Par1 phosphorylation on S366 and Par1b-depedendent S453/4/5 phosphorylation promote 14-3-3 binding of IRSp53. (A) Diagram of Par1b-dependent phosphorylation sites in IRSp53. (B) In vitro kinase assays with WT and Par1b-KA using [³²P]γATP and IRSp53-CT (WT and point mutants) as substrates. (C) IRSp53-HA–transfected cells (WT, A4, and S453-5A) were transduced with control (c) or Par1b virus, and the HA IPs were probed for P-serine and subsequently for HA. (D) In vitro kinase assays with WT and Par1b-ATP* using [³⁵S]benzyl-ATPγS and full-length IRSp53 WT and mutants as substrates. Autoradiographs/Coomassie stain of IRSp53. (E) Triton X-100 lysates of cells coexpressing Par1b-WT or Par1b-ATP* and WT IRSp53-Myc or IRSp53 mutants were incubated with [³⁵S]benzyl-ATPγS. Autoradiographs and IB for IRSp53-Myc and Par1b. (F) Recombinant WT IRSp53 and mutant proteins subjected to in vitro kinase assays with Par1b-WT or Par1b-KA were affinity isolated on 14-3-3 beads and competitively eluted (top). IRSp53 input (middle) and Par1b levels (bottom) are also shown. (G) 14-3-3 binding of IRSp53 proteins from cell lysates. Note the lower expression of the S117A sample accounts for the weaker signal in the bound fraction. The graph shows SEM from six experiments (three experiments for S117A/S148A). (H) Co-IP of 14-3-3-HA with IRSp53-Myc proteins from cell lysates.