The intranuclear localization and function of YT521-B is regulated by tyrosine phosphorylation.

YT521-B is a ubiquitously expressed nuclear protein that changes alternative splice site usage in a concentration dependent manner. YT521-B is located in a dynamic nuclear compartment, the YT body. We show that YT521-B is tyrosine phosphorylated by c-Abl in the nucleus. The protein shuttles between nucleus and cytosol, where it can be phosphorylated by c-Src or p59(fyn). Tyrosine phosphorylation causes dispersion of YT521-B from YT bodies to the nucleoplasm. Whereas YT bodies are soluble in non-denaturing buffers, the phosphorylated, dispersed form is non-soluble. Non-phosphorylated YT521-B changes alternative splice site selection of the IL-4 receptor, CD44 and SRp20, but phosphorylation of c-Abl minimizes this concentration dependent effect. We propose that tyrosine phosphorylation causes sequestration of YT521-B in an insoluble nuclear form, which abolishes the ability of YT521-B to change alternative splice sites

MiRNA-26b overexpression in ulcerative colitis-associated carcinogenesis

Background: Longstanding ulcerative colitis (UC) bears a high risk for development of UC-associated colorectal carcinoma (UCC). The inflammatory microenvironment influences microRNA expression, which in turn deregulates target gene expression. microRNA-26b (miR-26b) was shown to be instrumental in normal tissue growth and differentiation. Thus, we aimed to investigate the impact of miR-26b in inflammation-associated colorectal carcinogenesis. Methods: Two different cohorts of patients were investigated. In the retrospective group, a tissue microarray with 38 samples from 17 UC/UCC patients was used for miR-26b in situ hybridization and quantitative reverse transcription polymerase chain reaction analyses. In the prospective group, we investigated miR-26b expression in 25 fresh-frozen colon biopsies and corresponding serum samples of 6 UC and 15 non-UC patients, respectively. In silico analysis, Ago2-RNA immunoprecipitation, luciferase reporter assay, quantitative reverse transcription polymerase chain reaction examination, and miR-26b mimic overexpression were employed for target validation. Results: miR-26b expression was shown to be upregulated with disease progression in tissues and serum of UC and UCC patients. Using miR-26b and Ki-67 expression levels, an UCC was predicted with high accuracy. We identified 4 novel miR-26b targets (DIP1, MDM2, CREBBP, BRCA1). Among them, the downregulation of the E3 ubiquitin ligase DIP1 was closely related to death-associated protein kinase stabilization along the normal mucosa-UC-UCC sequence. In silico functional pathway analysis revealed that the common cellular pathways affected by miR-26b are highly related to cancerogenesis and the development of gastrointestinal diseases. Conclusions: We suggest that miR-26b could serve as a biomarker for inflammation-associated processes in the gastrointestinal system. Because miR-26b expression is downregulated in sporadic colon cancer, it could discriminate between UCC and the sporadic cancer type. © 2015 Crohn's & Colitis Foundation of America, Inc

Differential effects of PKA-controlled CaMKK2 variants on neuronal differentiation

Regulation between protein kinases is critical for the establishment of signaling pathways/networks to orchestrate cellular processes. Besides posttranslational phosphorylation, alternative pre-mRNA splicing is another way to control kinase properties, but splicing regulation between two kinases and the effect of resulting variants on cells have not been explored. We examined the effect of the protein kinase A (PKA) pathway on the alternative splicing and variant properties of the Ca++/calmodulin-dependent protein kinase kinase 2 (CaMKK2) gene in B35 neuroblastoma cells. Inclusion of the exon 16 of CaMKK2 was significantly reduced by H89, a PKA selective inhibitor. Consistently, overexpressed PKA strongly promoted the exon inclusion in a CaMKK2 sequence-dependent way in splicing reporter assays. In vitro, purified CaMKK2 variant proteins were kinase-active. In cells, they were differentially phosphorylated by PKA. In RNA interference assays, CaMKK2 was required for forskolin-induced neurite growth. Interestingly, overexpression of the variant without exon 16 (−E16) promoted neurite elongation while the other one (+E16) promoted neurite branching; in contrast, reduction of the latter variant enhanced neurite elongation. Moreover, the variants are differentially expressed and the exon 16-containing transcripts highly enriched in the brain, particularly the cerebellum and hippocampus. Thus, PKA regulates the alternative splicing of CaMKK2 to produce variants that differentially modulate neuronal differentiation. Taken together with the many distinct variants of kinases, alternative splicing regulation likely adds another layer of modulation between protein kinases in cellular signaling networks