Ed making use of an antibody against ubiquitin. InVOLUME 288 ?Number 31 ?AUGUST 2,22462 JOURNAL OF BIOLOGICAL CHEMISTRYPhosphorylation of Tyr-823 Is Essential for c-Kit SignalingFIGURE two. The Y823F mutant of c-Kit mediates enhanced phosphorylation of Cbl concomitant with enhanced ubiquitination and degradation of c-Kit. A, Ba/F3-c-Kit and Ba/F3-c-Kit/Y823F cells have been serum-starved and stimulated with one hundred ng/ml SCF for the indicated times. 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. B, lysates from serum-starved Ba/F3-c-Kit and Ba/F3-c-Kit/Y823F cells had been immunoprecipitated with anti-c-Kit antibody. Ubiquitination (Ub) on the receptor was detected using anti-Ub antibody. C, receptor degradation was assessed by serum starvation of Ba/F3-c-Kit and Ba/F3-c-Kit/Y823F cells for 4 h at 37 inside the presence of one hundred g/ml of cycloheximide. Cells had been then stimulated with one hundred ng/ml SCF for indicated periods of time, and cells had been promptly placed on ice. Cell lysates had been prepared and subjected to immunoprecipitation with c-Kit antibodies, followed by immunodetection making use of c-Kit antibody. D, signal intensities from two independent experiments were quantified using Multi-Gauge software to calculate the percentage of receptor degradation.agreement with the Cbl phosphorylation information, ubiquitination of c-Kit Y823F is extremely sturdy right after two min of SCF stimulation but then declines quickly. In contrast, ubiquitination of wild-type c-Kit is weaker but increases more than a longer time period (Fig. 2B). Degradation of c-Kit was analyzed by starving Ba/F3 cells within the presence of cycloheximide, an inhibitor of protein synthesis, for 4 h, followed by immunoprecipitation and detection employing anti-c-Kit antibody. Degradation on the c-Kit receptor was analyzed for up to 30 min of SCF stimulation. We observed that in wild-type c-Kit-expressing cells, c-Kit is detectable for up to 30 min, whereas in cells expressing the Y823F mutant, c-Kit is degraded prior to 15 min of SCF stimulation (Fig. 2, C and D). A adjust in Activation Loop Tyr-823 to Phenylalanine Causes the Receptor to Internalize Faster–It is recognized that, collectively with the JM domain, the activation loop also plays a part in sustaining the receptor in an inactivated state. Phosphorylation of Tyr-823 and of tyrosine residues within the JM domain is important to preserve the receptor in an active state. Furthermore, preceding reports show that Cbl, as well as regulating the degradation of receptor tyrosine kinases, also regulates their internalization. As a result, we analyzed the pattern of receptor internalization in response to ligand activation.Formula of 877399-31-0 Ba/F3 cells had been serum-starved inside the presence of cycloheximide for 4 h and stimulated with SCF for the indicated periods of time.Formula of 2-Bromo-1-cyclohexylethan-1-one Cells had been stained using a phycoerythrin-labeled anti-c-Kit antibody, and surface expression was determined by flow cytometry (Fig.PMID:33529468 3A). As an alternative strategy, we verified the rate of internalization of cell surface c-Kit compared using the total c-Kit by Western blotting (Fig. three, B and C). The data clearly demonstrate that the Y823F mutant of c-Kit is additional quickly internalized than wild-type c-Kit.AUGUST two, 2013 ?VOLUME 288 ?NUMBERMutation of Activation Loop Tyrosine 823 Has No Impact on Kinase Activity–Activation of c-Kit by its ligand triggers activation of a lot of downstream signal transduction molecules. Some of these molec.