Detection of phosphorylated T and B cell antigen receptor species by Phos-tag SDS- and Blue Native-PAGE

Abstract

Detection of phospho-proteins and differently phosphorylated forms of the same protein are important in understanding cell behaviour. One novel method is Phos-tag SDS-PAGE. A dinuclear Mn 2+ complex that binds to phosphate groups (the Phos-tag) is covalently attached to the polyacrylamide gel matrix. Thus, phosphorylated proteins are retarded in their migration and can be distinguished from their non-phosphorylated counterparts. We applied Phos-tag SDS-PAGE to the analysis of the ζ, CD3ɛ and CD3δ subunits of the T cell antigen receptor (TCR-CD3). Pervanadate stimulation generated six different phospho-ζ and each two different CD3ɛ and CD3δ forms. This corresponds to the phosphorylatable tyrosines on their cytoplasmic tails. The phosphorylation pattern was compatible with random phosphorylation events. Further, we showed that the Phos-tag technology can be applied to Blue Native (BN)-PAGE. This extends the applicability to the analysis of native protein complexes. Upon pervanadate stimulation the TCR-CD3 complex was predominantly detected as two distinct phospho-complexes. In contrast, the B cell antigen receptor (BCR) appeared as one phospho-form. Thus, Phos-tag BN-PAGE is useful for the analysis of different phosphorylation states of multiprotein complexes.