FIG. 5.

Chk1 is also phosphorylated on serine 345 in vivo. 293 cells were transfected with plasmids expressing myc-Chk1 (wild type), myc-Chk1(S317A), myc-Chk1(S345A), and myc-Chk1(S357A, S366A). Cells were incubated with ³²P-labeled inorganic phosphate in the presence of HU. Radiolabeled Chk1 (wild type and mutants) was digested with trypsin followed by Asp-N endopeptidase, and peptides were subjected to two-dimensional phosphopeptide mapping. Arrows indicate the direction of the first and second dimensions, and the origin is located where the two arrows meet.