Pak1p phosphorylates Rlc1p in vitro and in vivo, and Pak1p localization to the actomyosin ring is dependent on Rlc1p. (A) Recombinant Pak1p (GST-Pak1p 98 kD) was incubated with MBP, Rlc1p (GST-Rlc1p 47 kD), or Rlc1p-S35AS36A in the presence of γ-[³²P]ATP. The left panel shows Pak1p autophosphorylation and Pak1p phosphorylation of wild-type (wt) Rlc1p but not the Rlc1p-SA mutant. The right panel shows Pak1p and Rlc1p proteins in this assay. (B) 2D SDS-PAGE resolution of Rlc1p-Myc (arrowheads) in early anaphase cells of wild-type (top) and pak1-M460G strains (bottom). (C) Cell images of Pak1p-GFP in prometaphase cells (arrows) and in interphase cells (asterisks) of the nda3-KM311 rlc1–null strain at a nonpermissive temperature. These cells were also fixed and stained with phalloidin Alexa Fluor 488. Bars, 5 μm.