Calcium/calmodulin-dependent Serine Protein Kinase Expression Research Articles

363 A CRISPR/dCas9 Epigenetic Therapuetic Approach for CASK-Related MICPCH

OBJECTIVES/GOALS: CASK-related microcephaly with pontine and cerebellar hypoplasia (MICPCH) is a rare X-linked neurodevelopmental disorder caused by mutations in calcium/calmodulin-dependent serine protein kinase (CASK). We aim to rescue CASK expression via an CRISPR/dCas9 epigenetic therapeutic and create iPSC-based CASK relevant in vitro model systems. METHODS/STUDY POPULATION: As females have two X-chromosomes, disease causing mutations present with a 50/50 expression of mutant and wildtype, due to the mosaicism caused by random X-chromosome inactivation (XCI). This project will adapt an established CRISPR/dCas9 epigenetic approach to rescue expression from the silenced, wild-type CASK allele. We aim to accomplish this through testing different dCas9 orthologues and a guide RNA screen targeting the CASK promoter. Constructs will be tested for optimal targeting efficacy in vitro and assessed via RT-qPCR. Additionally, epigenetic modifications from our approach will be analyzed through bisulfite sequencing. We also aim to apply this epigenetic rescue technology in disease relevant cell lines and eventually in engineered patient mutation iPSC-derived neurons. RESULTS/ANTICIPATED RESULTS: Our results show the ability to target CASK and assess gene expression changes with CRISPR/dCas9 paired with an epigenetic modifier and transcriptional activator. Additionally, our fibroblast model with nonpathogenic single nucleotide polymorphisms within CASKallow for allele specific analysis of our targeted reactivation. We anticipate that following an increase of CASK expression, there would be a decrease in region specific promoter methylation. Further, with the identification of clinically described disease-causing point mutations that result in a loss of function of CASK protein, induction of the mutant sequence onto a healthy cell background will result in a similar reduction of CASK protein in our cell model. DISCUSSION/SIGNIFICANCE: This project will demonstrate the first therapeutic avenue for CASK-related MICPCH, and the potential to utilize targeted X-reactivation as a platform approach for X-linked disorders. Further, investigation of smaller dCas9 orthologues prepares our approach for future translational applications such as packaging into AAV for delivery.

CASK regulates Notch pathway and functions as a tumor promoter in pancreatic cancer

Calcium/calmodulin-dependent serine protein kinase (CASK), a member of membrane-associated guanylate kinase (MAGUK) super-family, is implicated in regulating cell proliferation, cytoskeletal remodeling, and cell metastasis. Our study aimed to investigate the effect of CASK on the malignant behaviors of pancreatic cancer cells and to determine the signaling pathway involved. CASK expression in pancreatic cancer tissues based on the TCGA database was analyzed using GEPIA online tool. The overall survival (OS) and disease-free survival (DFS) in patients with pancreatic cancer based on CASK expression was also analyzed using GEPIA. KEGG pathway enrichment analysis was used to show the association of 1522 CASK-related genes and signaling pathways. The expression of CASK, Notch1 and Hey1 was detected by Western blot. Cell proliferation, colony number, invasion, and apoptosis were detected by CCK-8, colony formation assay, Transwell invasion assay, and flow cytometry analysis, respectively. Results showed that CASK was upregulated in pancreatic cancer tissues and cells. Pancreatic cancer patients with high CASK expression showed shorter OS and DFS than patients with low CASK expression. KEGG pathway enrichment analysis proved that CASK and 1522 CASK-associated genes were primarily associated with the Notch pathway. CASK silencing inhibited cell proliferation, colony formation ability, and invasion and elicited apoptosis in pancreatic cancer cells. Additionally, we confirmed that CASK silencing inhibited the Notch pathway in pancreatic cancer cells. Overexpression of Notch1 resisted the anti-tumor functions of CASK knockdown in pancreatic cancer cells. In conclusion, CASK knockdown suppressed the malignant behaviors of pancreatic cancer cells by inactivating the Notch pathway.

