GAK is phosphorylated by c-Src and translocated from the centrosome to chromatin at the end of telophase

<p>Cyclin G-associated kinase (GAK) harbors a consensus phosphorylation motif (Y412) for c-Src; however, its physiological significance remains elusive. Here, we show that GAK is phosphorylated by c-Src not only at Y412 but also at Y1149. An anti-GAK-pY412 antibody recognized the shifted band of GAK during M phase. Immunofluorescence (IF) showed that GAK-pY412/pY1149 signals were present in the nucleus during interphase, translocated to chromosomes at prophase and prometaphase, moved to centrosomes at metaphase, and finally translocated to chromosomes at the end of telophase, when nuclear membrane formation was almost complete. These subcellular movements of GAK resemble those of DNA licensing factors. Indeed, mass spectrometry identified mini-chromosome maintenance (MCM) 3, an essential component of the DNA licensing system, as one of the association partners of GAK; immunoprecipitation-mediated Western blotting confirmed their association <i>in vivo</i>. These results suggest that the c-Src_GAK_MCM axis plays an important role in cell cycle progression through control of the DNA replication licensing system.</p

Gefitinib and Luteolin Cause Growth Arrest of Human Prostate Cancer PC-3 Cells via Inhibition of Cyclin G-Associated Kinase and Induction of miR-630

<div><p>Cyclin G-associated kinase (GAK), a key player in clathrin-mediated membrane trafficking, is overexpressed in various cancer cells. Here, we report that GAK expression is positively correlated with the Gleason score in surgical specimens from prostate cancer patients. Embryonic fibroblasts from knockout mice expressing a kinase-dead (KD) form of GAK showed constitutive hyper-phosphorylation of the epidermal growth factor receptor (EGFR). In addition to the well-known EGFR inhibitors gefitinib and erlotinib, the dietary flavonoid luteolin was a potent inhibitor of the Ser/Thr kinase activity of GAK <i>in vitro</i>. Co-administration of luteolin and gefitinib to PC-3 cells had a greater effect on cell viability than administration of either compound alone; this decrease in viability was associated with drastic down-regulation of GAK protein expression. A comprehensive microRNA array analysis revealed increased expression of miR-630 and miR-5703 following treatment of PC-3 cells with luteolin and/or gefitinib, and exogenous overexpression of miR-630 caused growth arrest of these cells. GAK appears to be essential for cell death because co-administration of gefitinib and luteolin to EGFR-deficient U2OS osteosarcoma cells also had a greater effect on cell viability than administration of either compound alone. Taken together, these findings suggest that GAK may be a new therapeutic target for prostate cancer and osteosarcoma.</p></div

Exogenous expression of miR-630 inhibits the growth of PC-3 cells.

<p>A, confirmation by qRT-PCR of the successful overexpression of exogenous miR-630 from the pEP-hsa-miR-630 expression vector in PC-3 cells. The data are represented as the mean ± SEM of n = 3 independent experiments. B, The growth rate of PC-3 cells transfected with the pEP-hsa-miR-630 expression vector. Cells were also transfected with GFP to monitor the successful expression of miR-630. The data are represented as the mean ± SEM of n = 3 independent experiments for each condition. <i>P</i><0.01 by a Student's <i>t</i>-test.</p

Luteolin and gefitinib induce death of PC-3 cells.

<p>A, the numbers of viable PC-3 cells 0, 24, 48, and 72 h after the addition of solvent alone (DMSO), 60 µM luteolin, 60 µM gefitinib, or 60 µM luteolin plus 60 µM gefitinib to the culture medium. The data are represented as the mean ± SEM of n = 3 independent experiments at each concentration. B, immunoblot analyses of GAK, AR, caspase-3, and α-tubulin (control) protein levels in PC-3 cells exposed to solvent alone (DMSO), 60 µM luteolin, 60 µM gefitinib, or 60 µM luteolin plus 60 µM gefitinib for 0–72 h. The arrowheads indicate reduced expression levels of GAK and AR in cells exposed to the drugs for 72 h. The arrows indicate the emergence of activated caspase-3, which suggested that gefitinib and luteolin induced apoptosis. C, FACS analysis of PC-3 cells PC-3 cells exposed to solvent alone (DMSO), 60 µM luteolin, 60 µM gefitinib, or 60 µM luteolin plus 60 µM gefitinib for 72 h. The arrowheads indicate the subG1 populations. D, the percentages of PC-3 cells in the indicated phases of the cell cycle after exposure to the same treatments for 72 h.</p

GAK is overexpressed in the nuclei of cancer cells.

