Collapsin response mediator protein-2 inhibits neuronal phospholipase D(2) activity by direct interaction.
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 PLD(2)-binding neuronal protein from rat brain cytosol. During the fractionation of rat brain cytosol by four-column chromatography, a 62-kDa PLD(2)-interacting protein was detected by PLD(2) 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 PLD(2) 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 PLD(2). Interestingly, CRMP-2 was found to potently inhibit PLD(2) activity in a concentration-dependent manner (IC(50) = 30 nm). Overexpression studies also showed that CRMP-2 is an in vivo inhibitor of PLD(2) in PC12 cells. Moreover, increasing the concentration of semaphorin 3A, one of the repulsive axon guidance cues, showed that PLD(2) activity can be inhibited in PC12 cells. Immunocytochemistry further revealed that PLD(2) 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 PLD(2), suggesting that this inhibitory mode of regulation may play a role in neuronal pathfinding during the developmental stage.
Lee
Sukmook
S
Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea.
Kim
Jung Hwan
JH
Lee
Chang Sup
CS
Kim
Jong Hyun
JH
Kim
Youndong
Y
Heo
Kyun
K
Ihara
Yasuo
Y
Goshima
Yoshio
Y
Suh
Pann-Ghill
PG
Ryu
Sung Ho
SH
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D009474:Neurons / Q000201:enzymology*; D016716:PC12 Cells; D010446:Peptide Fragments / Q000737:chemistry; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D000098855:Collapsin Response Mediator Protein 1
C485780:DPYSL4 protein, human; C485779:Dpysl4 protein, rat; D036341:Intercellular Signaling Peptides and Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D000098855:Collapsin Response Mediator Protein 1; C095114:collapsin response mediator protein-2; D005982:Glutathione Transferase; C105606:phospholipase D2; D010739:Phospholipase D