JMJD5, a Jumonji C domain-containing dioxygenase, is important for embryonic development and cancer growth. Here, we show that JMJD5 is up-regulated by hypoxia and is crucial for hypoxiainduced cell proliferation. JMJD5 interacts directly with pyruvate kinase muscle isozyme (PKM)2 to modulate metabolic flux in cancer cells. The JMJD5-PKM2 interaction resides at the intersubunit interface region of PKM2, which hinders PKM2 tetramerization and blocks pyruvate kinase activity. This interaction also influences translocation of PKM2 into the nucleus and promotes hypoxiainducible factor (HIF)-1α-mediated transactivation. JMJD5 knockdown inhibits the transcription of the PKM2-HIF-1α target genes involved in glucose metabolism, resulting in a reduction of glucose uptake and lactate secretion in cancer cells. JMJD5, along with PKM2 and HIF-1α, is recruited to the hypoxia response element site in the lactate dehydrogenase A and PKM2 loci and mediates the recruitment of the latter two proteins. Our data uncover a mechanism whereby PKM2 can be regulated by factor-bindinginduced homo/heterooligomeric restructuring, paving the way to cell metabolic reprogram.Warburg effect | aerobic glycolysis | breast cancer | cancer metabolism J MJD5 is a Jumonji C domain-containing dioxygenase shown to be involved in lysine demethylation (1-3) and hydroxylation functions (4). Although the exact cellular substrates and functions of JMJD5 remain unclear, JMJD5 was shown to positively regulate cyclin A1 but negatively regulate p53 and p21 (1-3). Knockdown of JMJD5 in Michigan Cancer Foundation (MCF)-7 cells inhibits cell proliferation (1), and JMJD5 −/− embryos showed severe growth retardation, resulting in embryonic lethality at the midgestation stage (3). These data, together with its general overexpression in tumor tissues, implicate a role of JMJD5 in carcinogenesis. In this paper, we define a role of JMJD5 in regulating tumor metabolism under normoxic and hypoxic conditions through its interaction with pyruvate kinase muscle isozyme (PKM)2.One of the hallmarks of cancer cells is their altered metabolism, referred to as aerobic glycolysis, or the Warburg effect (5). This generally involves an increased uptake of glucose, use of intracellular glucose to pyruvate via glycolysis, and the conversion into lactate in the presence of sufficient oxygen. Along this metabolic flux, PKM1 or its spliced variant, PKM2, which dephosphorylates phosphoenolpyruvate (PEP) into pyruvate, the last step of glycolysis, is an important signal integrator whose activities determine the cytosolic level of pyruvate, thereby affecting subsequent metabolic flow to lactate, tricarboxylic acid cycle or biosynthetic pathway (6). Enzymatically, PKM2, an embryonic isoform found abundantly in tumor cells, is less active than PKM1, which allows the accumulation of glycolytic intermediates and diversion into biosynthetic pathways, demanded by rapid-proliferating cells.As a pivotal regulator of tumor metabolism, PKM2's activity is further modulated by allosteric regulation vi...

Photodynamic therapy (PDT) is a kind of photochemo-therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, the mechanisms involved in the death of human epidermoid carcinoma A431 cells triggered by PDT with Photofrin (a clinically approved photosensitizer) were characterized. Photofrin distributes dynamically in A431 cells; the plasma membranes and Golgi complex are the main target sites of Photofrin after a brief (3 h) and prolonged (24 h) incubation, respectively. Cells with differentially localized Photofrin displayed distinct death phenotypes in response to PDT. The effects of PDT on cells with plasma membrane-localized Photofrin were further studied in details. Cells stopped proliferating post PDT at Photofrin dose >7 micro g/ml, and at higher dose (28 micro g/ml) plasma membrane disruption and cell swelling were observed immediately after PDT. Dramatic alterations of several important signaling events were detected in A431 cells post Photofrin-PDT, including (i) immediate formation of reactive oxygen species (ROS), (ii) rapid activation of c-Jun N-terminal kinase, (iii) delayed activation of caspase-3 and cleavage of polyADP-ribose polymerase and p21-activated kinase 2, and (iv) loss of mitochondrial membrane potential. Intriguingly, the characteristics of typical apoptosis such as phosphatidylserine externalization and DNA fragmentation were not detected in the cell death process caused by this PDT regime. In conclusion, our results show that when plasma membranes are the main targets, Photofrin-PDT can lead to instant ROS formation and subsequent activation of downstream signaling events similar to those elicited by many apoptotic stimuli, but the damage of plasma membranes renders the death phenotype more necrosis like.

