Characterization of hPRP4 Kinase Activation: Potential Role in Signaling

doi:10.1006/bbrc.2000.2651

Biochemical and Biophysical Research Communications

Volume 271, Issue 2, 10 May 2000, Pages 456-463

https://doi.org/10.1006/bbrc.2000.2651 Get rights and content

Abstract

Mitogen-activated protein kinases (MAPKs) and cyclin-dependent kinases (CDKs) are important proline-directed Ser/Thr kinases that play distinct roles in cell differentiation and proliferation. hPRP4 (pre-mRNA processing gene), a human homologue of S. pombe Prp4, is a recently isolated CDK-like kinase with homology to MAPKs. Little is known about the mRNA processing function of hPRP4 or about the signaling pathways with which it is associated. hPRP4 is expressed in a variety of human tissues with the highest expression in the brain, lung and liver. In this paper, we characterize the activation of hPRP4 in COS-7 cells and show that hPRP4 also possesses a transcription factor activation function. hPRP4 is activated by epidermal growth factor (EGF) or forskolin treatment, but not tetradecanoyl phorbol acetate (TPA) nor ultraviolet (UV) irradiation. Activated hPRP4 phosphorylates residue Thr-417 on Elk-1 resulting in Elk-1 activation. This site of Elk-1 phosphorylation is distinct from that of other MAPKs. Coexpression of hPRP4 with an Elk-1 reporter construct causes trans activation of the reporter. These findings suggest that hPRP4, a CDK-like kinase related to MAPKs, may play a distinct role in signal transduction in addition to its role in mRNA processing.

