Fig. 3. hKIS phosphorylates p27Kip1 on S10. (A) Two-dimensional tryptic phosphopeptide mapping of wild-type and mutant p27Kip1. p27Kip1, p27(S10A) or p27(T187A) were expressed in HEK 293 cells and metabolically labelled with [32P]orthophosphate in the presence of 50 µM lactacystin. The recombinant proteins were immunoprecipitated with p27Kip1 C19 antibody and subjected to 2D tryptic phosphopeptide mapping. Major phosphopeptides are numbered 1 and 2. Phosphopeptides containing S10 are indicated by arrows. An asterisk indicates the origin of migration, and arrows show the directions of separation by thin layer chromatography (TLC) and electrophoresis. (B) Mouse polyclonal antibodies recognize phosphorylated p27Kip1 on S10. A western blot of recombinant p27Kip1 phosphorylated by hKIS was performed with a p27S10-p antibody absorbed with non- phosphorylated recombinant p27Kip1 protein (Control, lane 2) or S10-p p27Kip1 peptide (lane 1). Phosphorylated p27Kip1 was incubated with (+) or without (–) CIAP, and a western blot with p27S10-p antibody was performed (lanes 3 and 4). The same blot was stripped and reprobed with a monoclonal p27Kip1 K25020 antibody (lanes 5 and 6). Recombinant p27Kip1 was phosphorylated by hKIS in the presence of [γ-32P]ATP incubated with (+, lane 8) or without CIAP (–, lane 7). HEK 293 cell lysates were analysed on SDS–PAGE and a western blot was performed using the p27S10-p antibody (upper panel, lanes 9 and 10). The same blot was stripped and reprobed with a p27Kip1 C19 antibody (lower panel, lanes 9 and 10). (C) hKIS phosphorylates p27Kip1 on S10. Recombinant p27Kip1 protein was incubated with immunoprecipitated, in vitro-transcribed and -translated hKIS (+, lane 2), and a western blot with p27S10-p antibody was performed. The same blot was stripped and reprobed with a p27Kip1 K25020 antibody (lanes 1 and 2, lower panel).