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Clik1: a novel kinase targeted to actin stress fibers by the CLP-36 PDZ-LIM protein

Haartman Institute and Helsinki University Central Hospital, Biomedicum Helsinki, P.O. Box 63, 00014 University of Helsinki, Finland

View larger version (39K): [in a new window]   Fig. 1. Characterization of the Clik1 kinase. (A) The amino-acid sequence of Clik1, demonstrating the kinase domain (underlined), the EST contig (double underlined), potential nuclear localization signals (dotted boxes) and the conserved lysine residue (asterisk) that was mutated to methionine to generate Myc-K98M. The GenBank Accession number is AF195026. (B) Immunoprecipitates from U2OS cells transfected with Myc-tagged Clik1 (Myc-Clik1) or with an ATP-binding site mutant Myc-tagged Clik1 (Myc-K98M) were subjected to an in vitro kinase reaction followed by SDS-PAGE, transfer to a nitrocellulose membrane and autoradiography (IP-kinase). Subsequently anti-Myc western blot analysis was used to visualize the 46 kDa Myc-Clik1 protein (IP-Western).

View larger version (77K): [in a new window]   Fig. 5. The C-terminal LIM domain of CLP-36 is essential for Clik1 relocalization. U2OS cells expressing EGFP-CLP-36 and myc-Clik1 (A,B,I,J), EGFP-CLP-36 and the kinase-deficient myc-ClikK98M (C,D), a C-terminal mutant lacking the LIM domain EGFP-CLP1-231 and myc-Clik1 (E,F) or EGFP-CLP-36 alone (G,H) were immunostained with mouse monoclonal anti-myc to detect Clik1 (B,F) and ClikK98M (D) or were immunostained with Texas-Red-conjugated phalloidin (H,J) to demonstrate actin filaments. Bars, 15 µm. Anti-CLP-36 (upper panel) and anti-Myc (lower panel) western blot analyses were used to control the expression of fusion proteins used for immunofluorescence studies (K).

View larger version (30K): [in a new window]   Fig. 2. Clik1 associates with CLP-36. (A) A yeast two-hybrid screen from a human fetal liver cDNA library with LexA-Clik1 identified 37 clones of which 36 represented CLP-36. Three representative clones (38, 51 and 47) with their PDZ (grey) and LIM (black) domains are shown. (B) To demonstrate the association of CLP-36 with cellular Clik1, 300 micrograms of U2OS cell extracts overexpressing Myc-Clik1 or vector control were incubated with GST-CLP-36 or GST proteins. Subsequently proteins associated with the GST proteins were purified and analyzed by anti-Myc western blotting (anti-Myc), demonstrating that CLP-36 associated with Myc-Clik1. Ponceau staining (lower panel) was used to control levels of GST-CLP-36 and GST proteins. The faster migrating bands in Ponceau staining in GST-CLP-36 lanes are caused by degradation of the purified protein. The input panel demonstrates amounts of Clik1 in 50 µg of extracts subsequently used for the pull-down assay.

View larger version (75K): [in a new window]   Fig. 3. Nuclear Clik1 is relocalized when coexpressed with CLP-36. U2OS osteosarcoma cells were transfected with the following expression vectors: Myc-Clik1 (A-C), HA-CLP-36 (D-F), Myc-Clik1 and HA-CLP-36 (G-I), Myc-CDK7 and HA-CLP-36 (J-L). The subcellular localization of Clik1, CDK7 and CLP-36 was analyzed by double-label immunofluorescence with a mouse monoclonal anti-Myc (A,D,G,J) and a rabbit polyclonal CLP-36 antibody (B,E,H,K). Hoechst staining was used to visualize nuclei (C,F,I,L). Bars, 15 µm.

View larger version (90K): [in a new window]   Fig. 4. CLP-36 targets Clik1 kinase to the actin stress fiber. U2OS osteosarcoma cells expressing Myc-Clik1 together with HA-CLP-36 (A-C) or HA-ALP (D-F) were treated with Triton X-100 (Triton) prior to fixation. Subsequently coverslips were analyzed by immunofluorescence with monoclonal anti-Myc to detect Myc-Clik1 (A,D) and with rabbit polyclonal CLP or ALP antibodies to detect CLP (B) and ALP (E), and Hoechst staining was used to visualize nuclei (C,F). Bars, 15 µm.

View larger version (56K): [in a new window]   Fig. 6. Both Clik1 and CLP-36 are expressed in adult testis. (A) Human multiple-tissue northern blot filters containing the indicated mRNAs (Clontech, no. 7760-1, 7766-1 and 7757-1) were probed with Clik1. The positions of the 7.5 kb mRNA standards are indicated on the right. (B) Western blot analysis from adult mouse testis extract with rabbit polyclonal CLP-36 antibody: total testis extract (lysate), anti-CLP-36 immunoprecipitate from testis extract (IP, -CLP-36) and blocked anti-CLP-36 immunoprecipitate (IP, block). (C) Frozen adult mouse testis sections were fixed with ethanol and stained with rabbit polyclonal anti-CLP-36 or with blocked anti-CLP-36 (inset). The white arrow indicates the peritubular contractile cells surrounding the seminiferous tubuli, and the white arrowhead indicates the elongating spermatids. Bar, 40 µm.