title: The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins.
abstract: We describe a structural domain common to proteins related to human calmodulin-regulated spectrin-associated protein1 (CAMSAP1). Analysis of the sequence of CAMSAP1 identified a domain near the C-terminus common to CAMSAP1 and two other mammalian proteins KIAA1078 and KIAA1543, which we term a CKK domain. This domain was also present in invertebrate CAMSAP1 homologues and was found in all available eumetazoan genomes (including cnidaria), but not in the placozoan Trichoplax adherens, nor in any nonmetazoan organism. Analysis of codon alignments by the sitewise likelihood ratio method gave evidence for strong purifying selection on all codons of mammalian CKK domains, potentially indicating conserved function. Interestingly, the Drosophila homologue of the CAMSAP family is encoded by the ssp4 gene, which is required for normal formation of mitotic spindles. To investigate function of the CKK domain, human CAMSAP1-enhanced green fluorescent protein (EGFP) and fragments including the CKK domain were expressed in HeLa cells. Both whole CAMSAP1 and the CKK domain showed localization coincident with microtubules. In vitro, both whole CAMSAP1-glutathione-s-transferase (GST) and CKK-GST bound to microtubules. Immunofluorescence using anti-CAMSAP1 antibodies on cerebellar granule neurons revealed a microtubule pattern. Overexpression of the CKK domain in PC12 cells blocked production of neurites, a process that requires microtubule function. We conclude that the CKK domain binds microtubules and represents a domain that evolved with the metazoa.
authors.affiliation: Department of Biosciences, University of Kent, Canterbury, Kent, United Kingdom. a.j.baines@kent.ac.uk
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Baines
authors.forename: Paola A
authors.initials: PA
authors.lastname: Bignone
authors.forename: Mikayala D A
authors.initials: MD
authors.lastname: King
authors.forename: Alison M
authors.initials: AM
authors.lastname: Maggs
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Bennett
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Pinder
authors.forename: Gareth W
authors.initials: GW
authors.lastname: Phillips
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D017124:Conserved Sequence; D006367:HeLa Cells; D006801:Humans; D016013:Likelihood Functions; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016501:Neurites / Q000378:metabolism; D016716:PC12 Cells; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013049:Spectrin / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D002147:Calmodulin; D013049:Spectrin