Figure 4.

Identification of Jade-1S site S377 phosphorylated in the presence of over-expressed CK1α. A) MS2 spectrum demonstrating Serine 377 phosphorylation on the Jade-1S fragment 373-394. B) Quantitative phosphoproteomic analysis of Jade phosphorylation change in the absence and presence of CK1α in whole cells. The S377 site is marked by an asterisk. Only high-quality (class I) sites are shown, with all Jade-1S sites labeled accordingly. C) Schematic illustrating the amino acid sequences of the N-terminal CK1α phosphorylation site (S18/S20, underlined) corresponding to a putative Polobox-binding domain, and the C-terminal S377 motif corresponding to a PLK1 phosphorylation consensus motif. Green represents shared amino acid sequences while red represents those that differ (X = any amino acid; Φ = hydrophobic amino acid; p = phosphorylated). D and E) 293T cells were transiently transfected with constructs as indicated and processed as whole cell lysates 24 hours later. Expression of V5.Jade-1 was heavily modified by overexpressed F.PLK1 (D). This effect was not seen when V5.Jade-1 was over-expressed with a kinase-dead mutant of F.PLK1 (E). IB, immunoblot. WCL, whole cell lysate.