Figure 1.

GCP6 binds directly to keratins in vitro. (A) 6xHIS-tagged GCP6 expressed as a stable transfectant in U2OS (human osteosarcoma) cells was detected by anti-HIS tag antibody in centrosomes identified by γ-tubulin immunofluorescence (arrows). Bar, 10 μm. (B) The same HIS-tagged GCP6 expressed in U2OS cells was purified by Ni²⁺ column, washed at 200 mM (lane 1), eluted at 350 mM imidazole (lane 2), and analyzed by immunoblot with an anti-HIS tag antibody. The same eluate as in lane 2 was subjected to phosphorylation with 250 μCi/ml [γ-³²P]ATP in the presence (+, lane 3) or absence (−, lane 4) of 10 U/ml Cdk1/cyclin B and analyzed by SDS-PAGE and PhosphorImager. The HIS-tagged protein eluted from the Ni²⁺ column was also analyzed by immunoblot with anti-GCP6 antibody (lane 5). The same antibody preincubated (+, lane 6) or not (−, lane 7) with the corresponding antigen peptide was used on blots from total extract of CACO-2 cells (50 μg total protein/lane). The arrow points at the apparent molecular weight of 190 kDa in all blots. Standards, 200, 128, 80, and 40 kDa. (C) The same purified full-length HIS-tagged GCP6 was phosphorylated with 10 U/ml Cdk1 but in the presence (+, lane 2) or absence (−, lane 1) of 1 mM cold ATP. These preparations of full-length GCP6 were used to overlay nitrocellulose sheets previously blotted with a mixture of purified CACO-2 keratins (K8, K19, and K18, 20 μg of total protein/lane) and blocked with BSA. After washes, the sheets were developed with an anti-HIS tag Ab (lanes 1 and 2). Then, the same sheets were sequentially stripped off and reprobed with anti-K8 (lanes 3 and 4), K19 (lanes 5 and 6), and K18 (lanes 7 and 8) monoclonal antibodies as load controls. One aliquot of the same preparation (10 ¼g) was run, blotted on a separate lane (lane 9), and stained with Ponceau S red dye. Standards, 80, 40, and 31 kDa. (D) A suspension of highly purified, native intermediate filaments from CACO-2 cells (top) or BSA as control (bottom) were loaded onto nitrocellulose sheets in a dot blot apparatus (10 ¼g/dot). After extensive washes and blocking protein binding with BSA, the dots were overlaid with the purified HIS-tagged GCP6, phosphorylated (+) or not (−) with Cdk1 as described above. The membranes were then washed and developed for chemiluminescence with the anti-HIS tag antibody. (E) For pull-down, highly purified native polymerized IFs (K8–K18 and K8–K19) were covalently coupled to CNBr-activated Sepharose and incubated in the presence of the same purified Cdk1-phosphorylated (+) or not (−) GCP6. After washes, the beads were eluted in sample buffer and analyzed by immunoblot with anti-HIS tag antibody.