Missing In Metastasis(MIM), A Putative Metastasis Suppressor,Promotes Cortactin But Inhibits N-WASP Mediated Actin Polymerization

Objective Actin polymerization and depolymerization plays a critical role in cell migration, cell adhesions and endocytosis. However, How extracellular signaling co-adjusted polymerization and depolymerization by adaptor proteins in the cell is unclear. The aim of this paper is to investigate the effect of a protein MIM( Missing in metastasis, a putative metastasis suppressor) on cell migration, discuss possible mechanisms. Methods Expressing level of MIM in cell was detected with RT-PCR; NIH3T3 , HUVE(Human umbilical vein endothelia) and HL-60 (Human acute promyelocytic leukemia)cells were infected with retroviruses encoding GFP-MIM,GFP or mutant of MIM; Cell migration was measured in Transwell chambers; The interaction of proteins in the cell and tyrosine phosphorylation of MIM were tested by immunopricipitated and immunoblotted; the binding affinity of purified MIM and other proteins were measured by pull down assay; Colocalization of GFP-MIM and cortactin in cell and change of cell shape were inspected under a fluorescent microscope; Kinetics of actin polymerization was monitored by measuring the increasing in pyrene fluorescence ; Morphology of actin filaments was detected under inverted fluorescence microscope . Results (1)Expressing level of MIM in normal cells were higher than that in tumor metastasis cells. (2)overexpression of GFP-MIM inhibited markedly the cell motility in response to PDGF in NIH3T3 cells, sphingosine 1 phosphate in HUVEC and MCP-1(Monocyte chemotactic protein-1) in HL-60cells(3T3 cells: 122±16.8 vs 196±21.4%, P<0.01; HUVEC: 148±22.3 vs 264±42.3%, P<0.01; HL-60: 69±12.5 vs 148±15.9%, P<0.01). (3) MIM underwent a rapid tyrosine phosphorylation and associates transiently with cortactin, an Arp2/3 activator, upon PDGF stimulation. (4) In the cell MIM colocalizes with cortactin in the punctate structures and cell periphery. (5) Recombinant full-length MIM protein binds to cortactin through the interaction between the cortactin SH3 domain and the MIM proline rich domain (PRD). (6) In vitro actin polymerization analysis demonstrates that full-length MIM enhanced significantly cortactin/Arp2/3 complex mediated assembly of non-branched actin filaments in a manner dependent upon both its interaction with cortactin and N-terminal sequences (32.4±2.1/field vs 20.2±2.3/field, P<0.001),. Under the same condition, however, MIM inhibits dramatically N-WASP-VCA mediated actin polymerization and actin branching as well (10.1±1.1% vs 79.3±5.6%, P<0.01). (7) GFP-MIM induces filopodium formation in 3T3cells (percent of cells with filopodia: 46±3.8% vs 16±2.3%, P<0.01). (8) interestingly, overexpression of MIM mutant that was unable to interfere with the function of VCA enhanced cell migration, whereas a mutant deficient in cortactin binding inhibited further PDGF mediated cell motility. Conclusion This study implies a novel mechanism for cell motility involving different regulation of distinct types of Arp2/3 complex activators.