The Role Of CASK-Id1 Signal Pathway On Cell Proliferation

CASK(calcium/calmodulin-dependent serine protein kinase)is a member of the membrane associated guanylate kinase(MAGUK) family, a group of conserved cytoskeletal proteins. CASK form scaffolds for protein networks at cell membranes, and play important role in construction of cytoskelecton, formation of cell junctions, signal transduction and regulation on gene expression.Our previous studies have shown that guanylate kinase (GUK) domain of hCASK interact with Id1 by yeast two-hybrid method. CASK and Id1 were coprecipitated in western blotting detection and distributed co-localized at cytoplasmThe Id family of helix-loop-helix (HLH) proteins are thought to affect the balance between cell growth and differentiation by negatively regulating the function of basic helix-loop-helix (bHLH) transcription factors. Id family has a constellation of cellular functions including proliferation and cell cycle progression, migration and invasiveness, cell fate determination, but it is not well elucidated on the upstream signal of Id protein.A large body of evidence has shown that bHLH transcription factors are involved in the transcription of pl6 and p21.We deduced that CASK might regulate these genes by impacting Idl/bHLH transcription factor binding.To identify this hypothesis, we established a CASK knockdown model of ECV304 cell line with RNA interference technique.Methods1. Cell culture: Human cell line ECV304 were cultured at 37C /5% CO2in Ml99 medium supplemented with 100IU/ml penicillin, 10ug/ml streptomycin, and 10% heat-inactivated fetal calf serum(FCS).2. Construction of recombinant siRNA expression plasmids: (1) Choose appropriate 19bp sequence on the transcript of target gene and blast to eliminate any target sequences with significant homology to other coding sequences. The oligonucleotides were synthesized by Shanghai Bioasia corporation. (2) Annealing of each pair of oligonucieotides (3) Double digestion of vector by endonuclease (4)Ligation of annealed oligonucleotide inserts to linearized vector.(5) Transformation of DH5 a (6) Identificationof recombinant plasmids by sequencing.3. Cell Transfection: (1) Optimizing the transfection condition according to manufacture's introductions, choosing the best ratio of FuGene6 to plasmid DNA. (2) Plate the ECV304 cells one day before the transfection experiment.When cells were 60-80% confluent, transfected cells according to the optimal condition.4. Dual-Luciferase Reporter 1000 Assay :ECV304 cells were transfected by siLuciferase(target to firefly luciferase) plasmid and pGL-3promoter plasmid/pRL plasmid. Collected the cell lysate and measure the firefly luciferase and renilla luciferase activity.5. Western blotting and immuno-coprecipitation: Two or three days after transfection, cells were washed with PBS and collected by scraping. They were lysed in ice-cold RIPA buffer and centrifuged. The supernatant was used for protein concentration determination by BCA-200 method. The proteins were resolved on 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, and incubated with the appropriate antibodies. The peroxidase-based detection was performed with Chemiluminescence Reagent (Pierce) according to the manufacturer's instructions.6. RT-PCR: Total RNA was prepared from ECV304 cells 48h after transfection using the Tripure reagent according to the manufacturer's protocol. RT-PCR amplification products were resolved by 1 % agarose gel.7. Growth rate and cell cycle analysis:The cells were seeded in 24 well plates and transfected with siCASK or pBS/U6 vector by using FuGene 6. On different time points after transfection, cells were trypsinized and counted with a hemocytometer. All samples were done in tripicate and every sample counted was repeated at least 6 times. ECV304 cells were seeded in 6.0cm dishes with M199 medium free of serum. The cells were synchronized in cell cycle 24h later. Then the cells were transfected with siCASK or pBS/U6 vector with FuGene6 reag...