ABSTRACT
Large-scale proteomic and functional analysis of isolated pseudopodia revealed the Lim, actin, and SH3 domain protein (Lasp-1) as a novel protein necessary for cell migration, but not adhesion to, the extracellular matrix (ECM). Lasp-1 is a ubiquitously expressed actin-binding protein with a unique domain configuration containing SH3 and LIM domains, and is overexpressed in 8-12% of human breast cancers. We find that stimulation of nonmotile and quiescent cells with growth factors or ECM proteins facilitates Lasp-1 relocalization from the cell periphery to the leading edge of the pseudopodium, where it associates with nascent focal complexes and areas of actin polymerization. Interestingly, although Lasp-1 dynamics in migratory cells occur independently of c-Abl kinase activity and tyrosine phosphorylation, c-Abl activation by apoptotic agents specifically promotes phosphorylation of Lasp-1 at tyrosine 171, which is associated with the loss of Lasp-1 localization to focal adhesions and induction of cell death. Thus, Lasp-1 is a dynamic focal adhesion protein necessary for cell migration and survival in response to growth factors and ECM proteins.
Subject(s)
Cell Movement/genetics , Focal Adhesions/metabolism , Homeodomain Proteins/metabolism , Neoplasm Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Pseudopodia/metabolism , Actins/biosynthesis , Animals , Apoptosis/drug effects , Apoptosis/physiology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carcinoma/genetics , Carcinoma/metabolism , Cell Survival/genetics , Cytoskeletal Proteins , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/pharmacology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Focal Adhesions/genetics , Growth Substances/metabolism , Growth Substances/pharmacology , Homeodomain Proteins/genetics , LIM Domain Proteins , Mice , NIH 3T3 Cells , Neoplasm Metastasis/genetics , Neoplasm Proteins/genetics , Phosphorylation , Protein Transport/drug effects , Protein Transport/genetics , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins c-abl/metabolism , Pseudopodia/geneticsABSTRACT
Nonmotile cells extend and retract pseudopodia-like structures in a random manner, whereas motile cells establish a single dominant pseudopodium in the direction of movement. This is a critical step necessary for cell migration and occurs prior to cell body translocation, yet little is known about how this process is regulated. Here we show that myosin II light chain (MLC) phosphorylation at its regulatory serine 19 is elevated in growing and retracting pseudopodia. MLC phosphorylation in the extending pseudopodium was associated with strong and persistent amplification of extracellular-regulated signal kinase (ERK) and MLC kinase activity, which specifically localized to the leading pseudopodium. Interestingly, inhibition of ERK or MLC kinase activity prevented MLC phosphorylation and pseudopodia extension but not retraction. In contrast, inhibition of RhoA activity specifically decreased pseudopodia retraction but not extension. Importantly, inhibition of RhoA activity specifically blocked MLC phosphorylation associated with retracting pseudopodia. Inhibition of either ERK or RhoA signals prevents chemotaxis, indicating that both pathways contribute to the establishment of cell polarity and migration. Together, these findings demonstrate that ERK and RhoA are distinct pathways that control pseudopodia extension and retraction, respectively, through differential modulation of MLC phosphorylation and contractile processes.
