Vectors for the Generation of FLAG®- or EGFPTagged cDNA Constructs and EGFP-Tagged Antisense RNA Constructs

Jakobi, Rolf; Moertl, Erin; Koeppel, Mark A.

doi:10.2144/01303bm02

Cited by 3 publications

(3 citation statements)

References 7 publications

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“…The bicistronic retroviral expression vector pRetroIRES-GFP 2 was generated from RetroTet RTRg(Ϫ)gfp (Dr. Helen M. Blau, Stanford University). The cloning vector pKoz/EGFP and the expression vector pExpress/EGFP were generated as described previously (26,27). pMT107 and pMT123 expression vectors for His-ubiquitin (His-Ub) and HA-ubiquitin (HA-Ub), respectively, were gifts from Dr. D. Bohmann, Rochester University.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, we examined whether full-length PAK-2 and caspase-activated PAK-2 differ in their subcellular localization. The localization of recombinant PAK-2 was examined using EGFP fusion constructs (18,26). The EGFP fusion proteins and EGFP alone were transiently expressed in 293T cells, and subcellular localization was monitored by fluorescence microscopy.…”

Section: Pak-2p34 Is Degraded By the 26 S Proteasomementioning

confidence: 99%

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Caspase-activated PAK-2 Is Regulated by Subcellular Targeting and Proteasomal Degradation

Jakobi

McCarthy

Koeppel

et al. 2003

Journal of Biological Chemistry

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p21-activated protein kinases (PAKs) are a family of serine/threonine protein kinases that are activated by binding of the p21 G proteins Cdc42 or Rac. The ubiquitous PAK-2 (␥-PAK) is unique among the PAK isoforms because it is also activated through proteolytic cleavage by caspases or caspase-like proteases. In response to stress stimulants such as tumor necrosis factor ␣ or growth factor withdrawal, PAK-2 is activated as a fulllength enzyme and as a proteolytic PAK-2p34 fragment. Activation of full-length PAK-2 stimulates cell survival, whereas proteolytic activation of PAK-2p34 is involved in programmed cell death. Here we provide evidence that the proapoptotic effect of PAK-2p34 is regulated by subcellular targeting and degradation by the proteasome. Full-length PAK-2 is localized in the cytoplasm, whereas the proteolytic PAK-2p34 fragment translocates to the nucleus. Subcellular localization of PAK-2 is regulated by nuclear localization and nuclear export signal motifs. A nuclear export signal motif within the regulatory domain prevents nuclear localization of fulllength PAK-2. Proteolytic activation removes most of the regulatory domain and disrupts the nuclear export signal. The activated PAK-2p34 fragment contains a nuclear localization signal and translocates to the nucleus. However, levels of activated PAK-2p34 are tightly regulated through ubiquitination and degradation by the proteasome. Inhibition of degradation by blocking polyubiquitination results in significantly increased levels of PAK-2p34 and as a consequence, in stimulation of programmed cell death. Therefore, nuclear targeting and inhibition of degradation appear to be critical for stimulation of the cell death response by PAK-2p34.

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Section: Methodsmentioning

confidence: 99%

Section: Pak-2p34 Is Degraded By the 26 S Proteasomementioning

confidence: 99%

Caspase-activated PAK-2 Is Regulated by Subcellular Targeting and Proteasomal Degradation

Jakobi

McCarthy

Koeppel

et al. 2003

Journal of Biological Chemistry

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“…In addition, EGFP-, Myc-, HA-, or FLAG-tagged recombinant proteins can be specifically immunoprecipitated from cell lysates with antibodies against the EGFP, Myc, HA, or FLAG tag. Previously, we have reported pKoz/M-FLAG and pKoz/ EGFP cloning vectors for the generation of FLAG-or EGFP-tagged cDNA constructs (10). The pKoz vectors are suitable for cloning of genes that are toxic in E. coli because prokaryotic transcription termination signals prevent nonspecific basal expression in E. coli.…”

Section: Mammalian Expression Vectors For Epitope Tag Fusion Proteinsmentioning

confidence: 99%

Mammalian Expression Vectors for Epitope Tag Fusion Proteins that Are Toxic in E. coli

