Abstract
Invadopodia, actin-based protrusions of invasive carcinoma cells that focally activate extracellular matrix-degrading proteases, are essential for the migration and intravasation of tumor cells during dissemination from the primary tumor. We have previously shown that cortactin phosphorylation at tyrosine residues, in particular tyrosine 421, promotes actin polymerization at newly-forming invadopodia, promoting their maturation to matrix-degrading structures. However, the mechanism by which cells regulate the cortactin tyrosine phosphorylation-dephosphorylation cycle at invadopodia is unknown. Mena, an actin barbed-end capping protein antagonist, is expressed as various splice-isoforms. The MenaINV isoform is upregulated in migratory and invasive sub-populations of breast carcinoma cells, and is involved in tumor cell intravasation. Here we show that forced MenaINV expression increases invadopodium maturation to a far greater extent than equivalent expression of other Mena isoforms. MenaINV is recruited to invadopodium precursors just after their initial assembly at the plasma membrane, and promotes the phosphorylation of cortactin tyrosine 421 at invadopodia. In addition, we show that cortactin phosphorylation at tyrosine 421 is suppressed by the phosphatase PTP1B, and that PTP1B localization to the invadopodium is reduced by MenaINV expression. We conclude that MenaINV promotes invadopodium maturation by inhibiting normal dephosphorylation of cortactin at tyrosine 421 by the phosphatase PTP1B.
Highlights
Phosphorylation of cortactin at tyrosine 421 recruits the sodium-hydrogen exchanger NHE1, which increases the local intracellular pH and triggers F-actin severing by releasing active cofilin from cortactin[23,24]
VASP staining was used to control for offtarget effects and β-actin was used as a loading control. (H) Representative images of MTLn3 cells in (G) or expressing eGFP alone, plated on Alexa Fluor 405 labeled gelatin in complete media, immuno-stained for invadopodium markers as in (B). (I) Quantification of mature invadopodia per cell (Tks5- and cortactinrich puncta co-localizing with degradation holes) of cells shown in H) (n > 90 cells/condition; n > 40 fields/ condition; three independent experiments)
Based on the findings presented here, we propose a model in which MenaINV localizes to the invadopodium precursor and increases cortactin phosphorylation at tyrosine 421 to promote invadopodium maturation by interfering with PTP1B (Fig. 9)
Summary
Phosphorylation of cortactin at tyrosine 421 recruits the sodium-hydrogen exchanger NHE1, which increases the local intracellular pH and triggers F-actin severing by releasing active cofilin from cortactin[23,24]. Cortactin phosphorylation recruits Nck1/N-WASP to stimulate Arp2/3 activity, which acts synergistically with cofilin severing of actin filaments to promote actin polymerization and invadopodium protrusion[17,22,25]. Expression of the MenaINV isoform, identified by an alternatively-included exon that inserts a 19 amino-acid sequence just downstream of the EVH1 domain, is associated with tumor metastasis and progression in mouse models of breast carcinoma[43,44]. Expression of Mena with the 11a exon is reduced in the invasive and disseminating subpopulation of breast carcinoma cells[40] This is interesting because expression of Mena11a is associated with suppression of invasion and dissemination[48,51]. Over-expression of MenaINV produces tumor cells that are less cohesive and demonstrate increased invasion, intravasation and lung metastasis in mouse models of breast cancer[44,45,51]. Despite the close relationship between MenaINV expression and tumor invasiveness, the mechanism underlying MenaINV–dependent enhancement of invadopodium stability and function has not been explored
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