Y. B, the Mo7e and Kasumi cell lines expressing endogenous c-Kit and Ba/F3 cells expressing c-Kit and c-Kit/Y823F were lysed and subjected to immunoprecipitation with anti-c-Kit antibody. c-Kit expression levels have been detected by Western blotting. C, Ba/F3 cells had been stably transfected together with the Ba/F3-c-Kit and Ba/F3-c-Kit/Y823F constructs. Cells have been serumstarved overnight at 37 and stimulated by SCF for the indicated time periods. Cell lysates were prepared, immunoprecipitated with anti-c-Kit antibody, and analyzed by Western blotting. Total receptor phosphorylation was detected using phosphotyrosine (pY) antibody, and c-Kit was utilized as a loading handle.there is either a conformational change within the Y823F mutant or that the mutant c-Kit stability is altered. Ubiquitination and Degradation Are Far more Fast inside the Y823F Mutant Than in Wild-type c-Kit–It has been shown previously that receptor tyrosine kinases, upon ligand stimulation, undergo monoubiquitination or polyubiquitination that targets them to be internalized and degraded inside the lysosomes or the proteasomes, respectively (1). Prior studies demonstrate that Cbl, which belongs to a family members of ubiquitin E3 ligases, is important for ubiquitination of c-Kit (1, 7, eight). In addition, Cbl can either directly interact with c-Kit at Tyr-568 and Tyr-936 (1) or indirectly via the adapter protein Grb2 (eight). This leads to monoubiquitination of c-Kit and targets it for degradation in lysosomes. We wanted to investigate no matter whether mutation of Tyr-823 within the activation loop of c-Kit affects the phosphorylation of Cbl, which could result in alterations in ubiquitination that, in turn, could affect receptor degradation. Cbl was immunoprecipitated from both wild-type and Y823F mutant c-Kit following ligand stimulation, and the extent of phosphorylation of Cbl was analyzed. The results demonstrate clearly that Cbl is phosphorylated currently immediately after two min. of SCF stimulation in each c-Kit WT and c-Kit-Y823F but that the mutant is unable to sustain the phosphorylation (Fig. 2A). To assess the degree of ubiquitination, c-Kit was immunoprecipitated, and Western blotting was performed using an antibody against ubiquitin. InVOLUME 288 Number 31 AUGUST two,22462 JOURNAL OF BIOLOGICAL CHEMISTRYPhosphorylation of Tyr-823 Is Crucial for c-Kit SignalingFIGURE two. The Y823F mutant of c-Kit mediates elevated phosphorylation of Cbl concomitant with increased ubiquitination and degradation of c-Kit. A, Ba/F3-c-Kit and Ba/F3-c-Kit/Y823F cells had been serum-starved and stimulated with 100 ng/ml SCF for the indicated occasions. Cells had been lysed, and lysates have been immunoprecipitated (IP) with anti-Cbl antibody, followed by Western blotting with phosphotyrosine antibodies and having a c-Kit antibody.Polymyxin B B, lysates from serum-starved Ba/F3-c-Kit and Ba/F3-c-Kit/Y823F cells had been immunoprecipitated with anti-c-Kit antibody.Sacubitril Ubiquitination (Ub) from the receptor was detected making use of anti-Ub antibody.PMID:23443926 C, receptor degradation was assessed by serum starvation of Ba/F3-c-Kit and Ba/F3-c-Kit/Y823F cells for four h at 37 within the presence of one hundred g/ml of cycloheximide. Cells had been then stimulated with 100 ng/ml SCF for indicated periods of time, and cells were immediately placed on ice. Cell lysates had been ready and subjected to immunoprecipitation with c-Kit antibodies, followed by immunodetection using c-Kit antibody. D, signal intensities from two independent experiments were quantified employing Multi-Gauge application to calculate the percentage of receptor d.