Technical NoteDetermination of protein phosphorylation in FcεRI-activated human mast cells by immunoblot analysis requires protein extraction under denaturing conditions
Introduction
As with other members of the immunoglobulin receptor superfamily, the signal transduction cascade following FcεRI aggregation in mast cells is largely regulated by a series of protein phosphorylation steps catalyzed by tyrosine, serine/threonine and/or mixed acting kinases (Kinet, 1999). The phosphorylation status of specific regulatory residues within signaling molecules correlates well with the event(s) mediated by these proteins (Zhang et al., 2000). Thus, antibodies that recognize phosphorylated peptide sequences, corresponding to activation motifs specific to these signaling molecules, can be utilized to monitor-specific cellular signal transduction events. As these antibodies can be used to probe whole cell lysates, this avoids the requirement for immunoprecipitation steps. Furthermore, these antibodies avoid interpretational problem where both inhibitory and activation residues are phosphorylated within the same molecule.
Unfortunately, mast cells contain a high content of both granule associated proteases and phosphatases (Metcalfe et al., 1997). Indeed, 25% or more of total mast cell protein mass is accounted for by proteases (Schwartz et al., 1981). This presents a significant problem during cell lysing and protein extraction as the signaling proteins are rapidly exposed to these proteases and can rapidly degrade. As human mast cells contain approximately 10 times more granule contents than do other models of mast cell function such as the RBL 2H3 cells (unpublished observations), this is more of a concern for studies conducted in human mast cells compared to other cell types. The data presented in this communication demonstrate significant proteolysis of certain intracellular signaling proteins taking place when cells are extracted with nonionic detergents such as Triton-X-100. This resulted in compromised results in studies utilizing anti-phosphotyrosine and activation state antibodies. This problem, however, can be largely circumvented by rapidly extracting the cellular proteins under denaturing conditions. These observations would be pertinent to signaling studies conducted in any cell type expressing high concentrations of proteases.
Section snippets
Methods
Human mast cells (HuMCs) were obtained from peripheral blood CD34+ cells as described (Kirshenbaum et al., 1999). The cells were maintained in culture for 8–10 weeks by which time, the population was between 98% and 100% mast cells. The cells were sensitized overnight in culture media containing chimeric human Fc anti-4-hydroxy-3-nitrophenylacetyl (NP)-specific IgE (NP-IgE) (Serotec, Raleigh, NC) (1 μg/ml). Following rinsing in HEPES buffer (Lin et al., 1991) containing BSA (0.04%), cells were
Results and discussion
To examine total protein phosphorylation, control and activated HuMCs were lysed using either Triton-X-100 (1%) lysis buffer or denaturing lysis buffer. From Fig. 1a, it can be seen that, although an increase in tyrosine phosphorylation was observed following cell activation under both extraction conditions, a greater recovery of tyrosine phosphorylated proteins, particularly those of 25 and 72–76 kDa, was observed in both untreated and treated cells extracted with the denaturing lysis buffer.
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