RhoA function in lamellae formation and migration is regulated by the alpha6beta4 integrin and cAMP metabolism.
Clone A colon carcinoma cells develop fan-shaped lamellae and exhibit random migration when plated on laminin, processes that depend on the ligation of the alpha6beta4 integrin. Here, we report that expression of a dominant negative RhoA (N19RhoA) in clone A cells inhibited alpha6beta4-dependent membrane ruffling, lamellae formation, and migration. In contrast, expression of a dominant negative Rac (N17Rac1) had no effect on these processes. Using the Rhotekin binding assay to assess RhoA activation, we observed that engagement of alpha6beta4 by either antibody-mediated clustering or laminin attachment resulted in a two- to threefold increase in RhoA activation, compared with cells maintained in suspension or plated on collagen. Antibody-mediated clustering of beta1 integrins, however, actually suppressed Rho A activation. The alpha6beta4-mediated interaction of clone A cells with laminin promoted the translocation of RhoA from the cytosol to membrane ruffles at the edges of lamellae and promoted its colocalization with beta1 integrins, as assessed by immunofluorescence microscopy. In addition, RhoA translocation was blocked by inhibiting phosphodiesterase activity and enhanced by inhibiting the activity of cAMP-dependent protein kinase. Together, these results establish a specific integrin-mediated pathway of RhoA activation that is regulated by cAMP and that functions in lamellae formation and migration.
O'Connor
K L
KL
Division of Cancer Biology, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Nguyen
B K
BK
Mercurio
A M
AM
D000954:Antigens, Surface / Q000302:isolation & purification / Q000378:metabolism*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002465:Cell Movement / Q000502:physiology*; D003094:Collagen / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism / Q000648:ultrastructure; D000242:Cyclic AMP / Q000378:metabolism*; D003599:Cytoskeleton; D006801:Humans; D039161:Integrin alpha6beta4; D016023:Integrins / Q000302:isolation & purification / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D020742:rhoA GTP-Binding Protein / Q000302:isolation & purification / Q000378:metabolism*
D000954:Antigens, Surface; D039161:Integrin alpha6beta4; D016023:Integrins; D007797:Laminin; C121346:laminin 1; D003094:Collagen; D000242:Cyclic AMP; D020742:rhoA GTP-Binding Protein