Profilin1 regulates PI(3,4)P ₂ and lamellipodin accumulation at the leading edge thus influencing motility of MDA-MB-231 cells

Abstract

Profilin1, a ubiquitously expressed actin-binding protein, plays a critical role in cell migration through actin cytoskeletal regulation. Given the traditional view of profilin1 as a promigratory molecule, it is difficult to reconcile observations that profilin1 is down-regulated in various invasive adenocarcinomas and that reduced profilin1 expression actually confers increased motility to certain adenocarcinoma cells. In this study, we show that profilin1 negatively regulates lamellipodin targeting to the leading edge in MDA-MB-231 breast cancer cells and normal cells; profilin1 depletion increases lamellipodin concentration at the lamellipodial tip (where it binds Ena/VASP), and this mediates the hypermotility. We report that the molecular mechanism underlying profilin1's modulation of lamellipodin localization relates to phosphoinositide control. Specifically, we show that phosphoinositide binding of profilin1 inhibits the motility of MDA-MB-231 cells by negatively regulating PI(3,4)P 2 at the membrane and thereby limiting recruitment of lamellipodin [a PI(3,4)P 2 -binding protein] and Ena/ VASP to the leading edge. In summary, this study uncovers a unique biological consequence of profilin1-phosphoinositide interaction, thus providing direct evidence of profilin1's regulation of cell migration independent of its actin-related activity.T he ubiquitously expressed cytoskeleton-modulating protein profilin1 influences multiple processes involved in cell motility, making it a challenge to elucidate the exact molecular mechanism that controls migration. At least one major function of profilin1 is to regulate actin polymerization. Profilin1 regenerates actin monomers from disassembling filament networks by facilitating the exchange of ATP for ADP on G-actin. By further inhibiting spontaneous nucleation of G-actin, profilin1 causes an accumulation of profilin1/ATP-G-actin pool available for polymerization. Because profilin1 also has an affinity for poly-Lproline sequences, it binds to almost all major actin nucleating and F-actin elongating proteins that contain proline-rich domains [e.g., N-WASP (neuronal Wiskott-Aldrich syndrome protein), Ena (enabled)/VASP (vasodilator stimulated phosphoprotein), and formins], and this allows profilin1-mediated recruitment of ATP-G-actin to these proteins, enhancing actin polymerization (1, 2). In addition, profilin1 binds to plasma membrane presumably through its interactions with various phosphoinositides (3). Profilin1 binds to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], phosphatidylinositol-3,4-bisphosphate [PI(3,4)P 2 ], and phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5) P 3 ]), at least in vitro (4). Based on PI(4,5)P 2 binding, it has been proposed that the phosphoinositide binding site of profilin1 overlaps with its actin-binding site (5), and to some extent spans a second region neighboring the polyproline binding site (6). This has prompted speculation that the major role of phosphoinositide binding of profilin1 would be to inhibit its interaction with act...

Epigenetic regulation of Smad2 and Smad3 by profilin-2 promotes lung cancer growth and metastasis

“…Profilin-1 can function as a negative regulator for the growth and migration of breast cancer cells [19][20][21][22][23] . However, a recent report has suggested the opposite roles of profilin-1 and profilin-2 in the migration and invasion of breast cancer cells 24 .…”

Parallel up-regulation of the profilin gene family following independent domestication of diploid and allopolyploid cotton ( Gossypium )

“…This is achieved, in part, by the interaction of profilin with the formin class of actin nucleators to promote actin polymerization and elongation (22)(23)(24). However, profilin also binds to phosphoinositides and other proteins (25,26), suggesting that it plays a broad role in modulating the actin cytoskeleton.…”

A Phosphatidylinositol Lipids System, Lamellipodin, and Ena/VASP Regulate Dynamic Morphology of Multipolar Migrating Cells in the Developing Cerebral Cortex

“…In HeLa cells, transfection of the catalytic domain-deleted SHIP2 and silencing of SHIP2 inhibited cell spreading and lamellipodia formation, respectively (Prasad et al, 2001;Prasad and Decker, 2005). In addition, Profilin1 inhibits MDA-MB-231 breast cancer cell motility by negatively regulating PI(3,4)P 2 at the membrane, thereby limiting the recruitment of Lpd and Ena/VASP proteins to the leading edge (Bae et al, 2010).…”

“…To detect Lpd, VASP, and NeuroD, coverslips were incubated at 70°C in 10% HistoVT One (Nacalai) for 20 min before treatment with the primary antibody for antigen retrieval. PI(3,4)P 2 staining using anti-PI(3,4)P 2 mouse IgG (1:800 echelon; in 5% BSA/PBS) was performed as described previously (Bae et al, 2010). Briefly, the cells were fixed and permeabilized with 3.7% formaldehyde/0.1% glutaraldehyde in 0.15 mg/ml saponin solution for 1 h at 37°C, followed by overnight incubation with the primary antibody at 4°C.…”

“…To investigate the distribution of PI(3,4)P 2 in multipolar cells, we labeled the multipolar cells in the MAZ with an mCherry expression vector, dissociated the cells, cultured them from E16.5 for 12 h, and then performed immunocytochemistry using a specific antibody for PI(3,4)P 2 (Bae et al, 2010). As a result, immunoreactivity was found in the growth cone-like structures and limited parts of the soma and processes (Fig.…”