Abstract
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.
Highlights
Kinase activity of Rho-associated protein kinases (ROCK) II, an important regulator of cell migration, is controlled by its endogenous inhibitor collapsin response mediator protein (CRMP)-2
To further map the interaction site of CRMP-2 splice variant (CRMP-2L) for ROCK II in detail, polypeptides derived from CRMP-2L
Our current data demonstrate that glycogen synthase kinase 3 (GSK3) phosphorylation of CRMP-2 is important for regulation of the interaction between ROCK II and CRMP-2
Summary
Kinase activity of ROCK II, an important regulator of cell migration, is controlled by its endogenous inhibitor CRMP-2. 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. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant fulllength and catalytic domain of ROCK II These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. We identified a novel endogenous ROCK II inhibitor, collapsin response mediator protein (CRMP)-2, whose impact on the kinase activity can modulate carcinoma cell adhesion and migration, for example [21]. We report that GSK3 partly regulates the interaction between CRMP-2 and ROCK II by phosphorylating CRMP-2 This phosphorylation of CRMP-2 is involved in the regulation of human colon carcinoma cell invasion and haptotactic migration. Inhibition of ROCK-mediated migration is found to be facilitated by binding of the unique N terminus of CRMP-2L alone to the kinase domain of ROCK II
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