Miyuki Nomura

Protein phosphatase Dusp26 associates with KIF3 motor and promotes N-cadherin-mediated cell-cell adhesion

Recent studies have demonstrated essential functions for KIF3, a microtubule-directed protein motor, in subcellular transport of several cancer-related proteins, including the β-catenin–cadherin(s) complex. In this study, we report identification of the protein-phosphatase Dusp26 as a novel regulator of the KIF3 motor. Here we undertake yeast two-hybrid screening and identify Kif3a, a motor subunit of the KIF3 heterotrimeric complex, as a novel Dusp26-binding protein. Co-immunoprecipitation and colocalization experiments revealed that Dusp26 associates not only with Kif3a, but also with Kap3, another subunit of the KIF3 complex. Dephosphorylation experiments in vitro and analysis using mutant forms of Dusp26 in intact cells strongly suggested that Dusp26 is recruited to the KIF3 motor mainly by interaction with Kif3a, and thereby dephosphorylates Kap3. Forced expression of Dusp26, but not its catalytically inactive mutant, promoted distribution of β-catenin/N-cadherin, an established KIF3 cargo, to cell–cell junction sites, resulting in increased cell–cell adhesiveness. We also showed that Dusp26 mRNA expression was downregulated in human glioblastoma samples. These results suggest previously unidentified functions of Dusp26 in intracellular transport and cell–cell adhesion. Downregulation of Dusp26 may contribute to malignant phenotypes of glioma.

Nuclear Inhibitor of Protein Phosphatase-1 (NIPP1) Directs Protein Phosphatase-1 (PP1) to Dephosphorylate the U2 Small Nuclear Ribonucleoprotein Particle (snRNP) Component, Spliceosome-associated Protein 155 (Sap155) *□

Pre-mRNA splicing entails reversible phosphorylation of spliceosomal proteins. Recent work has revealed essential roles for Ser/Thr phosphatases, such as protein phosphatase-1 (PP1), in splicing, but how these phosphatases are regulated is largely unknown. We show that nuclear inhibitor of PP1 (NIPP1), a major PP1 interactor in the vertebrate nucleus, recruits PP1 to Sap155 (spliceosome-associated protein 155), an essential component of U2 small nuclear ribonucleoprotein particles, and promotes Sap155 dephosphorylation. C-terminally truncated NIPP1 (NIPP1-ΔC) formed a hyper-active holoenzyme with PP1, rendering PP1 minimally phosphorylated on an inhibitory site. Forced expression of NIPP1-WT and -ΔC resulted in slight and severe decreases in Sap155 hyperphosphorylation, respectively, and the latter was accompanied with inhibition of splicing. PP1 overexpression produced similar effects, whereas small interfering RNA-mediated NIPP1 knockdown enhanced Sap155 hyperphosphorylation upon okadaic acid treatment. NIPP1 did not inhibit but rather stimulated Sap155 dephosphorylation by PP1 in vitro through facilitating Sap155/PP1 interaction. Further analysis revealed that NIPP1 specifically recognizes hyperphosphorylated Sap155 thorough its Forkhead-associated domain and dissociates from Sap155 after dephosphorylation by associated PP1. Thus NIPP1 works as a molecular sensor for PP1 to recognize phosphorylated Sap155.

CDC25A mRNA levels significantly correlate with Ki-67 expression in human glioma samples

Cell division cycle 25 (CDC25) phosphatases are cell-cycle regulatory proteins which are overexpressed in a significant number of human cancers. This study evaluated the role of CDC25 phosphatases in human glioma proliferation. Upregulation of CDC25A was observed in human glioma specimens and human glioma cell lines. Comparison of expression levels of CDC25A and CDC25B messenger ribonucleic acid (RNA) to Ki-67 labeling index in glioma tissues found that Ki-67 labeling index was significantly correlated with the expression of CDC25A, but not with that of CDC25B. Depletion of CDC25A by small interfering RNA and inhibition of CDC25 suppressed cell proliferation and induced apoptosis in glioma cell lines, indicating that CDC25A is a potential target for the development of new therapy for glioma.

MKP-7, a JNK phosphatase, blocks ERK-dependent gene activation by anchoring phosphorylated ERK in the cytoplasm

MAPK phosphatase-7 (MKP-7) was identified as a JNK-specific phosphatase. However, despite its high specificity for JNK, MKP-7 interacts also with ERK. We previously showed that as a physiological consequence of their interaction, activated ERK phosphorylates MKP-7 at Ser-446, and stabilizing MKP-7. In the present study, we analyzed MKP-7 function in activation of ERK. A time-course experiment showed that both MKP-7 and its phosphatase-dead mutant prolonged mitogen-induced ERK phosphorylation, suggesting that MKP-7 functions as a scaffold for ERK. An important immunohistological finding was that nuclear translocation of phospho-ERK following PMA stimulation was blocked by co-expressed MKP-7 and, moreover, that phospho-ERK co-localized with MKP-7 in the cytoplasm. Reporter gene analysis indicated that MKP-7 blocks ERK-mediated transcription. Overall, our data indicate that MKP-7 down-regulates ERK-dependent gene expression by blocking nuclear accumulation of phospho-ERK.

