doi:10.1016/j.tice.2004.10.004
Copyright © 2004 Elsevier Ltd All rights reserved.
Cyclin-dependent kinase activating kinase/Cdk7 co-localizes with PKC-ι in human glioma cells
References and further reading may be available for this article. To view references and further reading you must
purchase this article.
Elona Bicakua, b, Rekha Patela, b and Mildred Acevedo-Duncana, b, 
, 
aDepartment of Chemistry, University of South Florida, USA
bJames A. Haley Veterans Hospital, 13000 Bruce B. Downs Blvd. VAR 151, Tampa, FL 33612, USA
Received 5 April 2004;
revised 11 October 2004;
accepted 19 October 2004.
Available online 25 January 2005.
Abstract
Cyclin-dependent kinase activating kinase (CAK) is a trimeric complex composed of cdk7, cyclin H and MAT1. CAK/cdk7 functions as a master cell cycle regulator by phosphorylating cyclin-dependent kinases for cell cycle progression. We have previously reported that protein kinase C-ι (PKC-ι) associates with CAK/cdk7. In this investigation, immunofluorescence confocal microscopy was used to provide further evidence for the co-localization of PKC-ι with CAK/cdk7. PKC-ι was labeled with Alexa Fluor 488 (green fluorescent dye) and CAK/cdk7 was labeled with Alexa Fluor 555 (red fluorescent dye). The fusion of the red and green fluorescent colors produced a yellow color, which was used to quantify co-localization of PKC-ι and CAK/cdk7. Confocal microscopy revealed the co-localization of PKC-ι with CAK/cdk7 in both the cytoplasm and nucleus of U-373 MG cells
Keywords: CAK; cdk7; PKC-ι; Cell cycle; Glioma; U-373MG
Abbreviations: PKC, protein kinase C; U-373MG, human glioma U-373MG cell line; GO/G1, quiescence/gap 1; S; DNA synthesis phase; G2M, gap 2 and mitosis; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; PI, propidium iodide; EDTA, ethylenediamine-tetraacetic acid; EGTA, ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; DTT, dithiothreitol; PMSF, phenyl-methylsulfonly fluoride; Par-4; prostate apoptosis response-4; LIP, lambda interacting protein; CAK, cyclin-dependent kinase activating kinase; MAT1, ‘menage a trois’ 1 (a 36 kDa component of CAK); cdK7, cyclin-dependent kinase 7; TFIIH, transcription factor IIH; ZIP or p62, zeta interacting protein
 |
Fig. 1. Co-localization of CAK/cdk7 with PKC-ι. Panels A–D depicts control immunofluorescence micrographs of U-373MG cells. These micrographs show control number 6 from Table 1. Cells were incubated only with secondary antibodies. Panel A shows fluorescence from Alexa Fluor 555 (red fluorescent; Molecular Probes, cat# A-21429, 20 μg/mL) corresponding to secondary antibody used to detect CAK/cdk7. Panel B illustrates Alexa Fluor 488 (green fluorescent; Molecular Probes, cat# A-11029, 20 μg/mL) corresponding to secondary antibody used to detect PKC-ι. Panel C depicts both Alexa Fluor 555 (red fluorescent) and Alexa Fluor 488 (green fluorescent). Panel D illustrates the co-localization scattered diagram. Region 1 on the co-localization scattered diagram (bottom, right square) shows fluorescence corresponding the secondary antibody used to label cdk7. Region 2 on the co-localization scattered diagram (left rectangle) shows fluorescence corresponding the secondary antibody used to label PKC-ι, and region 3 shows the co-localization between these two. Regions 1–3 contain few pixels demonstrating that there is minimum non-specific labeling. U-373MG cells were incubated with primary antibodies: (E) CAK/cdk7 (Santa Cruz biotechnology, cat# sc529, 4 μg/mL) and (F) PKC-ι (Santa Cruz Biotechnology, cat# sc17837, 6 μg/mL). Secondary antibodies used to detect (E) CAK/cdk7 were Alexa Fluor 555 (Molecular Probes, cat# A = 21429, 20 μg/mL) and (F) PKC-ι, Alexa Fluor 488 (Molecular Probes, cat# A-11029, 20 μg/mL). Panel G shows transposed images of red and green fluorescence (orange color) demonstrating co-localization of CAK/cdk7 with PKC-ι. Panel H: Region 1 on the co-localization diagram (bottom, right square) shows labeling of CAK/cdk7, region 2 (left rectangle) shows labeling of PKC-ι, and region 3 pixels show presence of co-localization between CAK/cdk7 and PKC-ι. Identical results were obtained in a minimum of two other experiments for control immunofluorescence micrographs. Results from co-localizations results represent a minimum of three independent experiments. Magnification for all micrographs was ×630.
 |
Fig. 2. CAK/cdk7 associates with PKC-ι. Western blot analysis of the physical association between CAK/cdk7 and PKC-ι. Lysates from U-373MG (300 μg) were prepared at the indicated times as described in Section 2 and immunoprecipitated with rabbit antibodies to cdk7 (5 μg/22 μL); SC-529) and rocked overnight at 4 °C. Immunecomplexes were incubated at 4 °C for 1 h with 100 μg/100 μL of anti-rabbit IgG agarose and washed twice with lysis buffer. Proteins in the CAK/cdk7 immunoprecipitates were subjected to Western blotting with antibodies against (A) PKC-ι (P20520; Transduction Lab., Lex., KY, USA) or (B) cdk7 (SC-7344) at a 1/800 dilution. (C) Western blot analysis of actin in U-373MG cells throughout the cell cycle. Western blot shown is representative of numerous Western blots also performed with U-373MG cells to verify that protein loading (80 μg) and protein integrity are equal. Western blots were probed for actin with a monoclonal antibody to actin (Santa Cruz Biotechnology; SC-8432) at a 5:1000 dilution. Secondary antibodies were obtained from Accurate (JOM000003, Westbury, NY, USA) and used at 1:15000 dilution. Control mock immunoprecipitations included U-373MG cell extracts and anti-rabbit IgG agarose alone (lane 1), rabbit IgGs (2.4 μg/6 μL; 02-6102, Zymed, San Francisco, CA, USA) plus anti rabbit IgG agarose (lane 2). Western blots represent results from a minimum of three independent experiments.
Table 1.
Immunofluorescent controls tested on U-373MG glioma cellsa
a For indirect immunofluorescence microscopy U-373MG cells were grown on Lab-Tek slides for 24 h as described in Section
2. The antibody titers were; anti-cdk7 (sc-529, Santa Cruz Biotechnology) 4 μg/mL, anti-PKC-ι (sc-17837, Santa Cruz Biotechnology) 6 μg/mL, Alexa Fluor 555 (A-21429, Molecular Probes) 20 μg/mL, and Alexa Fluor 488 (A-11029) 20 μg/mL.
Corresponding author. Tel.: +1 813 972 2000x6967; fax: +1 813 972 7623.
Abstract
Purchase PDF (301 K)
E-mail Article
Add to my Quick Links
Cited By in Scopus (3)
Purchase PDF (1586 K)
