TY - JOUR

T1 - Phosphorylation and mRNA splicing of collapsin response mediator protein-2 determine inhibition of rho-associated protein kinase (ROCK) II function in carcinoma cell migration and invasion

AU - Morgan-Fisher, Marie

AU - Couchman, John R

AU - Yoneda, Atsuko

PY - 2013/10/25

Y1 - 2013/10/25

N2 - 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.

AB - 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.

KW - Animals

KW - Breast Neoplasms

KW - Carcinoma

KW - Cell Line, Tumor

KW - Cell Movement

KW - Colonic Neoplasms

KW - Female

KW - Glycogen Synthase Kinase 3

KW - Humans

KW - Intercellular Signaling Peptides and Proteins

KW - Neoplasm Invasiveness

KW - Neoplasm Proteins

KW - Nerve Tissue Proteins

KW - Phosphorylation

KW - Protein Binding

KW - Protein Structure, Tertiary

KW - RNA Splicing

KW - RNA, Messenger

KW - RNA, Neoplasm

KW - Rats

KW - rho-Associated Kinases

U2 - 10.1074/jbc.M113.505602

DO - 10.1074/jbc.M113.505602

M3 - Journal article

C2 - 24036111

VL - 288

SP - 31229

EP - 31240

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 43

ER -