Collapsin response mediator protein-2 inhibits neuronal phospholipase D-2 activity by direct interaction
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- Collapsin response mediator protein-2 inhibits neuronal phospholipase D-2 activity by direct interaction
- Lee, S; Kim, JH; Lee, CS; Kim, JH; Kim, YD; Heo, K; Ihara, Y; Goshima, Y; Suh, Pann-Ghill; Ryu, SH
- GROWTH CONE COLLAPSE; ADP-RIBOSYLATION FACTOR; C-TERMINAL DOMAINS; KINASE-C; PC12 CELLS; ACTIN POLYMERIZATION; SEMAPHORIN-III; D ACTIVATION; RAT-BRAIN; ASSOCIATION
- Issue Date
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
- JOURNAL OF BIOLOGICAL CHEMISTRY, v.277, no.8, pp.6542 - 6549
- Although the functional significance of neuronal phospholipase D (PLD) is being recognized, little is known about its regulatory role in neuronal cells. To elucidate the regulatory mechanism of neuronal PLD, we investigated PLD2-binding neuronal protein from rat brain cytosol. During the fractionation of rat brain cytosol by four-column chromatography, a 62-kDa PLD2-interacting protein was detected by PLD2 overlay assay and identified as collapsin response mediator protein-2 (CRMP-2), which controls neuronal axon guidance and outgrowth. Using bacterially expressed glutathione S-transferase fusion proteins, we found that two regions (amino acids 65-192 (the phagocytic oxidase domain) and 724-825) of PLD2 and a single region (amino acids 243-300) of CRMP-2 are required for the direct binding of both proteins. A co-immunoprecipitation study in COS-7 cells also showed an in vivo interaction between CRMP-2 and PLD2. Interestingly, CRMP-2 was found to potently inhibit PLD2 activity in a concentration-dependent manner (IC50 = 30 nM). Overexpression studies also showed that CRMP-2 is an in vivo inhibitor of PLD2 in PC12 cells. Moreover, increasing the concentration of semaphorin 3A, one of the repulsive axon guidance cues, showed that PLD2 activity can be inhibited in PC12 cells. Immunocytochemistry further revealed that PLD2 is co-localized with CRMP-2 in the distal tips of neurites, its possible action site, in differentiated PC12 cells. Taken together, our results indicate that CRMP-2 may interact directly with and inhibit neuronal PLD2, suggesting that this inhibitory mode of regulation may play a role in neuronal pathfinding during the developmental stage.
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