MYO18A is found as a novel PAK2 binding partner via βPIX/GIT1. MYO18A-depleted cells showed dramatic changes in shape, actin stress fiber and membrane ruffle formation, and displayed increases in the number and size of focal adhesions and a decrease in cell migration, suggesting an important role of MYO18A in regulating epithelial cell migration.

The PAK2/βPIX/GIT1 (p21-activated kinase 2/PAK-interacting exchange factor-β/G protein-coupled receptor kinase-interactor 1) complex has been shown to distribute to both membrane ruffles and focal adhesions of cells, where it plays an important role in regulating focal adhesion turnover. However, the detailed mechanism underlying this regulation is largely unknown. We previously reported that MYO18Aα interacts via its carboxyl terminus with the PAK2/βPIX/GIT1 complex through direct binding to βPIX, and that knockdown of MYO18Aα in epithelial cells causes accumulation of the complex in focal adhesions and decreased cell migration ability (Hsu et al., 2010). The current study characterized the detailed MYO18Aα-βPIX interaction mechanism and the biological significance of this interaction. We found that deletion of the carboxyl-terminal globular domain of MYO18Aα profoundly altered the cellular localization of βPIX and inhibited cell migration. βPIX interacts through its most carboxyl-terminus, PAWDETNL (639-646), with MYO18Aα and partially colocalized with MYO18Aα in membrane ruffles of cells, whereas βPIX(1-638), a mutant with deletion of PAWDETNL, accumulated in focal adhesions. Both focal adhesion numbers and area in βPIX(1-638)-expressing cells were greater than those in cells expressing wild-type βPIX(FL). Further experiments using deletion mutants of MYO18A and βPIX showed that disruption of MYO18A-βPIX interaction not only impaired cell motility but also decreased Rac1 activity. Collectively, our data unravel the interaction regions between MYO18A and βPIX and provide evidence for the critical role of this interaction in regulating cellular localization of βPIX, Rac1 activity, and adhesion and migration in epithelial cells.

BACKGROUND A previous comparative tissue proteomics study by the authors of the current study led to the identification of caldesmon (CaD) as one of the proteins associated with cervical metastasis of oral cavity squamous cell carcinoma (OSCC). In the current investigation, the authors focused on the potential functions of CaD in patients with OSCC. METHODS CaD expression was examined in tissue samples from 155 patients using immunohistochemical analysis. The expression of CaD variants was determined by Western blot analysis and reverse transcriptase‐polymerase chain reaction. In addition, the specific effects of CaD gene overexpression and silence were determined in OSCC cell lines. RESULTS CaD expression was found to be significantly higher in tumor cells from metastatic lymph nodes compared with primary tumor cells, and was nearly absent in normal oral epithelia. Higher CaD expression was found to be correlated with positive N classification, poor differentiation, perineural invasion, and tumor depth (P = .001, P = .029, P = .001, and P = .031, respectively). In survival analyses, OSCC patients with higher CaD expression were found to have poorer prognosis with regard to disease‐specific survival and disease‐free survival (P = .003 and P = .014, respectively). Multivariate analyses further indicated that higher CaD expression was an independent predictor of disease‐specific survival (P = .043). Serum CaD levels were found to be significantly higher in patients with OSCC, but this finding was not associated with clinicopathological manifestations. Data obtained from in vitro suppression, rescue, and overexpression of CaD in OEC‐M1 cells indicated that CaD promotes migration and invasive processes in OSCC cells. CONCLUSIONS The findings of the current study collectively suggest that the low‐molecular‐weight CaD expression in OSCC tumors is associated with tumor metastasis and patient survival. Cancer 2013;119:4003–4011. © 2013 American Cancer Society.

