c-Abl-mediated Phosphorylation of WAVE3 Is Required for Lamellipodia Formation and Cell Migration

Abstract

The activity of the Wiskott-Aldrich syndrome-related WAVE3 protein is critical for the regulation of the Arp2/3-dependent cytoskeleton organization downstream of Rac-GTPase. The Ableson (Abl) non-receptor tyrosine kinase is also involved in the remolding of actin cytoskeleton in response to extracellular stimuli. Here we show that platelet-derived growth factor stimulation of cultured cells results in WAVE3-Abl interaction and localization to the cell periphery. WAVE3-Abl interaction promotes the tyrosine phosphorylation of WAVE3 by Abl, and STI-571, a specific inhibitor of Abl kinase activity, abrogates the Abl-mediated phosphorylation of WAVE3. We have also shown that Abl targets and phosphorylates four tyrosine residues in WAVE3 and that the Abl-dependent phosphorylation of WAVE3 is critical for the stimulation of lamellipodia formation and cell migration. Our results show that the activation of WAVE3 to promote actin remodeling is enhanced by the c-Ablmediated tyrosine phosphorylation of WAVE3.The actin cytoskeleton plays an important role in cell shape and cell motility (1). Members of the Wiskott-Aldrich syndrome protein (WASP) 2 family that include WASP, neuronal-WASP (N-WASP) and WAVE1, -2, and -3 play crucial roles in actin polymerization through the activation of the actin-related protein (Arp) 2/3 complex (2-4). All five members of the WASP family of proteins contain a conserved C terminus verprolin (V), cofilin (C), and acidic (A) region (VCA domain), which binds to the Arp2/3 complex and stimulates its actin polymerization activity (5). The regulation of the activity of both WASP and N-WASP is thought to be achieved by their inclusion in inactive conformations through intramolecular interaction between the GTPase-binding domain and the VCA region (2, 6, 7). The binding of CDC42, or SH3-containing proteins such as WISH and NCK to the proline-rich domain of N-WASP/WASP alleviates their inhibition by disrupting this intramolecular interaction, leading to the activation of N-WASP and WASP (8 -11). On the other hand, the activity of the WAVE proteins is believed to be regulated by their inclusion in a protein complex that also contains PIR121/NAP1/ Abi/HSPC300, which keeps them in an inactive state (12-16). This inhibition can be lifted by active small GTPases such as GTP-Rac (13), leading to the translocation of the complex to sites of active actin polymerization (14, 16).Phosphorylation of the WASP/WAVE proteins has been shown to regulate their activity (17-21). Phosphorylation of N-WASP/WASP proteins by non-receptor tyrosine kinases, such as ACK1 enhances the ability of WASP to stimulate actin polymerization (22), whereas growth factor stimulation of cultured cells results in hyper-phosphorylation of the WAVE proteins as well as a delay in their mobility in SDS-PAGE (23). WAVE1 was shown to be phosphorylated by the cyclindependent kinase 5 (24), whereas phosphorylation of WAVE2 has recently been shown to involve the non-receptor tyrosine kinase c-Abl (25), resulting in both cases in the stimulation ...

Activation of Abl Family Kinases in Solid Tumors

“…1). Moreover, Mader et al 100 demonstrated that Arg localized to invadopodia and promoted EGFR/Src/cortactin-mediated matrix degradation, actin polymerization, invadopodia formation, and Matrigel transwell invasion of MDA-MB-231 cells, and Sossey-Alaoui and colleagues 101 showed that c-Abl not only promotes matrix degradation but also stimulates PDGF-directed motility of MDA-MB-231 cells via c-Abl-dependent phosphorylation of WAVE3 (Fig. 1).…”

“…One possibility may involve staining tumor serial sections with antibodies directed against (1) c-Abl and Arg to examine overall expression, (2) c-Abl/Arg substrates (e.g., Crk/CrkL, cortactin, WAVE3, galectin-3) 83,100,101,103,123,125,126 to indirectly examine kinase activity, and (3) proteins known to act downstream of c-Abl/Arg in a particular cancer type (e.g., STAT3, p38, p68, ERK5). 83,86,89,127 Alternatively, for tumor types containing low levels of stromal tissue and other non-tumor-associated cells, direct assessment of activity could be determined by kinase assay on tumor homogenates if enough sample is available.…”

Multifunctional Abl kinases in health and disease

“…Abl kinases are transiently activated by growth factors that stimulate RTKs and lead to actin cytoskeleton reorganization that is required for the formation of lamellipodial protrusions, membrane dorsal ruffles, cell migration, invasion, cell scattering and/or tubulogenesis (Boyle et al, 2007;Li et al, 2015;Plattner et al, 2003Plattner et al, , 1999. Abl-dependent regulation of cytoskeletal dynamics is mediated by the phosphorylation of target proteins, such as cortactin that is required for platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles (Boyle et al, 2007), as well as actin nucleatingpromoting factors, such as N-WASP and WAVE proteins required for membrane protrusions (Burton et al, 2005;Miller et al, 2004;Sossey-Alaoui et al, 2007;Stuart et al, 2006). Abl kinases also transduce signals from cell surface receptors, such as integrins, cadherins, chemokine receptors and the T cell receptor (TCR), to reorganize the cytoskeleton.…”

Organization of F-Actin via Concerted Regulation of Kette by PTP61F and dAbl

“…Thus, a phosphatase is capable of controlling the extent and duration of signaling events via synergistic dephosphorylation of a kinase and its substrate. It is also important to note that, in addition to the direct regulation of Kette, PTP61F may participate in control of the actin cytoskeleton indirectly through inactivation of dAbl, which may phosphorylate SCAR/ WAVE for regulation of cell spreading, lamellipodium formation, and cell migration, as evidenced in mammals (43,46). If this is the case, to terminate such signaling cascades, PTP61F may simply dephosphorylate dAbl, thus downregulating the activity of SCAR/WAVE indirectly.…”