Formation of a scattered phenotype is a central problem in understanding both branching morphogenesis and cancer cell metastatic phenotypes. Using PY proteomics, we have identified candidate cell scattering-modifying proteins downstream of the oncogenic nonreceptor tyrosine kinase Src. Our data support a central biological role for AnxA2 phosphorylation on Tyr23 in the regulation of cell scattering and EMT-mediated processes. Thus, our results indicate that tyrosine phosphorylation of AnxA2 by itself enhances cell spreading and protrusiveness and induces cell scattering in 2D and HGF-independent branching morphogenesis in 3D. Importantly, shRNA-mediated knockdown of AnxA2 inhibited cell spreading, scattering, and normal cystogenesis. The AnxA2 tyrosine phosphorylation-driven events occurred in association with an altered F-actin cytoskeletal rearrangement in association with activation of the cofilin pathway.
Besides AnxA2, also other v-Src targets were identified. Most of these identified, differentially phosphorylated proteins are (in)directly associated with v-Src-induced cytoskeletal restructuring. Ezrin is phosphorylated on two residues, of which at least Src-induced phosphorylation of Y145 is required for the regulation of cytoskeletal reorganization prior to cell scattering (29
). v-Src caused at least the phosphorylation of Y477 in MDCK cells (M. de Graauw et al., unpublished data), but the cell biological role of this phosphorylation needs further evaluation. In addition, vinculin is a direct target of Src kinase and mutation of the tyrosine residue results in impaired cell spreading (37
). The close family members AnxA1 and AnxA2, which both bind F-actin, belong to the first proteins phosphorylated in relation to v-Src kinase activation. While AnxA1 has been shown to be phosphorylated by the epidermal growth factor receptor, AnxA2 has previously been identified as a direct target for Src kinase (22
). The combined phosphorylation of these different cytoskeleton-associated proteins may be required for the well-coordinated reorganization and/or turnover of the actin cytoskeletal network, thereby facilitating cell scattering. However, for a large portion, the Y23E-AnxA2 phenotype mimics the v-Src-induced phenotype, suggesting that AnxA2 is not only the major tyrosine-phosphorylated protein but also a crucial player in v-Src-induced scattering.
AnxA2 is found at the membrane-actin cytoskeleton interface and is associated with sites of active actin remodeling, like rocketing macropinosomes (14
), actin-rich pedestals (38
), and lamellipodia (26
). Our data indicate that AnxA2 phosphorylation is important in actin remodeling. While Y23E-AnxA2-expressing cells showed a reduction in actin stress fiber formation, cells depleted of AnxA2 become enriched in stress fibers (Fig. ) (14
) and are defective in the formation of a polarized cyst with an actin-rich apical domain (Fig. ) (18
). AnxA2 localizes to regions of active actin remodeling enriched in the plasma membrane lipid phosphatidylinositol-4,5-bisphosphate PtdIns(4,5)P2
). The architecture of the PtdIns(4,5)P2
binding site in AnxA2 is not precisely known but depends, at least in part, on its unique N-terminal domain since the C-terminal AnxA2 core domain does not efficiently compete with full-length AnxA2 for PtdIns(4,5)P2
). Since Tyr23 of AnxA2 is located within this region, phosphorylation apparently influences its binding to these PtdIns(4,5)P2
sites, most likely through altered protein conformation, thereby directing AnxA2 to cellular microenvironments with high actin remodeling activity and targeting other cytoskeletal and/or morphogenesis-regulating proteins, including Cdc42 and the Par6/aPKC complex (18
AnxA2-mediated actin remodeling is related to its ability to influence the phosphorylation status (i.e., functional activation) of the actin-severing protein cofilin. We propose that Y23E-AnxA2-induced cell scattering is directly related to cofilin dephosphorylation. Firstly, two independent constructs of the phosphorylation-mimicking cofilin S3E mutant (HA and GFP tagged) inhibited cell scattering while phospho-defective S3A did not affect scattering. Secondly, pharmacological inhibition of the ROCK/cofilin pathway with Y27632 caused cofilin dephosphorylation (Fig. ), loss of F-actin stress fibers and cellular tension, and increased MDCK cell scattering and uncontrolled branching morphogenesis (unpublished observation;6
). Importantly, depletion of AnxA2 inhibited Y27632-induced cofilin dephosphorylation (Fig. ) and cell scattering (unpublished observations), indicating a role for AnxA2 in the regulation of cofilin phosphorylation. Thirdly, the Rho/ROCK/LIMK pathway has been described as a major upstream effector pathway of cofilin modulation (31
). Despite the fact that AnxA2 binds RhoA and thereby regulates RhoA activation in Caco-2 cells (2
), in our studies with MDCK cells, Y23E-AnxA2 did not affect the activation status of RhoA upstream of cofilin. Nevertheless, expression of a constitutively active LIMK (HA-LIMK-508EE) significantly reduced the number of scattered Y23E-AnxA2 cells (Fig. ), suggesting that the pool of nonphosphorylated cofilin that drives the AnxA2-mediated scattering can be targeted by LIMK. Unfortunately, the potential activation of LIMK due to Y23E-AnxA2 expression could not be determined since no good working antibody was available. Together, these data support the notion that AnxA2 affects the rate of phosphorylated cofilin turnover, and thereby cell scattering, possibly by suppressing LIMK activity in a RhoA-independent manner or by activation of the cofilin phosphatase Slingshot. This needs further investigation.
Alternatively, AnxA2 may influence cofilin phosphorylation more directly via competition of binding to actin monomers. Cofilin affects actin polymerization through its ability to sequesters actin monomers in a phosphorylation-dependent manner (4
). It disassembles F-actin from the rear of the actin network to recycle actin monomers to the leading edge for further rounds of polymerization, thus driving lamellipodium formation. Also, AnxA2 binds and sequesters G-actin directly, thereby regulating actin filament turnover, most likely through monomer sequestration and barbed-end capping activities (14
). Phosphorylation of AnxA2 possibly affects its binding to monomers, explaining the diminished formation of stress fibers in Y23E-AnxA2 cells. In contrast, AnxA2 knockdown decreases the actin filament turnover, causing the formation of thick stress fibers. Since AnxA2 also has the ability to bind to the plasma membrane, it is able to deliver actin monomers directly to the cell cortex, where they are required for rapid polymerization.
Protein tyrosine kinases, including epidermal growth factor receptor family members and Src kinases, play an important role in cancer development and progression. This is often related to a mesenchymal/dedifferentiated phenotype of the tumor cells and a high metastatic potential. Interestingly, AnxA2 expression is elevated in a subset of metastatic breast tumor cells with a mesenchymal phenotype (M. de Graauw, A. M. Cleton-Janssen, and B. van de Water, unpublished observations). Therefore, the combination of high protein tyrosine kinase activity and high AnxA2 expression may ensure increased AnxA2 tyrosine phosphorylation, thereby driving cell scattering and supporting tumor metastasis formation. Consequently, upstream and downstream molecular programs that control and mediate the function of tyrosine-phosphorylated AnxA2 may be novel targets for therapeutic intervention.