Cask methylation involved in the injury of insulin secretion function caused by interleukin1-β.

Islet inflammation severely impairs pancreatic β‐cell function, but the specific mechanisms are still unclear. Interleukin1‐β (IL‐1β), an essential inflammatory factor, exerts a vital role in multiple physio‐pathologic processes, including diabetes. Calcium/calmodulin‐dependent serine protein kinase (CASK) is an important regulator especially in insulin secretion process. This study aims to unveil the function of CASK in IL‐1β–induced insulin secretion dysfunction and the possible mechanism thereof. Islets of Sprague‐Dawley (SD) rats and INS‐1 cells stimulated with IL‐1β were utilized as models of chronic inflammation. Insulin secretion function associated with Cask and DNA methyltransferases (DNMT) expression were assessed. The possible mechanisms of IL‐1β‐induced pancreatic β‐cell dysfunction were also explored. In this study, CASK overexpression effectively improved IL‐1β‐induced islet β‐cells dysfunction, increased insulin secretion. DNA methyltransferases and the level of methylation in the promoter region of Cask were elevated after IL‐1β administration. Methyltransferase inhibitor 5‐Aza‐2’‐deoxycytidine (5‐Aza‐dC) and si‐DNMTs partially up‐regulated CASK expression and reversed potassium stimulated insulin secretion (KSIS) and glucose‐stimulated insulin secretion (GSIS) function under IL‐1β treatment in INS‐1 and rat islets. These results reveal a previously unknown effect of IL‐1β on insulin secretion dysfunction and demonstrate a novel pathway for Cask silencing based on activation of DNA methyltransferases via inducible nitric oxide synthase (iNOS) and modification of gene promoter methylation.

CASK, APBA1, and STXBP1 collaborate during insulin secretion

Calcium/calmodulin-dependent serine protein kinase (CASK) knockdown reduces insulin vesicle docking to cell membranes. Here, we explored CASK interactions with other proteins during insulin secretion. Using co-immunoprecipitation, liquid chromatography-mass spectrometry and bioinformatic analysis, we identified that CASK, Adapter protein X11 alpha (APBA1), and Syntaxin binding protein 1 (STXBP1) formed tripartite complex during insulin secretion. CASK enhanced APBA1–STXBP1 interaction and mediated their traffic from cytoplasm to plasma membrane during insulin release. High fatty acid stimulation decreased insulin secretion along with CASK, APBA1, and STXBP1 expression; Cask overexpression enhanced CASK/APBA1/STXBP1 tripartite complex function, and may thereby rescue lipotoxicity-induced insulin-release defects. Collectively, our results illustrated the function of CASK in insulin granules exocytosis, which broadens the underlying mechanism of insulin secretion and highlights the clinical potential of CASK as a drug target of type 2 Diabetes Mellitus (T2DM).

