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doi: 10.1242/10.1242/jcs.00170

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Identification of CfNek, a novel member of the NIMA family of cell cycle regulators, as a polypeptide copurifying with tubulin polyglutamylation activity in Crithidia

Stefan Westermann^* and Klaus Weber

Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Am Fassberg 11, 37077 Goettingen, Germany
^* Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA

View larger version (31K): [in a new window]   Fig. 1. A modified purification scheme for tubulin polyglutamylase identifies Crithidia p54. (A) Flow chart of the purification scheme using hydroxyapatite chromatography on a CHT-2 column as the final step. (B) Aliquots (30 µl) of successive fractions (500 µl) eluting between 100 and 200 mM sodium phosphate from the CHT-2 column were subjected to SDS-PAGE and proteins were visualised by silver staining. The first lane shows marker proteins with molecular masses in kDa. A 10 µl aliquot of each fraction was also tested for tubulin glutamylation activity using the standard assay (upper panel). Note that a 54 kDa polypeptide copurifies with the glutamylation activity (arrow).

View larger version (33K): [in a new window]   Fig. 2. Cloning the p54 cDNA. (A) Schematic representation of the p54 cDNA and the cloning strategy by 5' and 3' RACE (see Materials and Methods for details). SL denotes the spliced leader sequence, UAP is a universal amplification primer. In frame stop codons are depicted by asterisks. (B) Deduced amino acid sequence of Crithidia p54. Amino acids identified from direct peptide sequencing are underlined. The cDNA and amino acid sequences have been deposited in GenBank under accession no. AJ494838. (C) Schematic representation of the Crithidia p54 (CfNek) domain structure in comparison to the original NIMA protein from Aspergillus nidulans.

View larger version (85K): [in a new window]   Fig. 3. (A) Amino acid sequence alignment of the kinase domains of CfNek and other NIMA-related kinases. The alignment was generated using PILEUP (GCG-package) and the output was shaded with Macboxshade. Residues conserved in >60% of the proteins are shaded in black, simililar residues are shaded in light grey. The eleven kinase subdomains are denoted above the sequence. Sequences used: Leishmania major LNK-1 (Q9NKT3), Trypanosoma brucei NrkA (Q08942), Aspergillus nidulans NIMA (P11837), Homo sapiens Nek2 (P51955), Mus musculus Nek1 (P51954) and Nek3 (Q9R0A5), Tetrahymena pyriformis TpNrk (O76134), Chlamydomonas reinhardtii FA2 (AF479588). The CfNek sequence is from Fig. 2B. (B) Phylogenetic tree derived from the alignment in A. Using the PILEUP output the tree was generated with the GCG-programs DISTANCE and GROWTREE.

View larger version (23K): [in a new window]   Fig. 4. The tubulin polyglutamylase preparation displays ß-casein phosphorylation activity. 10 µl aliquots of successive fractions from the final hydroxyapatite chromatography were assayed for tubulin polyglutamylation activity while 5 µl aliquots were used in kinase assays with ß-casein as substrate. The kinase reactions were run on an SDS-gel and analysed by autoradiography. Note that the ß-casein phosphorylation activity exactly coelutes from the CHT-2 column with the tubulin glutamylation activity.

View larger version (70K): [in a new window]   Fig. 5. Expression of recombinant His-tagged CfNek in Crithidia. (A,B) Immunofluorescence microscopy of Crithidia cells transfected with His-CfNek (A) or vector only (B). The cells were fixed with methanol and stained with a His-antibody (red) and with Hoechst blue to visualise DNA. The immunofluorescence images were overlaid with the corresponding phase contrast images to allow easier localisation. Note that only His-CfNek-expressing cells show a staining of the flagellar attachment zone/basal body. Bar, 5 µm. (C) Immunoblot analysis of total cell extracts from His-CfNek-expressing and control cells. Proteins were separated on a 10% polyacrylamide gel, blotted and stained with anti-His antibody. Only the extract from CfNek-expressing cells shows a corresponding band of 56 kDa.

View larger version (19K): [in a new window]   Fig. 6. Purification and analysis of His-tagged CfNek from Crithidia. (A) Purification of His-CfNek over a Ni-column. 10% polyacrylamide gel stained with silver of the thrombin eluate from wild-type (WT) or CfNek-expressing cells. Note that a contaminating triple band of about 98 kDa is present in both eluates while only the CfNek eluate contains the expected 56 kDa polypeptide. (B) 5 µl aliquots of the thrombin eluates from A were assayed for tubulin polyglutamylation activity. Only the CfNek eluate displays significant tubulin polyglutamylation activity. (C) 5 µl aliquots of the thrombin eluates from A were used in kinase assays with ß-casein as substrate. The kinase reactions were run on an SDS-gel and analyzed by autoradiography. Again, only the CfNek eluate catalyses the incorporation of radioactve phosphate into ß-casein. (D) 5 µl aliquots of the CfNek eluate from A were used in different glutamylation assays with the indicated components. Glutamylation activity is only observed with CfNek, tubulin and ATP in the reaction mixture.

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