FKBP133: A novel mouse FK506-binding protein homolog alters growth cone morphology

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Abstract

FK506-binding proteins are the peptidyl prolyl cis–trans isomerases that are involved in various intracellular events. We characterized a novel mouse FK506-binding protein homolog, FKBP133/KIAA0674, in the developing nervous system. FKBP133 contains a domain similar to Wiskott–Aldrich syndrome protein homology region 1 (WH1) and a domain homologous to FK506-binding protein motif. FKBP133 was predominantly expressed in cerebral cortex, hippocampus, and peripheral ganglia at embryonic day 18.5. FKBP133 protein was distributed in the axonal shafts and was partially co-localized with F-actin in the growth cones of dorsal root ganglion neurons (DRG). The number of filopodia was increased in the DRG neurons overexpressing FKBP133. In contrast, the overexpression of a mutant deleted the WH1 domain reduced the growth cone size and the number of filopodia. Furthermore, the neurons overexpressing FKBP133 became significantly resistant to Semaphorin-3A induced collapse response. These results suggest that FKBP133 modulates growth cone behavior with the WH1 domain.

Section snippets

Experimental procedures

Materials. Antibodies were used: a rabbit polyclonal anti-HA antibody Y-11 from Santa Cruz Biotech (Santa Cruz, CA, US), anti-α-tubulin mAb (DM1A) from ICN (Costa Mesa, CA, US), secondary antibodies conjugated Alexa 488 or 594 and Rhodamine–Phalloidin from Invitrogen-Molecular Probes (Eugene, OR, US), and Horseradish peroxidase-conjugated secondary antibodies from Amersham (Arlington Heights, IL, US).

Plasmid construction. To construct mouse FKBP133 expression vector, the coding region of

FKBP133/KIAA0674 encodes a Wiskott–Aldrich syndrome protein homology region 1 and a FK506 binding motif

We have recently identified a mouse homolog of human KIAA0674 protein (AB014574) as an antigen cross-reacting with anti-Plexin-A4 polyclonal antibody [18]. The mouse Riken cDNA C430014M02 (GenBank Accession No. BC060651) encodes the full length of the protein. We assigned the open reading frame of BC060651 from 41 to 3691 bp. The coding region encodes 1216 amino acid polypeptides and the calculated molecular weight is 133-kDa (Fig. 1A). Due to the acidic isoelectric point (pKi = 4.8) of the

Discussion

In the present study, we demonstrate predominant expression of FKBP133 in the developing nervous system (Fig. 2, Fig. 3) and subcellular localization of the protein in the axons and in the growth cones (Fig. 3). The signal of in situ hybridization at E18.5 is thought to reflect FKBP133 mRNA expression in neuronal cells, because small numbers of glial cells have been generated at the stage. FKBP133 mRNA exhibited limited expression in the neuronal cell bodies of the hippocampus and of the

Acknowledgments

We thank Dr. Nagase (Kazusa DNA Institute) for providing human KIAA0674 clone. We also thank Dr. Usui for critical reading of the manuscripts. This work was supported by Grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to F.N. and Y.G.); Grants-in-Aid from CREST (Core Research for Evolutional Science and Technology) of JST (Japan Science and Technology Corporation) (to Y.G. and K.T.); Yokohama Medical Foundation (to O.N. and Y.G.); The Yokohama City

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