Expression of CASK Antibody in Non-Mucinous Colorectal Adenocarcinoma

AbstractBackground: Colorectal cancers are one of the leading causes of cancer related morbidity and mortality worldwide. In Egypt was 4% in the year 2012 ranking as the sixth cancer in Egypt and representing 53% of GI malignancy. It is obvious that there is no single determining factor for the process of carcinogenesis or spread of colorectal carcinoma. There are many proposed contributing factors, including disturbances in Epithelial-Mesenchymal Transition (EMT) and loss of regulation of adhesion molecules and polarity proteins. Cal-cium/calmodulin-dependent serine protein kinase (CASK) belongs to the Membrane-Associated Guanylate Kinase (MAGUK) family. The role of CASK in Colorectal cancer hasn't been yet fully understood.Aim of Study: To evaluate the expression of Calci-um/calmodulin-dependent serine protein kinase (CASK) in non-mucinous colorectal adenocarcinoma and to evaluate its correlation with variable clinicopathological factors.Material and Methods: It is retrospective descriptive study is conducted in the Department of Pathology, Faculty of Medicine, Suez Canal University Teaching Hospital. It include 42 archived paraffin blocks of non-mucinous colorectal adenocarcinoma of patients who underwent surgical excision without previous chemotherapy or radiation, 15 adenomatous polyps and 5 normal mucosa samples. The clinical and path-ological data of studied patients are taken from medical records, pathology referral reports and pathology reports. Patients with incomplete data excluded from the study. Re-viewing the data of survival from the archived registry files at department of Oncology, Suez Canal University Teaching Hospital. Sections cut into 5um thick sections and mounted on positive charged slides for immunohistochemical staining using CASK Antibody (S56A-50): Mouse Monoclonal, Novus Biologicals, Catalogue no. NBP1-47648. Staining intensity graded according to the Allred score on a 0-3 scale.Data Analysis: The relation between CASK antibody staining intensity in the studied biopsies and other clinico-pathological parameters including (age, gender, histopatho-logical tumor features) was evaluated using Pearson's x2 test. The level of statistical significance (p-value) was set at Results: The study revealed that CASK protein had mainly cytoplasmic and cytoplasmic-membranous pattern of expres-sion. The CASK protein was overexpressed in the majority of CRC samples with 85.7% of cases showing moderate to strong expression, while only 14.3% of cases displayed minimal faint expression. On the other hand, CASK expression was low in the majority (53.3%) of adenoma samples. Addi-tionally, adenomas with high grade dysplasia showed stronger staining intensity for CASK protein and higher percentage of positive cases than the ones with low grade dysplasia by IHC. As regard to the association between CASK protein overex-pression and clinicopathological prognostic factors we found that CASK demonstrated significantly higher expression in tumor samples with early stages (I/II) rather than advanced stage (III/IV). Low grade tumors showed higher percentage of positive cases and stronger intensity of staining for CASK protein than high grade tumors. Expression of CASK protein showed no statistically significant correlation with patients' gender or age respectively. There was also no significant correlation with tumor site, gross tumor size, histologic tumor border configuration, lymphovascular invasion or distant metastasis.Conclusion: CASK protein was overexpressed in the majority of non-mucinous colorectal adenocarcinoma (CRC) cases with mainly cytoplasmic and cytoplasmic-membranous localization. However, its expression was significantly less in adenomas with a possible association with the grade of dysplasia; giving positive pattern in highly dysplastic cases suggesting a role in the progression of adenomas into carci-nomas. CASK overexpression also associated with both TNM stage of tumors and their histologic grade. CASK was signif-icantly overexpressed in early stage and low grade tumors rather than tumors with advanced stage and higher histological grades. This suggests that CASK protein is a good prognostic factor and might contribute in tumor confinement and local-ization.

Lateral Membrane-Specific MAGUK CASK Down-Regulates Na V 1.5 Channel in Cardiac Myocytes

Mechanisms underlying membrane protein localization are crucial in the proper function of cardiac myocytes. The main cardiac sodium channel, NaV1.5, carries the sodium current (INa) that provides a rapid depolarizing current during the upstroke of the action potential. Although enriched in the intercalated disc, NaV1.5 is present in different membrane domains in myocytes and interacts with several partners. To test the hypothesis that the MAGUK (membrane-associated guanylate kinase) protein CASK (calcium/calmodulin-dependent serine protein kinase) interacts with and regulates NaV1.5 in cardiac myocytes. Immunostaining experiments showed that CASK localizes at lateral membranes of cardiac myocytes, in association with dystrophin. Whole-cell patch clamp showed that CASK-silencing increases INa in vitro. In vivo CASK knockdown similarly increased INa recorded in freshly isolated myocytes. Pull-down experiments revealed that CASK directly interacts with the C-terminus of NaV1.5. CASK silencing reduces syntrophin expression without affecting NaV1.5 and dystrophin expression levels. Total Internal Reflection Fluorescence microscopy and biotinylation assays showed that CASK silencing increased the surface expression of NaV1.5 without changing mRNA levels. Quantification of NaV1.5 expression at the lateral membrane and intercalated disc revealed that the lateral membrane pool only was increased upon CASK silencing. The protein transport inhibitor brefeldin-A prevented INa increase in CASK-silenced myocytes. During atrial dilation/remodeling, CASK expression was reduced but its localization remained unchanged. This study constitutes the first description of an unconventional MAGUK protein, CASK, which directly interacts with NaV1.5 channel and controls its surface expression at the lateral membrane by regulating ion channel trafficking.

