CKIδ interacts with the microtubule plus-end–binding proteins EB1 and p150glued. (A) Still images from time-lapse imaging (Video 5) showing conjugate formation between a Jurkat cell expressing GFP-CKIδ (green) and an SEE-pulsed Raji cell (blue). Insets correspond to higher magnifications of the centrosomal region. Asterisks mark fiberlike extrusions near the centrosome. Bars: 10 µm; (inset) 1 µm. (B) In vitro microtubule-pelleting assay. Pure tubulin was incubated in the presence of GTP without (−) or with (+) taxol and then added to cytoplasmic extracts of Jurkat cells transfected with GFP-CKIδ. High-speed supernatants (S) and pellets (P) were collected and immunoblotted with antibodies as indicated. WB, Western blotting. (C) A schematic view of key functional domains of CKIδ: kinase, autoinhibitory (Longenecker et al., 1998), and centrosome-targeting (Greer and Rubin, 2011) domains. Sequence alignments of the extreme C termini of two CKIδ isoforms and CKIε are shown below. Acidic and basic amino acids are in red and blue, respectively. Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl, Xenopus laevis; Dr, Danio rerio. (D) Cytoplasmic cell extracts (CCE) of Jurkat cells were subjected to pull-down assays with GST, GST-EB1 (EB1), or GST-EB3 (EB3) and were immunoblotted with antibodies as indicated. Recombinant GST products are shown in Ponceau S staining below. (E) Jurkat cells were unconjugated (−) or conjugated (+) with anti-CD3–coated beads. Cytoplasmic cell extracts were processed for immunoprecipitation (ip) with random IgG (ip con) or anti-p150glued (ip p150glued) antibodies and immunoblotted with the indicated antibodies. (F) Cytoplasmic cell extracts of Jurkat cells were mock depleted with random IgG (−) or immunodepleted of p150glued (+). Depleted extracts were then processed for pull-down assays with GST or GST-EB1 (EB1) and immunoblotted with the indicated antibodies. Recombinant GST products are visible in Ponceau S staining below.
