Morgan-Fisher, Marie5; Couchman, John R6; Yoneda, Atsuko7
1 Section of Molecular Pathology, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet2 Couchman Group, BRIC Research Groups, BRIC, Københavns Universitet3 Graduate School of Health and Medical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet4 Couchman Group, BRIC, Faculty of Health and Medical Sciences, Københavns Universitet5 Graduate School of Health and Medical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet6 Couchman Group, BRIC, Faculty of Health and Medical Sciences, Københavns Universitet7 Section of Molecular Pathology, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, Københavns Universitet
The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.
Journal of Biological Chemistry, 2013, Vol 288, Issue 43, p. 31229-40