Multiplexed drug-based selection and counterselection genetic manipulations in Drosophila.

The power of Drosophila melanogaster as a model system relies on tractable germline genetic manipulations. Despite Drosophila's expansive genetics toolbox, such manipulations are still accomplished one change at a time and depend predominantly on phenotypic screening. We describe a drug-based genetic platform consisting of four selection and two counterselection markers, eliminating the need to screen for modified progeny.

Plakophilin3 increases desmosome assembly, size and stability by increasing expression of desmocollin2.

Previous reports show that the desmosomal plaque protein plakophilin3 (PKP3) is essential for desmosome formation. Here, we report that PKP3 over-expression decreases calcium dependency for de novo desmosome formation and makes existing cell-cell adhesion junctions more resilient in low calcium medium due to an increase in desmocollin2 expression. PKP3 overexpression increases the stability of other desmosomal proteins independently of the increase in DSC2 levels and regulates desmosome formation and stability by a multimodal mechanism affecting transcription, protein stability and cell border localization of desmosomal proteins.

ERK1/2 can feedback-regulate cellular MEK1/2 levels.

Signal transduction of the Raf/MEK/ERK pathway is regulated by various feedback mechanisms. Given the greater molar ratio between Raf-MEK than between MEK-ERK in cells, it may be possible that MEK1/2 levels are regulated to modulate Raf/MEK/ERK activity upon pathway stimulation. Nevertheless, it has not been reported whether MEK1/2 expression can be subject to a feedback regulation.

Sp1 regulates Raf/MEK/ERK-induced p21(CIP1) transcription in TP53-mutated cancer cells.

We previously reported that the upregulation of mortalin, an Hsp70 family chaperone, is important for B-Raf(V600E) tumor cells to bypass p21(CIP1) expression, which is activated as a tumor-suppressive mechanism in response to aberrant MEK/ERK activation (Wu et al., 2013). Interestingly, mortalin depletion induced p21(CIP1) transcription not only in wild-type TP53 but also in TP53-mutated B-Raf(V600E) cancer cells, suggesting the presence of an additional mechanism for p21(CIP1) regulation.

A mortalin/HSPA9-mediated switch in tumor-suppressive signaling of Raf/MEK/extracellular signal-regulated kinase.

Dysregulated Raf/MEK/extracellular signal-regulated kinase (ERK) signaling, a common hallmark of tumorigenesis, can trigger innate tumor-suppressive mechanisms, which must be inactivated for carcinogenesis to occur. This innate tumor-suppressive signaling may provide a potential therapeutic target. Here we report that mortalin (HSPA9/GRP75/PBP74) is a novel negative regulator of Raf/MEK/ERK and may provide a target for the reactivation of tumor-suppressive signaling of the pathway in cancer.

E-cadherin and plakoglobin recruit plakophilin3 to the cell border to initiate desmosome assembly.

A decrease in the levels of the desmosomal plaque protein, plakophilin3 (PKP3), leads to a decrease in desmosome size and cell-cell adhesion. To test the hypothesis that PKP3 is required for desmosome formation, the recruitment of desmosomal components to the cell surface was studied in the PKP3 knockdown clones. The PKP3 knockdown clones showed decreased cell border staining for multiple desmosomal proteins, when compared to vector controls, and did not form desmosomes in a calcium switch assay.