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First published online 27 May 2003
doi: 10.1242/jcs.00501

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A STAT-regulated, stress-induced signalling pathway in Dictyostelium

Tsuyoshi Araki1,*, Masatsune Tsujioka1,*, Tomoaki Abe1, Masashi Fukuzawa1, Marcel Meima1, Pauline Schaap1, Takahiro Morio2, Hideko Urushihara2, Mariko Katoh2, Mineko Maeda3, Yoshimasa Tanaka2, Ikuo Takeuchi4 and Jeffrey G. Williams1,{ddagger}

1 School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dow Street, Dundee, DD1 5EH, UK
2 Institute of Biological Sciences, University of Tsukuba,Tsukuba, Ibaraki 305-8572, Japan
3 Department of Biology, Osaka University, Machikaneyama 1-16, Toyonaka, Osaka 560-0043, Japan
4 Novartis Foundation (Japan) for the Promotion of Science, Roppongi, Minato-ku, Tokyo 106-0032, Japan



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  		Fig. 1. Activation and nuclear accumulation of Dd-STATc by osmotic stress. Ax2 cells were allowed to develop for 4 hours in shaken suspension, were harvested and then divided into two. One portion was incubated in KK2 containing 200 mM sorbitol and the other portion was incubated in KK2 containing 100 nM DIF. At the indicated times, one aliquot was harvested and the specific tyrosine phosphorylation level of Dd-STATc was determined by western transfer (A). At the same time, a separate aliquot was analysed immunohistochemically to determine the proportion of cells showing nuclear enrichment of Dd-STATc (B). The results are shown as the mean±s.d. but the error bars at 5, 10 and 15 minutes for the sorbitol treated sample are so small as to be obscured by the filled boxes.

 


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  		Fig. 2. Dd-STATa shows neither osmotic stress or 8-bromo-cGMP-induced nuclear translocation. Ax2 cells were allowed to develop for 4 hours in shaken suspension and divided into five. One portion was incubated in KK2 alone, and the other portions were incubated in KK2 with the indicated additions (5 mM cAMP, 200 mM sorbitol, 100 nM DIF or 20 mM 8-bromo cGMP). After 3 minutes of incubation, two aliquots were removed. In one aliquot cells were analysed immunohistochemically to determine the proportion of cells showing nuclear enrichment of Dd-STATa (dark grey boxes), and in the other aliquot the proportion of cells showing nuclear enrichment of Dd-STATc was measured (light grey boxes). The results are shown as the mean±s.d.

 


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  		Fig. 3. Activation of Dd-STATc by heat shock and ATP depletion. (A) Ax2 cells were allowed to develop for four hours in shaken suspension at 21°C and then transferred to a heating block at 33°C. At the indicated times thereafter, aliquots were harvested and the specific tyrosine phosphorylation level of Dd-STATc was determined by western transfer. As a positive control for the induction, a portion of the cells was maintained at 21°C, DIF was added to a final concentration of 100 nM and an aliquot was analysed in parallel with the heat shock samples. The results are therefore quantitatively comparable and heat shock appears to be a more effective activator than DIF. (B) Ax2 cells were allowed to develop for 4 hours in shaken suspension and di-nitro phenol (DNP) was added to a final concentration of 50 µM. At the indicated times thereafter, aliquots were harvested and the specific tyrosine phosphorylation level of Dd-STATc was determined by western transfer. DIF was added to a final concentration of 100 nM and an aliquot was analysed in parallel with the oxidative shock samples. The results are therefore quantitatively comparable and oxidative shock is a much less effective inducer than DIF. (N.B. This experiment and the experiment described above, in Fig. 3A, are not directly comparable because they were performed at different times.)

 


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  		Fig. 4. Osmotic shock activation of Dd-STATc and of a Y to F mutant of Dd-STATc. GFP-STATc contains the entire Dd-STATc protein with GFP fused at its N-terminus, whereas in GFPSTATc(Y922F) the site of tyrosine phosphorylation at residue 922 is replaced with a phenylalanine residue (Fukuzawa et al., 2001Go). The two constructs were cloned into a vector containing a blasticidin cassette and transformed into Dd-STATc null cells generated using a hygromycin resistance cassette. The Dd-STATc gene displays a very high frequency of homologous recombination. Therefore, to prevent gene conversion/repair of the remnants of the endogenous Dd-STATc gene, the transformation was performed using REMI (restriction enzyme mediated integration); a technique that disfavours homologous recombination (Kuspa and Loomis, 1992Go). Western transfer using 7H3, the N-terminal-specific monoclonal antibody, showed that the GFP:STATc and GFP:STATc-YF cells express their respective fusion proteins at similar levels (data not shown). The GFP:STATc construct causes reversion of the Dd-STATc null phenotype; the transformant shows normal plaque morphology when plated on a bacterial lawn and loses its 'slugger' phenotype. By contrast, GFP:STATc-YF transformants remain as defective as their Dd-STATc null parent (data not shown). GFP:STATc and GFP:STATc-YF were subjected to shaken development as in Fig. 2 and then induced with 100 mM sorbitol for the indicated time.