High expression of CASK correlates with progression and poor prognosis of colorectal cancer.

Calcium/calmodulin-dependent serine protein kinase (CASK), which localizes at cell-cell adhesion sites and binds to the heparan sulfate proteoglycan syndecan-2, is involved in cell proliferation, cytoskeletal remodeling, and cell migration. To demonstrate the role of CASK in colorectal cancer (CRC) carcinogenesis, we examined the expression of CASK and its binding protein syndecan-2 in human CRC tissues. The expression of CASK was measured in CRC specimens and the controls from adenomas and normal mucosae by immunohistochemical staining and Western blot analysis. Syndecan-2 protein level was tested in CRC samples and the controls by Western blot analysis. The correlations between CASK expression and clinicopathological variables, including disease-free survival (DFS) and overall survival (OS), were analyzed. Compared to the controls, both CASK and syndecan-2 expression were enhanced in CRC tissues. Furthermore, high expression of CASK and syndecan-2 was significantly correlated with advanced tumor stage, lymphatic invasion, lymph node metastasis, vascular invasion, liver metastasis, and unresectable metastatic CRC. Survival analysis showed that patients with low CASK staining had a significantly better survival compared to patients with high CASK staining. In multivariate analysis, CASK overexpression, advanced tumor stage, lymph node metastasis, vasvular invasion, and liver metastasis were independent prognostic factors of poor DFS and OS. Our present study indicates that CASK overexpression is associated with an unfavorable prognosis. CASK is an independent prognostic factor for CRC, which suggests that it is a novel and crucial predictor for CRC metastasis.

Calcium/calmodulin-dependent serine protein kinase is involved in exendin-4-induced insulin secretion in INS-1 cells

ObjectiveExendin-4 (Ex-4) is an anti-diabetic drug that is a potent agonist of the glucagon-like peptide-1 (GLP-1) receptor. It has already been approved for the treatment of type 2 diabetes mellitus, but its underlying mechanisms of action are not fully understood. Calcium/calmodulin-dependent serine protein kinase (CASK), which plays a vital role in the transport and release of neurotransmitters in neurons, is expressed in pancreatic islet cells and β-cells. This study aimed to investigate whether CASK is involved in the insulin secretagogue action induced by Ex-4 in INS-1 cells. Material/MethodsA glucose-stimulated insulin secretion (GSIS) assay was performed with or without siRNA treatment against CASK. The expression level and location of CASK were evaluated by real-time PCR, western blotting and immunofluorescence. With the use of a protein kinase A (PKA) inhibitor or an exchange protein directly activated by cAMP-2 (Epac2) agonist, immunoblotting was performed to establish the signaling pathway through which Ex-4 alters CASK expression. ResultsKnock-down of CASK significantly attenuated the Ex-4-enhanced insulin release, and we showed that Ex-4 could increase transcription of CASK mRNA and expression of CASK protein but did not change the cellular location of CASK. A PKA inhibitor reduced the ability of Ex-4 to stimulate CASK expression, but an Epac2 agonist had no effect suggesting that regulation was mediated by the cAMP/PKA pathway. ConclusionOur study suggests that the stimulation of β-cell insulin secretion by Ex-4 is mediated, at least in part, by CASK via a novel signaling mechanism.