Baines Anthony J, Bignone Paola A, King Mikayala D A, Maggs Alison M, Bennett Pauline M, Pinder Jennifer C, Phillips Gareth W
Department of Biosciences, University of Kent, Canterbury, Kent, United Kingdom.
Mol Biol Evol. 2009 Sep;26(9):2005-14. doi: 10.1093/molbev/msp115. Epub 2009 Jun 9.
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.
我们描述了一种与人类钙调蛋白调节的血影蛋白相关蛋白1（CAMSAP1）相关的蛋白质共有的结构域。对CAMSAP1序列的分析确定了CAMSAP1与另外两种哺乳动物蛋白KIAA1078和KIAA1543在C端附近共有的一个结构域，我们将其命名为CKK结构域。该结构域也存在于无脊椎动物CAMSAP1同源物中，并且在所有可用的真后生动物基因组（包括刺胞动物）中都能找到，但在扁盘动物黏胶丝盘虫中不存在，在任何非后生动物生物体中也未发现。通过位点似然比方法对密码子比对进行分析，结果表明对哺乳动物CKK结构域的所有密码子都有强烈的纯化选择，这可能表明其功能保守。有趣的是，CAMSAP家族的果蝇同源物由ssp4基因编码，该基因是有丝分裂纺锤体正常形成所必需的。为了研究CKK结构域的功能，将人CAMSAP1增强型绿色荧光蛋白（EGFP）和包括CKK结构域的片段在HeLa细胞中表达。完整的CAMSAP1和CKK结构域都显示出与微管共定位。在体外，完整的CAMSAP1-谷胱甘肽-S-转移酶（GST）和CKK-GST都与微管结合。在小脑颗粒神经元上使用抗CAMSAP1抗体进行免疫荧光显示出微管模式。在PC12细胞中过表达CKK结构域会阻断神经突的产生，而这一过程需要微管功能。我们得出结论，CKK结构域与微管结合，代表了一个随着后生动物进化而来的结构域。
