Figure 5.

JAK2-dependent phosphorylation of PAK1 increases FLNa Ser2152 phosphorylation. A, 293T cells were cotransfected with plasmid encoding myc-FLNa, HA-PAK1 WT or vector, and JAK2 WT or kinase-inactive JAK2 K882E mutant (K–E). Myc-FLNa was IP'd from cell lysates with antimyc Ab. Proteins were resolved by SDS-PAGE followed by immunoblotting with the indicated Abs. B, T47D cells were transfected with plasmid encoding myc-FLNa, HA-PAK1 WT or vector, and constitutively active JAK2 V617F or kinase-inactive JAK2 K882E mutant (K–E) and processed as in panel A. The graph represents the densiometric analysis of the band obtained for phosphorylated FLNa normalized with total FLNa for each lane. Bars represent mean ± SE. * P < 0.05 compared with cells without overexpressed PAK1. n = 5. C, FLNa was overexpressed in T47D GFP cells, T47D PAK1 WT, or T47D PAK1 Y3F cells. The cells were treated with or without PRL. FLNa was IP'd and immunobloted with anti-pSer 2152 and reblotted with anti-FLNa. The graph represents densiometric analysis of the band obtained for phosphorylated FLNa normalized with total FLNa for each lane. Bars represent mean ± SE. * P < 0.05 compared with the same cells untreated with PRL. n = 3.