The structure of the 5′-end of the protein-tyrosine phosphatase PTPRJ mRNA reveals a novel mechanism for translation attenuation

Analysis of the human protein-tyrosine phosphatase (PTP) PTPRJ mRNA detected three in-frame AUGs at the 5′-end (starting at nt +14, +191 and +356) with no intervening stop codons. This tandem AUG arrangement is conserved between humans and the mouse and is unique among the genes of the classical PTPs. Until now it was assumed that the principal open reading frame (ORF) starts at AUG356. Our experiments showed that: (i) translation of the mRNA synthesized under the PTPRJ promoter starts predominantly at AUG191, leading to the generation of a 55 amino acid sequence preceding the signal peptide; (ii) the longer form is being likewise correctly processed into mature PTPRJ; (iii) the translation of the region between AUG191 and AUG356 inhibits the overall expression, a feature which depends on the sequence of the encoded peptide. Specifically, a sequence of 13 amino acids containing multiple arginine residues (RRTGWRRRRRRRR) confers the inhibition. In the absence of uORF these previously unrecognized characteristics of the 5′-end of the mRNA present a novel mechanism to suppress, and potentially to regulate translation

Similar kinetics of FLT3-DEP-1 complex formation and FLT3 autophosphorylation.

<p>Association of DEP-1 with FLT3 was measured by <i>in situ</i> PLA as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062871#pone-0062871-g001" target="_blank">Figure 1</a>. (<b>A</b>) Example images; DEP-1/FLT3 complexes as RCPs are shown in red, nuclei are depicted in blue and scale bars represent 20 µm for the overview images and 5 µm for the insets. (<b>B</b>) Cumulated data from 7 independent experiments (means ± SD, *p<0.05, **p<0.01 for difference from unstimulated sample by one-way ANOVA). (<b>C</b>) FLT3 was immunoprecipitated from THP-1 cells stimulated for the indicated timepoints and autophosphorylation was assessed by immunoblotting with anti-phosphotyrosine antibodies (pY100). FLT3 amounts were detected by reblot with anti-FLT3 antibodies. Note that for the FLT3 reblot only the section above the very abundant IgG band (star) is depicted. Two FLT3 species of 130 kDa and 150 kDa represent the immature high-mannose form and the mature, complex glycosylated form, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062871#pone.0062871-SchmidtArras2" target="_blank">[31]</a>. Only the 150 kDa form (arrows) becomes phosphorylated in a ligand-dependent manner. Weak bands of even higher molecular mass in the stimulated samples represent ubiquitinylated FLT3. Right panel: Quantification of multiple (n = 5) independent experiments. The pFLT3 signals were normalized to FLT3 amounts of the stripped and reprobed blots (means ± SD, *p<0.05, **p<0.01 for difference from unstimulated sample by one-way ANOVA).</p

FLT3 kinase inhibition and DEP-1 oxidation interfere with complex formation.

<p>Association of DEP-1 with FLT3 was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062871#pone-0062871-g001" target="_blank">Figure 1</a>. (<b>A</b>), (<b>B</b>) Cells were pretreated with the FLT3 kinase inhibitor compound 102 (Cpd.102, 1 µM) for 2 hours before FL stimulation (20 min). (<b>A</b>) Example images; DEP-1-FLT3 complexes as RCPs are shown in red, nuclei are depicted in blue and scale bars represent 20 µm for the overview images and 5 µm for the inset. (<b>B</b>) Quantification of 5 images per variant. (<b>C</b>) <i>In situ</i> PLA detection of complex formation between DEP-1 and FLT3 in either THP-1 cells or MV4-11 cells, harboring the oncogenic FLT3 variant FLT3 ITD. FL stimulation 20 min. (<b>D</b>) THP-1 cells were starved and pretreated with 1 mM H₂O₂ (5 min) before stimulation with FL (10 min) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062871#pone-0062871-g001" target="_blank">Figure 1</a> and <i>in situ</i> PLA detection. The data are representative for 3 experiments with consistent results.</p

DEP-1 regulates FL-stimulated FLT3 signaling. DEP-1 expression was stably downregulated in THP-1 cells by lentiviral transduction of shRNA.

<p>Control cells harbor a non-targeting shRNA construct. (<b>A</b>) FL-dependent ERK1/2 activation was assessed by immunoblotting of cell lysate samples with antibodies recognizing activated ERK1/2 (pERK1/2). Loading was analyzed by reblot with ERK1/2 antibodies. Representative experiment; the efficiency of stable DEP-1 knockdown is also shown (upper panel). (<b>B</b>) Quantitative data for 4 independent experiments. Numbers represent pERK1/2 signals normalized to ERK1/2 levels in the same sample. The value of control cells at 10 min was set to 1.0. Significance for the difference between responses of the two different cell pools was determined by two-way ANOVA.</p

Expression of protein-tyrosine phosphatases in Acute Myeloid Leukemia cells : FLT3 ITD sustains high levels of DUSP6 expression