Abrogation of protein phosphatase 6 promotes skin carcinogenesis induced by DMBA

Somatic mutations in the gene encoding the catalytic subunit of protein phosphatase 6 (Ppp6c) have been identified in malignant melanoma and are thought to function as a driver in B-raf- or N-ras-driven tumorigenesis. To assess the role of Ppp6c in carcinogenesis, we generated skin keratinocyte-specific Ppp6c conditional knockout mice and performed two-stage skin carcinogenesis analysis. Ppp6c deficiency induced papilloma formation with 7,12-dimethylbenz (a) anthracene (DMBA) only, and development of those papillomas was significantly accelerated compared with that seen following DMBA/TPA (12-O-tetradecanoylphorbol 13-acetate) treatment of wild-type mice. NF-κB activation either by tumor necrosis factor (TNF)-α or interleukin (IL)-1β was enhanced in Ppp6c-deficient keratinocytes. Overall, we conclude that Ppp6c deficiency predisposes mice to skin carcinogenesis initiated by DMBA. This is the first report showing that such deficiency promotes tumor formation in mice.

Characterization of a novel low-molecular-mass dual specificity phosphatase-4 (LDP-4) expressed in brain.

Dual-specificity phosphatases (DSPs), which dephosphorylate proteins at Ser/Thr as well as Tyr residues, are thought to be involved in critical signaling events such as control of MAP kinases (MAPKs). We have isolated the cDNA for a novel DSP and termed it low molecular mass DSP-4 (LDP-4). LDP-4 is composed of 211 amino acids with a predicted molecular mass of 23.9-kDa. Northern blot analysis using various mouse tissues showed that the LDP-4 transcript was expressed exclusively in brain. In situ hybridization showed that brain expression of LDP-4 was ubiquitous except for the hippocampus. When expressed in COS-7 cells, FLAG-tagged LDP-4 protein was present within the nucleus and Golgi apparatus. LDP-4 expression did not reduce phosphorylation levels of MAPKs, but rather evoked activation of JNK and p38.

The protein phosphatase 6 catalytic subunit (Ppp6c) is indispensable for proper post-implantation embryogenesis.

Ppp6c, which encodes the catalytic subunit of phosphoprotein phosphatase 6 (PP6), is conserved among eukaryotes from yeast to humans. In mammalian cells, PP6 targets IκBε for degradation, activates DNA-dependent protein kinase to trigger DNA repair, and is reportedly required for normal mitosis. Recently, Ppp6c mutations were identified as candidate drivers of melanoma and skin cancer. Nonetheless, little is known about the physiological role of Ppp6c. To investigate this function in vivo, we established mice lacking the Ppp6c phosphatase domain by crossing heterozygous mutants. No viable homozygous pups were born, indicative of a lethal mutation. Ppp6c homozygous mutant embryos were identified among blastocysts, which exhibited a normal appearance, but embryos degenerated by E7.5 and showed clear developmental defects at E8.5, suggesting that mutant embryos die after implantation. Accordingly, homozygous blastocysts showed significant growth failure of the inner cell mass (ICM) in in vitro blastocyst culture, and primary Ppp6c exon4-deficient MEFs showed greatly reduced proliferation. These results establish for the first time that the Ppp6c phosphatase domain is indispensable for mouse embryogenesis after implantation.

Loss of protein phosphatase 6 in mouse keratinocytes increases susceptibility to ultraviolet-B-induced carcinogenesis

We previously reported that deficiency in the gene encoding the catalytic subunit of protein phosphatase 6 (Ppp6c) predisposes mouse skin tissue to papilloma formation initiated by DMBA. Here, we demonstrate that Ppp6c loss acts as a tumor promoter in UVB-induced squamous cell carcinogenesis. Following UVB irradiation, mice with Ppp6c-deficient keratinocytes showed a higher incidence of skin squamous cell carcinoma than did control mice. Time course experiments showed that following UVB irradiation, Ppp6c-deficient keratinocytes upregulated expression of p53, PUMA, BAX, and cleaved caspase-3 proteins. UVB-induced tumors in Ppp6c-deficient keratinocytes exhibited a high frequency of both p53- and γH2AX-positive cells, suggestive of DNA damage. Epidemiological and molecular data strongly suggest that UVB from sunlight induces p53 gene mutations in keratinocytes and is the primary causative agent of human skin cancers. Our analysis suggests that PP6 deficiency underlies molecular events that drive outgrowth of initiated keratinocytes harboring UVB-induced mutated p53. Understanding PP6 function in preventing UV-induced tumorigenesis could suggest strategies to prevent and treat this condition.

