MBE Advance Access published online on June 9, 2009
Molecular Biology and Evolution, doi:10.1093/molbev/msp115
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Research Article |
The CKK domain (DUF1781) domain binds microtubules and defines the CAMSAP/ssp4 family of animal proteins
1 Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK
2 Centre for Biomedical Informatics, University of Kent, Canterbury, Kent, CT2 7NJ, UK
4 Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, UK
# Author for correspondence E-mail: A.J.Baines{at}kent.ac.uk, Telephone: + 44 (0) 1227 823462, Fax: + 44 (0) 1227 763912
Received for publication March 3, 2009. Revision received May 8, 2009. Accepted for publication May 18, 2009.
We describe a structural domain common to proteins related to human CAMSAP1 (calmodulin-regulated spectrin-associated protein1). Analysis of the sequence of CAMSAP1 identified a domain near the C-terminus common to CAMSAP1 and two other mammalian proteins KIAA1078 and KIAA1053, 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 non-metazoan 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-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-GST and CKK-GST bound to microtubules. Immunofluorescence using anti-CAMSAP1 antibodies on cerebellar granule neurons revealed a microtubule pattern. Over-expression 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.
Key Words: calmodulin • spectrin • cytoskeleton • DUF1781 domain • tubulin • microtubule • nerve axon
5 Present address: Pfizer Ltd, Ramsgate Road, Sandwich, Kent, CT13 9NJ.
Deceased 3 Present address: LRF Lymphoma Antigens Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Room 4A13 Level 4 Academic Block, John Radcliffe Hospital, Headington, Oxford, OX3 9DU.