2002

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The amplification from the crude lysates of the recombinant P. pastoris strains KM71 (pPIC9-MYO)-Mut S and GS115 (pPIC9-MYO)-Mut + , which contained the integration of the myostatin gene to chromosomal DNA, was completed. When Taq DNA polymerase was used for amplification, we observed either no or nonspecific amplification products. The positive control sample yielded the 831-bp specific fragment. When the HOTStar Taq DNA polymerase was used for the amplification, the 831-bp specific amplification product was visible. The wild-type gene AOX1 2.2-kb fragment was co-amplified in GS115-derived strains. The fragment is not visible in KM71-derived strains, because the gene is deleted from the parent strain. The highest yield was attained with 10 min of boiling at 80°C (Figure 1). Changing the volume of amplification mixture did not influence the result. No changes were found when the annealing temperature and number of amplification cycles were changed. Similar results were observed when the recombinant yeast P. pastoris strain KM71 (pPIC9-LEP)-Mut S with cloned gene for leptin was amplified using the same procedure. The results are demonstrated in Figure 2. In this experiment, a specific 912-bp product was observed as a positive result. No product from the 2.2-kb wild-type gene fragment was visible in these KM71-derived strains. From the presented results we can deduce that crude yeast lysates contain many PCR inhibitors, which decreases the specificity of the amplification and the amount of amplification product. In contrast to the amplification from the crude lysates of some bacterial cells, only some robust Taq DNA polymerases can overcome these inhibitors. However, fundamentally, the amplification from crude yeast lysates is possible, and the described method can be used as an inexpensive and quick alternative to isolation of chromosomal DNA from yeast cells.

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Elevated p21-Activated Kinase 2 Activity Results in Anchorage-Independent Growth and Resistance to Anticancer Drug–Induced Cell Death

et al. 2009

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p21-activated kinase 2 (PAK-2) seems to be a regulatory switch between cell survival and cell death signaling. We have shown previously that activation of full-length PAK-2 by Rac or Cdc42 stimulates cell survival, whereas caspase activation of PAK-2 to the proapoptotic PAK-2p34 fragment is involved in the cell death response. In this study, we present a role of elevated activity of full-length PAK-2 in anchorage-independent growth and resistance to anticancer drug-induced apoptosis of cancer cells. Hs578T human breast cancer cells that have low levels of PAK-2 activity were more sensitive to anticancer drug-induced apoptosis and showed higher levels of caspase activation of PAK-2 than MDA-MB435 and MCF-7 human breast cancer cells that have high levels of PAK-2 activity. To examine the role of elevated PAK-2 activity in breast cancer, we have introduced a conditionally active PAK-2 into Hs578T human breast cells. Conditional activation of PAK-2 causes loss of contact inhibition and anchorage-independent growth of Hs578T cells. Furthermore, conditional activation of PAK-2 suppresses activation of caspase 3, caspase activation of PAK-2, and apoptosis of Hs578T cells in response to the anticancer drug cisplatin. Our data suggest a novel mechanism by which full-length PAK-2 activity controls the apoptotic response by regulating levels of activated caspase 3 and thereby its own cleavage to the proapoptotic PAK-2p34 fragment. As a result, elevated PAK-2 activity interrupts the apoptotic response and thereby causes anchorage-independent survival and growth and resistance to anticancer drug-induced apoptosis.

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Vectors for the Generation of FLAG®- or EGFPTagged cDNA Constructs and EGFP-Tagged Antisense RNA Constructs

Cited by 3 publications

References 7 publications

Caspase-activated PAK-2 Is Regulated by Subcellular Targeting and Proteasomal Degradation

Caspase-activated PAK-2 Is Regulated by Subcellular Targeting and Proteasomal Degradation

Mammalian Expression Vectors for Epitope Tag Fusion Proteins that Are Toxic in E. coli

Elevated p21-Activated Kinase 2 Activity Results in Anchorage-Independent Growth and Resistance to Anticancer Drug–Induced Cell Death

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