<p>Western blot analysis of GAK, lamin A/C, and α-tubulin (control) in cytoplasmic (Cyt) and nuclear (Nuc) extracts from prostate cancer (PC-3 and LNCaP), breast cancer (MDA-MB231 and MCF-7) and cervical cancer (HeLaS3) cell lines. Lamin A/C was used as a nuclear marker. Two different anti-GAK antibodies were used. The arrows and arrowheads indicate the GAK bands identified primarily in the nuclear and cytoplasmic fractions, respectively.</p

GAK is overexpressed in radical prostatectomy specimens from prostate cancer patients.

<p>A–D, hematoxylin and eosin (H-E) staining (A, B) and anti-GAK immunostaining (C, D) of representative radical prostatectomy sections of cancerous (A, C) and normal (B, D) tissues from prostate cancer patients (n = 42). Scale bar = 50 µm. E, higher power magnification of the boxed area shown in C. Scale bar = 50 µm.</p

Loss of GAK activity leads to constitutive hyper-phosphorylation of the EGFR.

<p>A, western blot analysis of EGFR expression in WT (GAK-kd^+/+) and mutant (GAK-kd^−/−) MEFs. B, the effects of a Tyr-phosphatase inhibitor (50 mM Na₃VO₄) and Ser/Thr phosphatase inhibitors (50 mM NaF and 50 mM β-glycerophosphate, or 2.5 µM okadaic acid) on the inhibition of EGFR phosphorylation by λ-phosphatase (λPPase; 200 U). A, B, the arrows indicate the phosphorylated EGFR protein. Alpha-tubulin (α-Tub) was used as a loading control. (C, D) Western blot analysis of expression levels of the EGFR and ERK1/2 in WT (+/+) and GAK-kd (−/−) MEFs following EGF stimulation for the indicated times. Cycloheximide (50 µg/ml) was added to the culture medium 1 h prior to EGF (10 µg/ml) to inhibit novel protein synthesis. The tilted and horizontal arrows indicate the phosphorylated and hyper-phosphorylated EGFR bands, respectively. GAPDH was used as a loading control. C, the arrowheads indicate differential expression of phosphorylated ERK1/2 in WT and GAK-kd MEFs. D, NT, non-treated. E, immunostaining of the EGFR protein in WT (+/+) and mutant (−/−) MEFs treated with or without the proteasome inhibitor MG132 (50 µg/ml). Notable panels are encircled by turquoise and green lines. F, the numbers of EGFR-positive cells in WT and GAK-kd (Homo) cells in the presence or absence of MG132. The data are represented as the mean ± SEM of n = 3 independent experiments at each time point.</p

Statistical analysis of the relationship between the Gleason score and GAK immunostaining in the prostatectomy specimens.

<p><i>P</i><0.01.</p

Luteolin and erlotinib inhibit GAK activity similarly to gefitinib and SB203580.

<p>A–C, representative SDS-PAGE analysis of proteins subjected to <i>in vitro</i> kinase assays using ³²P-γATP, GAK (∼100 µg/ml) as the enzyme, PP2A B′γ (∼10 µg/ml) as the substrate, and the indicated concentrations of gefitinib, erlotinib, and SB203580 as inhibitors. The signals incorporated were detected by autoradiography and the amounts of proteins loaded were assessed by Coomassie Brilliant Blue (CBB) staining. The graphs show the intensity ratios of phosphorylated PP2A B′γ and GAK relative to that of non-treated samples. The data are represented as the mean ± SEM of n = 3 independent experiments at each concentration.</p

Working model showing the molecular mechanisms involved in the induction of growth arrest and apoptosis of PC-3 cells by luteolin and gefitinib.

<p>The arrows and T-shaped lines signify positive and negative actions, respectively. The thickness of each line is proportional to the strength of the denoted action. The dashed green line and arrow indicate that these actions were verified by another group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100124#pone.0100124-Lee1" target="_blank">[10]</a>. The red and blue arrows indicate up-regulation and down-regulation, respectively. Down-regulation of GAK leads to hyper-phosphorylation of tyrosine residues (pY) in EGFR.</p