB in 446A and 447A and compared the cell morphology between two cell lines before and after the chronic forskolin treatment.

Comments

Poster presentation at the 2025 Student Research Symposium.

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Apr 25th, 12:00 PM Apr 25th, 1:00 PM

Post – Translational regulation of ICER

The Inducible cAMP Early Repressor (ICER) is a group of isoforms that act as transcription factors of the cyclic AMP (cAMP) pathway, originating from the cAMP Response Element Modulator (CREM) gene. The cAMP pathway and its transcription factors are important for the development of BRAF/MEK treatment-resistant melanoma. Our laboratory has previously shown that ICER is regulated by post-translational mechanisms like phosphorylation and ubiquitination. We decided to examine whether the abnormal expression interferes with ICER’s role as a transcriptional repressor in melanoma cells. We performed a battery of expression assays to compare two cell lines – 447A expressing NHA-wtICER, a more aggressive tumour line, and 446A expressing EGFP, a less aggressive tumour line. We have performed Western blots measuring the expression levels of transgenic HA-ICER in total extract, and nuclear and cytosolic fractions of 447A cells. We further examined expression with immunocytochemistry. Both assays showed co-localization of ICER to the nucleus. Then, we used a dual luciferase assay to look for differences in PRKACA promoter activity between 446A and 447A. We quantified activity in the presence or absence of forskolin, a compound activating the cAMP pathway. Our results show that PRKACA promoter activity is lower in 447A cells compared to 446A. Additionally, PRKACA promoter activity decreases when the cAMP pathway is activated by chronic treatment with FSK. To further explore these results we have examined the relative phosphorylation level of CREB in 446A and 447A and compared the cell morphology between two cell lines before and after the chronic forskolin treatment.