A polyclonal, phospho-epitope-specific antibody (P-STM) was generated to detect the activated p21-activated kinase 2 (PAK2), based on the regulatory autophosphorylation site Thr(402) of PAK2 [Yu et al., 1998]. In this report, we show that this antibody can also recognize many phosphoproteins in mitotic HeLa and A431 cells. Signal of these phosphoproteins emerged after treating the cells with nocodazole and okadaic acid, and was highly detected in G2-M phase transition of HeLa cells released from double thymidine block. Immunofluorescence analysis revealed that P-STM strongly stained HeLa cells at prometaphase and metaphase, but not at interphase and anaphase. Interestingly, this staining pattern was almost identical to that obtained by staining with MPM2, a monoclonal antibody known to react with phosphoproteins in mitotic HeLa cells. However, the phosphoproteins detected by the two antibodies are quite different. Two-dimensional gel electrophoresis (2DE) and tryptic peptide fingerprint analysis by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry were employed to identify lamins A and C as two of the mitotic cell-specific phosphoproteins recognized by P-STM. Lamins A and C immunoprecipitated from nocodazole-treated cells, but not from untreated cells showed strong reactivity to P-STM, and this reactivity lost completely after protein phosphatase 2A treatment. In summary, our results show that P-STM represents a novel tool for detecting mitotic phosphoproteins, which are different from those recognized by MPM2, and that lamins A and C are the two prominent mitotic phosphoproteins detected by P-STM.

BackgroundLamins A and C, two major structural components of the nuclear lamina that determine nuclear shape and size, are phosphoproteins. Phosphorylation of lamin A/C is cell cycle-dependent and is involved in regulating the assembly–disassembly of lamin filaments during mitosis. We previously reported that P-STM, a phosphoepitope-specific antibody raised against the autophosphorylation site of p21-activated kinase 2, recognizes a number of phosphoproteins, including lamins A and C, in mitotic HeLa cells.ResultsHere, using recombinant proteins and synthetic phosphopeptides containing potential lamin A/C phosphorylation sites in conjunction with in vitro phosphorylation assays, we determined the lamin A/C phosphoepitope(s) recognized by P-STM. We found that phosphorylation of Thr-19 is required for generating the P-STM phosphoepitope in lamin A/C and showed that it could be created in vitro by p34cdc2/cyclin B kinase (CDK1)-catalyzed phosphorylation of lamin A/C immunoprecipitated from unsynchronized HeLa S3 cells. To further explore changes in lamin A/C phosphorylation in living cells, we precisely quantified the phosphorylation levels of Thr-19 and other sites in lamin A/C isolated from HeLa S3 cells at interphase and mitosis using the SILAC method and liquid chromatography-tandem mass spectrometry. The results showed that the levels of phosphorylated Thr-19, Ser-22 and Ser-392 in both lamins A and C, and Ser-636 in lamin A only, increased ~2- to 6-fold in mitotic HeLa S3 cells.ConclusionsCollectively, our results demonstrate that P-STM is a useful tool for detecting Thr-19-phosphorylated lamin A/C in cells and reveal quantitative changes in the phosphorylation status of major lamin A/C phosphorylation sites during mitosis.

Photodynamic therapy (PDT), a photochemotherapeutic regimen used to treat several diseases, including cancer, exerts its effects mainly through induction of cell death. Using human epidermoid carcinoma A431 cells as a model, we previously showed that distinct cell death types could be triggered by protocols that selectively delivered Photofrin (a clinically approved photosensitizer) to different subcellular sites (Hsieh et al. [2003] J Cell Physiol 194: 363-375]. Here, the responses elicited by PDT in A431 cells containing intracellular organelle-localized Photofrin were further characterized. Two prominent cell phenotypes were observed under these conditions: one characterized by perinuclear vacuole (PV) formation 2-8 h after PDT followed by cell recovery or shrinkage within 48 h, and a second characterized by typical apoptotic features appearing within 4 h after PDT. DCFDA-sensitive reactive oxygen species formed proximal to PVs during the response to PDT, covering areas in which both endoplasmic reticulum (ER) and the Golgi complex were located. Biochemical analyses showed that Photofrin-PDT also induced JNK activation and altered the protein secretion profile. A more detailed examination of PV formation revealed that PVs were derived from the ER. The alteration of ER structure induced by PDT was similar to that triggered by thapsigargin, an ER Ca(2+)-ATPase inhibitor that perturbs Ca(2+) homeostasis, suggesting a role for Ca(2+) in the formation of PVs. Microtubule dynamics did not significantly affect PV formation. This study demonstrates that cells in which intracellular organelles are selectively loaded with Photofrin mount a novel response to ER stress induced by PDT.