Protein-tyrosine phosphatases (PTPs) are important regulators of cellular signaling and changes in PTP activity can contribute to cell transformation. Little is known about the role of PTPs in Acute Myeloid Leukemia (AML). The aim of this study was therefore to establish a PTP expression profile in AML cells and to explore the possible role of FLT3 ITD (Fms-like tyrosine kinase 3 with internal tandem duplication), an important oncoprotein in AML for PTP gene expression. PTP mRNA expression was analyzed in AML cells from patients and in cell lines using a RT-qPCR platform for detection of transcripts of 92 PTP genes. PTP mRNA expression was also analyzed based on a public microarray data set for AML patients. Highly expressed PTPs in AML belong to all PTP subfamilies. Very abundantly expressed PTP genes include PTPRC, PTPN2, PTPN6, PTPN22, DUSP1, DUSP6, DUSP10, PTP4A1, PTP4A2, PTEN, and ACP1. PTP expression was further correlated with the presence of FLT3 ITD, focusing on a set of highly expressed dual-specificity phosphatases (DUSPs). Elevated expression of DUSP6 in patients harboring FLT3 ITD was detected in this analysis. The mechanism and functional role of FLT3 ITD-mediated upregulation of DUSP6 was then explored using pharmacological inhibitors of FLT3 ITD signal transduction and si/shRNA technology in human and murine cell lines. High DUSP6 expression was causally associated with the presence of FLT3 ITD and dependent on FLT3 ITD kinase activity and ERK signaling. DUSP6 depletion moderately increased ERK1/2 activity but attenuated FLT3 ITD-dependent cell proliferation of 32D cells. In conclusion, DUSP6 may play a contributing role to FLT3 ITD-mediated cell transformation

Expression of protein-tyrosine phosphatases in Acute Myeloid Leukemia cells: <it>FLT3 ITD sustains high levels of DUSP6 expression</it>

Abstract Protein-tyrosine phosphatases (PTPs) are important regulators of cellular signaling and changes in PTP activity can contribute to cell transformation. Little is known about the role of PTPs in Acute Myeloid Leukemia (AML). The aim of this study was therefore to establish a PTP expression profile in AML cells and to explore the possible role of FLT3 ITD (Fms-like tyrosine kinase 3 with internal tandem duplication), an important oncoprotein in AML for PTP gene expression. PTP mRNA expression was analyzed in AML cells from patients and in cell lines using a RT-qPCR platform for detection of transcripts of 92 PTP genes. PTP mRNA expression was also analyzed based on a public microarray data set for AML patients. Highly expressed PTPs in AML belong to all PTP subfamilies. Very abundantly expressed PTP genes include PTPRC, PTPN2, PTPN6, PTPN22, DUSP1, DUSP6, DUSP10, PTP4A1, PTP4A2, PTEN, and ACP1. PTP expression was further correlated with the presence of FLT3 ITD, focusing on a set of highly expressed dual-specificity phosphatases (DUSPs). Elevated expression of DUSP6 in patients harboring FLT3 ITD was detected in this analysis. The mechanism and functional role of FLT3 ITD-mediated upregulation of DUSP6 was then explored using pharmacological inhibitors of FLT3 ITD signal transduction and si/shRNA technology in human and murine cell lines. High DUSP6 expression was causally associated with the presence of FLT3 ITD and dependent on FLT3 ITD kinase activity and ERK signaling. DUSP6 depletion moderately increased ERK1/2 activity but attenuated FLT3 ITD-dependent cell proliferation of 32D cells. In conclusion, DUSP6 may play a contributing role to FLT3 ITD-mediated cell transformation.</p

Protein-tyrosine Phosphatase DEP-1 Controls Receptor Tyrosine Kinase FLT3 Signaling*

Fms-like tyrosine kinase 3 (FLT3) plays an important role in hematopoietic differentiation, and constitutively active FLT3 mutant proteins contribute to the development of acute myeloid leukemia. Little is known about the protein-tyrosine phosphatases (PTP) affecting the signaling activity of FLT3. To identify such PTP, myeloid cells expressing wild type FLT3 were infected with a panel of lentiviral pseudotypes carrying shRNA expression cassettes targeting different PTP. Out of 20 PTP tested, expressed in hematopoietic cells, or presumed to be involved in oncogenesis or tumor suppression, DEP-1 (PTPRJ) was identified as a PTP negatively regulating FLT3 phosphorylation and signaling. Stable 32D myeloid cell lines with strongly reduced DEP-1 levels showed site-selective hyperphosphorylation of FLT3. In particular, the sites pTyr-589, pTyr-591, and pTyr-842 involved in the FLT3 ligand (FL)-mediated activation of FLT3 were hyperphosphorylated the most. Similarly, acute depletion of DEP-1 in the human AML cell line THP-1 caused elevated FLT3 phosphorylation. Direct interaction of DEP-1 and FLT3 was demonstrated by “substrate trapping” experiments showing association of DEP-1 D1205A or C1239S mutant proteins with FLT3 by co-immunoprecipitation. Moreover, activated FLT3 could be dephosphorylated by recombinant DEP-1 in vitro. Enhanced FLT3 phosphorylation in DEP-1-depleted cells was accompanied by enhanced FLT3-dependent activation of ERK and cell proliferation. Stable overexpression of DEP-1 in 32D cells and transient overexpression with FLT3 in HEK293 cells resulted in reduction of FL-mediated FLT3 signaling activity. Furthermore, FL-stimulated colony formation of 32D cells expressing FLT3 in methylcellulose was induced in response to shRNA-mediated DEP-1 knockdown. This transforming effect of DEP-1 knockdown was consistent with a moderately increased activation of STAT5 upon FL stimulation but did not translate into myeloproliferative disease formation in the 32D-C3H/HeJ mouse model. The data indicate that DEP-1 is negatively regulating FLT3 signaling activity and that its loss may contribute to but is not sufficient for leukemogenic cell transformation