The CKK Domain (DUF1781) Binds Microtubules and Defines the CAMSAP/ssp4 Family of Animal Proteins
The CKK Domain (DUF1781) Binds Microtubules and Defines the CAMSAP/ssp4 Family of Animal Proteins
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DOI：
10.1093/molbev/msp115
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发表时间：
2009-09-01
影响因子：
10.7
通讯作者：
Phillips, Gareth W.
中科院分区：
文献类型：
--
作者：
Baines, Anthony J.;Bignone, Paola A.;Phillips, Gareth W.
我们描述了与人钙调蛋白调节血影蛋白相关蛋白 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 和其他两种哺乳动物蛋白 KIAA1078 和 KIAA1543 共有的 C 端附近的结构域，我们将其称为 CKK 结构域。该结构域也存在于无脊椎动物 CAMSAP1 同源物中，并在所有可用的真后生动物基因组（包括刺胞动物）中发现，但不存在于长生动物毛盘动物中，也不存在于任何非后生动物生物中。通过位点似然比方法对密码子比对的分析提供了对哺乳动物 CKK 结构域的所有密码子进行强纯化选择的证据，这可能表明保守的功能。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，在 HeLa 细胞中表达了人 CAMSAP1 增强型绿色荧光蛋白 (EGFP) 和包含 CKK 结构域的片段。整个 CAMSAP1 和 CKK 结构域均显示出与微管一致的定位。在体外，整个 CAMSAP1-谷胱甘肽-s-转移酶 (GST) 和 CKK-GST 均与微管结合。使用抗 CAMSAP1 抗体对小脑颗粒神经元进行免疫荧光显示微管模式。 PC12 细胞中 CKK 结构域的过度表达会阻止神经突的产生，而神经突的产生需要微管功能。我们得出的结论是，CKK 结构域结合微管，代表与后生动物一起进化的结构域。
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.