Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading.
Integrin-induced cell adhesion results in transmission of signals that induce cytoskeletal reorganizations and resulting changes in cell behavior. The cytoskeletal reorganizations are regulated by transient activation and inactivation of Rho GTPases. Previously, we identified mu-calpain as an enzyme that is activated by signaling across beta1 and beta3 integrins. We showed that it mediates cytoskeletal reorganizations in bovine aortic endothelial (BAE) and Chinese hamster ovary (CHO) cells and does so by acting upstream of Rac1 activation. Here we show that mu-calpain is also involved in inactivating RhoA during integrin-induced signaling. Cleavage of RhoA was detectable in BAE cells plated on an integrin substrate; it did not occur in cells plated on poly-l-lysine. Cleavage was inhibited by calpain inhibitors. In vitro, mu-calpain cleaved RhoA generating a fragment of the same size as in intact cells. The cleavage site was identified, an HA-tagged construct expressing calpain-cleaved RhoA generated, and the construct expressed in BAE and CHO cells. Calpain-cleaved RhoA inhibited integrin-induced stress fiber assembly and decreased cell spreading. Together, our data show that calpain cleaves RhoA and generates a form that inhibits integrin-induced stress fiber assembly and cell spreading.
Kulkarni
Sucheta
S
Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Goll
Darrel E
DE
Fox
Joan E B
JE
D000199:Actins / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001483:Base Sequence; D016466:CHO Cells; D002154:Calpain / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000502:physiology*; D004926:Escherichia coli; D006868:Hydrolysis; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D017931:DNA Primers; D016023:Integrins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D002154:Calpain; C449002:mu-calpain; D020742:rhoA GTP-Binding Protein