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Keywords:

  • cytoskeletal regulation;
  • cytoskeleton;
  • molecular evolution;
  • nerve cells;
  • protein domain;
  • protein function

Abstract

Thumbnail image of graphical abstract

Calmodulin regulated spectrin-associated protein 1 (CAMSAP1) is a vertebrate microtubule-binding protein, and a representative of a family of cytoskeletal proteins that arose with animals. We reported previously that the central region of the protein, which contains no recognized functional domain, inhibited neurite outgrowth when over-expressed in PC12 cells [Baines et al., Mol. Biol. Evol. 26 (2009), p. 2005]. The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins (Baines et al. 2009). In the central region, three short well-conserved regions are characteristic of CAMSAP-family members. One of these, CAMSAP-conserved region 1 (CC1), bound to both βIIΣ1-spectrin and Ca2+/calmodulin in vitro. The binding of Ca2+/calmodulin inhibited spectrin binding. Transient expression of CC1 in PC12 cells inhibited neurite outgrowth. siRNA knockdown of CAMSAP1 inhibited neurite outgrowth in PC12 cells or primary cerebellar granule cells: this could be rescued in PC12 cells by wild-type CAMSAP1-enhanced green fluorescent protein, but not by a CC1 mutant. We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin-binding to neurite outgrowth.

Knockdown of the cytoskeletal protein CAMSAP1 using siRNA inhibited NGF-induced (nerve growth factor) neurite outgrowth from PC12 cells, and axon production by cerebellar granule cells in culture. This activity is linked to a spectrin- and Ca2+/calmodulin-binding region (CC1), since over-expression of isolated CC1 inhibited neurite production from PC12 cells. We previously showed that CAMSAP1 binds microtubules at the C-terminal CKK domain. Our data indicates CAMSAP1 is a cytoskeletal interconnector required for neurite and axon production.