Ex vivo model of non‑small cell lung cancer using mouse lung epithelial cells

Lung cancer is the most common cause of cancer mortality, however, efficient methods to culture, expand and transform lung epithelial (LE) cells have not been established. In the present study, an efficient ex vivo method was applied to recapitulate lung carcinogenesis using mouse LE cells. A Matrigel-assisted three-dimensional culture was used to isolate and selectively expand LE cells from mouse lungs. Purified LE cells were passaged and expanded for at least 2 to 3 months while maintaining epidermal growth factor-dependence. LE cells were also easily transformed by genetic manipulations using retroviral vectors. A SV40 large-T antigen, suppressing p53 and pRB, plus an activated oncogene, such as KrasG12V or EGFRex19del, were required to transform LE cells. Transformed cells formed tumors resembling non-small cell lung cancer (NSCLC) in allograft models and exhibited strong oncogene addiction. This experimental system provided a unique model system to study lung tumorigenesis and develop novel therapeutics against NSCLC.

A metabolic vulnerability of small-cell lung cancer

Small-cell lung cancer (SCLC) accounts for 15-20% of lung cancers, and patient prognosis in this subset is poorer than in other types of lung cancer. Genomic studies in a large cohort of SCLC patients revealed simultaneous inactivation of the tumor suppressors TP53 and RB1 but few driver mutations have been therapeutically targeted [1]. Therefore, patients with SCLC have not benefited from recent advances in targeted therapy. We recently revisited glucose metabolism in cancer and identified the glycolytic enzyme PKM1 as a potential new target for SCLC. Enhanced glucose uptake is a cancer hallmark, as evidenced by the fact that most cancers including SCLC are effectively detected by a FDG-PET scan. Glucose taken up by tumor cells undergoes a series of glycolytic reactions to fuel other metabolic pathways such as the TCA cycle, the pentose-phosphate pathway and amino acid biosynthetic pathways (Figure 1). The end product of glycolysis is lactate, which was classically considered cellular waste. However, recent work reveals that lactate is not only released from but also actively taken up by cancer cells, particularly in in vivo settings, to fuel the TCA cycle after conversion back to pyruvate (see comment in reference [2]). It is also noteworthy that pyruvate can be generated from carbon source(s) other than glucose, such as glutamine. In these ways, glycolysis is linked to many metabolic pathways in an extensive and complex network. The glycolytic pathway is comprised of 10 steps, three of which are irreversible. One is conversion of phosphoenol-pyruvate (PEP) to pyruvate, which is catalyzed by pyruvate kinase (PK). Vertebrates express 4 PK isozymes encoded by 2 genes, PKM and PKLR. PKM is predominant in most cells, whereas PKs in hepatocytes, red blood cells and some kidney cells are encoded by PKLR. Importantly, PKM transcripts exist as 2 splicing variants that give rise to distinct proteins: PKM1, which is constitutively active and promotes glucose catabolism, and PKM2, which is activated only in response to increased levels of allosteric activator(s) such as fructose 1,6-bisphosphate, an upstream intermediate in glycolysis. The latter property ensures that PKM2 will maintain a low rate of PEP-to-pyruvate conversion from glucose relative to PKM1. Generally, expression of PKM1 and PKM2 is mutually exclusive in a given cell type. PKM isoform switching has received much attention in cancer metabolism since the discovery that PKM2, but not PKM1, is highly expressed in cancer cells and apparently favors tumor growth in some cell lines [3]. One conclusion was that high, non-regulatable PKM1 activity is not compatible with proliferation for unknown reasons, and thus, that PKM2 is the predominant isoform in dividing cells. Paradoxically, however, PKM2-knockout (KO) mice, which are deficient only in a PKM2-specific exon, show enhanced rather than decreased tumorigenesis [4]. Interestingly, PKM2-KO mice also display compensatory and partial expression of the more active isoform PKM1, although at varying levels [4]. In these contexts, it has been unclear whether PKM2 promotes or suppresses tumor growth. To address these questions, we developed two lines of knock-in (KI) mice, each expressing either PKM1 or PKM2 [5]. Mice from both lines developed normally and were fertile, strongly suggesting that PKM1 expression itself did not significantly perturb normal cell proliferation or differentiation. More importantly, evaluation of our models revealed that relatively high PKM1 activity, as compared to PKM2, confers metabolic advantages and promotes tumor growth in various experimental models. PKM1-expressing cells exhibited both higher glucose flux Editorial