Figure 4.

Cortactin acts downstream of Paks in IL-2Rβ endocytosis. (A) In vitro phosphorylation of cortactin by Pak1. Purified GST-Pak1 (1 μg) was incubated with 5 μg of purified GST-cortactin (GST-Cort) or GST and 5 μCi γ[³²P]ATP. Autoradiography (left), Coomassie blue gel (right). (B) Hep2β cells were transfected with myc-PID and GFP. (C) Hep2β cells were transfected with myc-PID and GFP-cortactin wild-type (Cort^WT). Cells were treated and analysed as described in Fig 1 (asterisks indicate the cells coexpressing the constructs). (D) Quantification of the endocytosis results was carried out as described in Fig 1C. (E) Hep2β cells were transfected with small interfering RNA (siRNA) against Pak1 (siRNAPak1) and/or Pak2 (siRNAPak2) or against an irrelevant protein (siRNAC) and transfected either with GFP-cortactin or GFP. Quantification of the endocytosis results was carried out as described in Fig 1C (mean±s.e.; n=50 cells in two independent experiments). GFP, green fluorescent protein; GST, glutathione-S-transferase; IL-2Rβ, interleukin-2 receptor β; Pak, p21-activated kinase; PID, Pak inhibitory domain; Rac1, Ras-related C3 botulinum toxin substrate 1; Tf, transferrin.