 


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  		Fig. 5. Activation and nuclear translocation of Dd-STATc in response to membrane-permeant cyclic nucleotide analogues. (A) The tyrosine phosphorylation of Dd-STATc was determined in untreated cells (cont), cells exposed to 200 mM sorbitol (sorbitol) and in cells exposed to the indicated concentrations of membrane-permeant cyclic nucleotide analogue. Induction was for 3 minutes and the samples were analysed as described in Fig. 2. (B) The activation and (C) the nuclear translocation of Dd-STATc in response to 20 mM 8-bromo-cAMP and 8-bromo-cGMP were determined as described in Fig. 2. The results in panel C are shown as the mean±s.d.

 


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  		Fig. 6. Osmotic stress-induced activation of Dd-STATc in Dictyostelium strains mutant in the cAMP- and cGMP-mediated stress-response pathways. The activation of Dd-STATc in response to sorbitol in the parental (Ax-2) strain and the indicated mutants was determined essentially as described in Fig. 2. However, after probing with CP22 (tyrP-STATc), the filter was reprobed with 7H3 (Total STATc) as described in Materials and Methods. The latter signal provides a convenient loading control. All the strains are published (see text), with the exception of the sgc-/dokA- strain. This was generated using as a start point the dokA- strain (Schuster et al., 1996Go), which was created using a G418 resistance cassette. An sgc blasticidin-based disruption cassette was created using a novel in vitro transposition technique (T. Abe and J. G. Williams, unpublished). PCR analysis shows that the mutated Dictyostelium cells contain a blasticidin-resistance cassette in the centre of the sgc gene, in the approximate position of the C1/C2 catalytic domains (Roeloffs et al., 2001). Consistent with this, we analysed osmotic stress induced guanylyl cyclase activation in the sgc-/dokA- strain (Roeloffs et al., 2000) and it is absent (data not shown).

 


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  		Fig. 7. Expression of the gapA and rtoA genes in parental and Dd-STATc null cells. Ax2 cells and a Dd-STATc null cells were subjected to development and induced with either sorbitol or DIF, as described in the legend to Fig. 1. After 15 minutes of induction total cellular RNA was isolated and analysed by northern transfer. The blot was hybridised sequentially with the indicated probes. Ig7 is a constitutively expressed RNA that serves as a loading control. The gapA mRNA migrates as a broad band at the expected size of 3.2kb (Adachi et al., 1997Go). The rtoA transcript is reported to be 1.6 kb and we also observed a band of 1.6kb when cells were growing or developing on a substratum (data not shown). However, when cells were developing in suspension we again observed a band of 1.6 kb, but there is an additional, higher molecular weight species of 2 kb. Because the two RNAs show parallel concentration changes we believe that the longer species is an RNA processing variant of the shorter species. There is a 430 nt intron in the rtoA gene and it has a very unorthodox (GG) splice donor site (Wood et al., 1996Go). The size difference between the longer and shorter transcripts (c. 2 kb minus c. 1.6 kb=0.4 kb) is therefore consistent with a splicing defect in the cells placed in suspension. The result shown is for a Dd-STATc null strain generated using a construct containing a blasticidin casette. We also analysed strains generated using a hygromycin disruption casette and, in this case, we compared homologous integrants (i.e. Dd-STATc disruptants) and non-homologous, random integrants from the same transformation (data not shown). This strategy corrects for any unsuspected genetic divergence between the parental strain and the null strain. Again, expression of gapA and rtoA in the Dd-STATc disruptants was unresponsive to osmotic stress (data not shown).

 


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  		Fig. 8. Expression of the gapA and rtoA genes in response to membrane permeant cyclic nucleotide analogues. Ax2 cells were subjected to development and induced with 8-bromo AMP or 8-bromo GMP as described in the legend to Fig. 5. After 15 and 30 minutes of induction, total cellular RNA was isolated and analysed by northern transfer as in Fig. 7.

 


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  		Fig. 9. A schematic representation of the regulatory roles fulfilled by Dd-STATc. This is a representation of the promoters of the two classes of gene known to be regulated by Dd-STATc. The promoter of the ecmA gene is under the negative control of Dd-STATc; the element(s) that normally direct ecmA expression only in pstA cells are repressed by Dd-STATc; such that in a Dd-STATC null strain they become ectopically active in the pstO cells (Fukuzawa et al., 2001Go). Activation of Dd-STATc in pstO cells is under the direct control of DIF. As we have shown, gapA and rtoA are regulated at two levels. They display semiconstitutive activity during growth and development and they are super-inducible, above this level, by hyperosmotic stress. We assume that different promoter elements are utilised for these different activities. We also know that gapA and rtoA are not induced by DIF, that is, the DIF and the stress signalling pathways do not display 'cross talk', and two possible explanations for this are presented in the text.

 




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