Tyrosyl phosphorylated PAK1 regulates breast cancer cell motility in response to prolactin through filamin A

Abstract

The p21-activated serine-threonine kinase (PAK1) is activated by small GTPase-dependent and -independent mechanisms and regulates cell motility. Both PAK1 and the hormone prolactin (PRL) have been implicated in breast cancer by numerous studies. We have previously shown that the PRL-activated tyrosine kinase JAK2 (Janus tyrosine kinase 2) phosphorylates PAK1 in vivo and identified tyrosines (Tyr) 153, 201, and 285 in the PAK1 molecule as sites of JAK2 tyrosyl phosphorylation. Here, we have used human breast cancer T47D cells stably overexpressing PAK1 wild type or PAK1 Y3F mutant in which Tyr(s) 153, 201, and 285 were mutated to phenylalanines to demonstrate that phosphorylation of these three tyrosines are required for maximal PRL-dependent ruffling. In addition, phosphorylation of these three tyrosines is required for increased migration of T47D cells in response to PRL as assessed by two independent motility assays. Finally, we show that PAK1 phosphorylates serine (Ser) 2152 of the actin-binding protein filamin A to a greater extent when PAK1 is tyrosyl phosphorylated by JAK2. Down-regulation of PAK1 or filamin A abolishes the effect of PRL on cell migration. Thus, our data presented here bring some insight into the mechanism of PRL-stimulated motility of breast cancer cells.

Figure 1.

Characterization of T47D cell lines stably expressing GFP alone, or GFP with myc-tagged PAK1 WT and PAK1 Y3F. A, T47D cells stably expressing GFP (lane 1), two clones stably expressing myc-PAK1 WT (lanes 2 and 3), two clones stably expressing myc-PAK1 Y3F (lanes 4 and 5), and parental T47D cells (lane 6) were lysed, and proteins were resolved by SDS-PAGE. Overexpressed proteins were visualized by immunoblotting with antimyc Ab (upper panel). The same membrane was stripped and reblotted with anti-PAK1 Ab to visualize endogenous (lanes 1 and 6) and overexpressed PAK1 (lanes 2–5) (middle panel). B, myc-PAK1 was IP'd with antimyc Ab from T47D PAK1 WT (lanes 1–2) or T47D PAK1 Y3F cells (lanes 3–4) transfected with vector (lanes 1 and 3) or constitutive active Rac1 V12 (lanes 2 and 4). IP'd myc-PAK1 was subjected to an in vitro kinase assay with H4 histone as a substrate (³²P incorporation into H4 histone indicated in upper panel), and probed with anti-PAK1 (middle panel) and anti-Rac1 (lower panel) Ab. All blots are representative of at least three experiments. C, T47D PAK1 WT and T47D PAK1 Y3F cells were treated with or without PRL. Myc-PAK1 was IP'd with antimyc Ab and subjected to the in vitro kinase assay as described in panel B. ³²P incorporation into PAK1 molecule (PAK1 autophosphorylation) is indicated in the middle panel. Relative PAK1 kinase activity was then normalized by the amount of IP'd PAK1 for each lane and plotted (right plot). Bars represent mean ± SE * P < 0 .05. n = 3. ctrl, Control.

Figure 2.

PAK1 WT enhances and PAK1 Y3F inhibits PRL-induced membrane ruffling. A, T47D cells stably expressing GFP alone [vector (vctr)], or GFP plus myc-tagged PAK1 WT or PAK1 Y3F were treated with or without PRL. Filamentous actin was visualized by staining with Texas Red-phalloidin (shown). Scale bar, 50 μm. C, MCF-7 cells were transfected with plasmid encoding GFP (vctr), myc-tagged PAK1 WT, or PAK1 Y3F and treated as in panel A. Scale bar, 50 μm. Arrows indicate representative ruffles in both transfected and untransfected cells, and asterisks denote transfected cells. B and D, Cells positive for GFP or antimyc were scored for the presence of ruffles for each experimental condition. The ruffling index as a number of ruffles per cell was counted. Bars represent mean ± SE. ^# P < 0.05 compared with cells expressing GFP (vctr) with the same treatment. Each experiment was repeated three times with 100 cells each time. n = 3 for each experimental condition.

Figure 3.

Effect of PRL-dependent pTyr-PAK1 on closure of wounded monolayer. Monolayers of T47D cells stably overexpressing GFP, PAK1 WT, or PAK1 Y3F were wounded and cultured without (white bar) or with (black bar) PRL. Bars represent mean ± SE. * P < .05 compared with the same cells untreated with PRL. Each experiment was repeated three times with 10 measurements each time. n = 3 for each experimental condition. Scale bar, 200 μm.

Figure 4.

Maximal migration of T47D cells in response to PRL requires tyrosyl phosphorylation of PAK1. A, Equal amount of T47D cells stably overexpressing GFP, PAK1 WT, or PAK1 Y3F were loaded into the upper part of the Boyden chamber. The number of cells that migrated to the lower surface of the chamber toward PRL (black bar) or buffer control (white bar) after 48 h were counted and plotted (B). Scale bar, 300 μm. C and D, T47D cells (C) or MCF-7 cells (D) were transfected with control or PAK1 siRNA, stimulated (black bars) or not (white bars) with PRL, and assessed for migration using the Boyden chamber assay as in panel A. Silencing efficiency was judged by immunoblotting with anti-PAK1 Ab 48 and 72 h after transfection. The expression levels of γ-tubulin (Tu) were used as an internal control (Ctrl). Bars represent mean ± SE. * P < 0.05 compared with the same cells untreated with PRL. n = 3 for each experimental condition.

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.

Figure 6.

FLNa is required for PAK1 function in PRL-induced cell migration. A, T47D GFP cells, T47D PAK1 WT, or T47D PAK1 Y3F cells were transfected with control (ctrl) or FLNa siRNA. Endogenous FLNa was immunoblotted with anti-FLNa in 72 h after transfection. The expression levels of γ-tubulin (Tu) were used as an internal control. B, FLNa was depleted from the indicated cells as described in panel A, and the cells were assessed for migration using the Boyden chamber assay with (black bars) or without (white bars) PRL. Bars represent mean ± SE. * P < 0.05 compared with the same cells untreated with PRL. n = 3 for each experimental condition.

Figure 7.

Schematic representation of the proposed working model. Initiation of PRL signaling involves PRL binding to PRL receptor (PRL-R) at the plasma membrane and activation of the tyrosine kinase JAK2, which, in turn, phosphorylates PRL-R. Phosphorylated tyrosines within the receptor and JAK2 recruit an array of signaling proteins, including PAK1. JAK2 tyrosyl phosphorylates PAK1 on Tyr(s) 153, 201, and 285, thereby increases PAK1 activities (both the serine/threonine kinase activity and ability to create potential protein-protein interactions) and stimulates phosphorylation of FLNa on Ser 2152. Phosphorylated FLNa has increased actin-regulating activity to stimulate cell migration and enhances PAK1 kinase activity by a positive feedback loop.