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Evaluation of cancer dependence and druggability of PRP4 kinase using cellular, biochemical, and structural approaches
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Citation Excerpt :
Furthermore, using HeLa as a mammalian cell model, it was demonstrated that PRP4 loss of function resulted in spindle assembly checkpoint failure (4). Among the substrates identified for PRP4 kinase are splicing factors SF2, PRPF6, and PRPF31 as well as KLF13 and ELK-1 transcription factors that are involved in numerous cellular functions independent of splicing (5, 8–10). These findings suggest a broader spectrum of PRP4 functions than reported previously, thus meriting further biological investigation of this kinase as a potential therapeutic target for drug discovery.
PRP4 kinase is known for its roles in regulating pre-mRNA splicing and beyond. Therefore, a wider spectrum of PRP4 kinase substrates could be expected. The role of PRP4 kinase in cancer is also yet to be fully elucidated. Attaining specific and potent PRP4 inhibitors would greatly facilitate the study of PRP4 biological function and its validation as a credible cancer target. In this report, we verified the requirement of enzymatic activity of PRP4 in regulating cancer cell growth and identified an array of potential novel substrates through orthogonal proteomics approaches. The ensuing effort in structural biology unveiled for the first time unique features of PRP4 kinase domain and its potential mode of interaction with a low molecular weight inhibitor. These results provide new and important information for further exploration of PRP4 kinase function in cancer.
Background: Little is known about the cancer dependence and druggability of PRP4.
Results: Significance of PRP4 catalytic activity is demonstrated, novel substrates are identified, and features of kinase domain structure are revealed.
Conclusion: PRP4 is required for cancer cell survival, displays substrate specificity, and is amenable to pharmacological inhibition.
Significance: Our results indicate that PRP4 is a potential drug target to pursue in cancer.
Curcumin induces radiosensitivity of in vitro and in vivo cancer models by modulating pre-mRNA processing factor 4 (Prp4)
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Removal of introns from pre-mRNA is catalyzed by the spliceosome, which is considered as an important mediator for the generation of functional proteins. Prp4 plays a central role in signal transduction and is essential for pre-mRNA processing by spliceosome activation [29]. Prp4K has been reported to be essential for stable tri-snRNP association during spliceosomal B complex formation in pre-mRNA splicing [30], but the exact molecular mechanism by which Prp4K mediates apoptosis upon curcumin treatment is not yet established.
Radiation therapy plays a central role in adjuvant strategies for the treatment of both pre- and post-operative human cancers. However, radiation therapy has low efficacy against cancer cells displaying radio-resistant phenotypes. Ionizing radiation has been shown to enhance ROS generation, which mediates apoptotic cell death. Further, concomitant use of sensitizers with radiation improves the efficiency of radiotherapy against a variety of human cancers. Here, the radio-sensitizing effect of curcumin (a derivative of turmeric) was investigated against growth of HCT-15 cells and tumor induction in C57BL/6J mice. Ionizing radiation induced apoptosis through ROS generation and down-regulation of Prp4K, which was further potentiated by curcumin treatment. Flow cytometry revealed a dose-dependent response for radiation-induced cell death, which was remarkably reversed by transfection of cells with Prp4K clone. Over-expression of Prp4K resulted in a significant decrease in ROS production possibly through activation of an anti-oxidant enzyme system. To elucidate an integrated mechanism, Prp4K knockdown by siRNA ultimately restored radiation-induced ROS generation. Furthermore, B16F10 xenografts in C57BL/6J mice were established in order to investigate the radio-sensitizing effect of curcumin in vivo. Curcumin significantly enhanced the efficacy of radiation therapy and reduced tumor growth as compared to control or radiation alone. Collectively, these results suggest a novel mechanism for curcumin-mediated radio-sensitization of cancer based on ROS generation and down-regulation of Prp4K.
Both mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 and phosphatidylinositide-3-OH kinase (PI3K)/Akt pathways regulate activation of E-twenty-six (ETS)-like transcription factor 1 (Elk-1) in U138 glioblastoma cells
2012, International Journal of Biochemistry and Cell Biology
Citation Excerpt :
Elk-1 can be activated by different pathways. Elk-1 is one of the main nuclear targets of activated MAPK/ERK1/2 after growth factor stimulation (Huang et al., 2000; Marais et al., 1993). Moreover, the PI3K/Akt pathway via the MAPK/ERK1/2 pathway activates Elk-1 in lung carcinoma cells to induce their proliferation (Zhang et al., 2006).
Epidermal growth factor (EGF) and its receptor (EGFR) have been shown to play a significant role in the pathogenesis of glioblastoma. In our study, the EGFR was stimulated with EGF in human U138 glioblastoma cells. We show that the activated mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 pathway phosphorylated the E twenty-six (ETS)-like transcription factor 1 (Elk-1) mainly at serine 383 residue. Mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, UO126 and ERK inhibitor II, FR180204 blocked the Elk-1 phosphorylation and activation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt pathway was also involved in the Elk-1 activation. Activation of the Elk-1 led to an increased survival and a proliferative response with the EGF stimulation in the U138 glioblastoma cells. Knocking-down the Elk-1 using an RNA interference technique caused a decrease in survival of the unstimulated U138 glioblastoma cells and also decreased the proliferative response to the EGF stimulation. The Elk-1 transcription factor was important for the survival and proliferation of U138 glioblastoma cells upon the stimulation of EGFR with EGF. The MAPK/ERK1/2 and PI3K/Akt pathways regulated this response via activation of the Elk-1 transcription factor. The Elk-1 may be one of the convergence points for pathways located downstream of EGFR in glioblastoma cells. Utilization of the Elk-1 as a therapeutic target may lead to a novel strategy in treatment of glioblastoma.
Cloning of Human PRP4 Reveals Interaction with Clk1
2001, Journal of Biological Chemistry
Citation Excerpt :
The hPRP4 is classified based on sequence similarity of the kinase domain into a member of CMGC group, which includes cyclin-dependent kinase family, mitogen-activated protein kinase family, glycogen synthase kinase family, and cdc2-like kinase (Clk) family (44). The region between the kinase subdomains VII and VIII of hPRP4 shows significant similarity to mitogen-activated protein kinases (5, 42, 45). Residues Thr-847 and Tyr-849 in the subdomain VIII (see Fig. 1 A) are in an equivalent position to the TPY sequence in JNK/SAPK.
Prp4 is a protein kinase ofSchizosaccharomyces pombe identified through its role in pre-mRNA splicing, and belongs to a kinase family including mammalian serine/arginine-rich protein-specific kinases and Clks, whose substrates are serine/arginine-rich proteins. We cloned human PRP4 (hPRP4) full-length cDNA and the antiserum raised against a partial peptide of hPRP4 recognized 170-kDa polypeptide in HeLa S3 cell extracts. Northern blot analysis revealed that hPRP4 mRNA was ubiquitously expressed in multiple tissues. The extended NH₂-terminal region of hPRP4 contains an arginine/serine-rich domain and putative nuclear localization signals. hPRP4 phosphorylated and interacted with SF2/ASF, one of the essential splicing factors. Indirect immunofluorescence analysis revealed that endogenous hPRP4 was distributed in a nuclear speckled pattern and colocalized with SF2/ASF in HeLa S3 cells. Furthermore, hPRP4 interacted directly with Clk1 on its COOH terminus, and the arginine/serine-rich domain of hPRP4 was phosphorylated by Clk1in vitro. Overexpression of Clk1 caused redistribution of hPRP4, from the speckled to the diffuse pattern in nucleoplasm, whereas inactive mutant of Clk1 caused no change of hPRP4 localization. These findings suggest that the NH₂-terminal region of hPRP4 may play regulatory roles under an unidentified signal transduction pathway through Clk1.
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^☆: Abbreviations used: Prp, pre-mRNA processing; hPRP4, human prp4; MAPKs, mitogen-activated protein kinases; ERKs, extracellular signal-regulated kinases; JNKs, Jun N-terminal kinases; EGF, epidermal growth factor; TPA, tetradecanoyl phorbol acetate; UV, ultraviolet; TCFs, ternary complex factors; SRF, serum response factor; SRE, serum response element; PCR, polymerase chain reaction; HA, hemagglutinin; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; SDS–PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SR, serine/arginine-rich.

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Regular ArticleCharacterization of hPRP4 Kinase Activation: Potential Role in Signaling☆

Abstract

J. Biol. Chem.

Cell

Trends Biochem. Sci.

Trends Biochem. Sci.

Biochem. Biophys. Res. Commun.

Methods Enzymol.

Methods Enzymol.

Methods Enzymol.

Cell

Curr. Opin. Genet. Dev.

FASEB J.

Mol. Reprod. Dev.

Philos. Trans. R. Soc. London B Biol. Sci.

Physiol. Rev.

Science

Genes Dev.

Science

FASEB J.

Science

Proc. Natl. Acad. Sci. USA

Mol. Cell Biol.

Regular Article
Characterization of hPRP4 Kinase Activation: Potential Role in Signaling☆