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First published online 27 February 2008
doi: 10.1242/dev.009936

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Evidence that DIF-1 and hyper-osmotic stress activate a Dictyostelium STAT by inhibiting a specific protein tyrosine phosphatase

Tsuyoshi Araki^1,*, Judith Langenick^1,*, Marianne Gamper^2,3, Richard A. Firtel² and Jeffrey G. Williams^1,

¹ University of Dundee, College of Life Sciences, Dow Street, Dundee DD1 5EH, UK.
² University of California, San Diego, Natural Sciences Building Room 6111, 9500 Gilman Drive, La Jolla, CA 92093-0380, USA.
³ Biomedical Research Foundation (SBF), Lauchefeld 31, CH-9548 Matzingen, Switzerland.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Analysis of STATc in slugs of a strain overexpressing PTP3 protein tyrosine phosphatase or its dominant-negative form. (A) PTP3 and slug morphology. The control slugs (left) are transformed with a myc-GFP overexpression construct, while those on the right overexpress PTP3. The arrows indicate points at which the slugs seem to have split apart. (B) PTP3 and STATc staining slugs derived from a non-transformed control and from cells transformed with myc:PTP3 or myc:PTP3CS, the dominant-negative form, were immunostained using a STATc antibody. In the control, STATc is nuclear enriched in the pstO region (double-headed arrow). In the slugs transformed with the PTP3 overexpressor, there is almost no detectable staining; in cells transformed with the dominant-negative PTP3, mutant PTP3 is nuclear enriched in all parts of the slug. (C) PTP3 and STATc activation level. Total cell lysate was prepared from slugs of parental Ax2 cells and PTP3OE cells. The samples were analysed using a tyrosine phosphorylation specific STATc antibody (CP22), total STATc antibody (7H3) and, as a loading control, a GSK-3 antibody.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Analysis of STATc activation by DIF-1 and osmotic stress in a PTP3 overexpressing strain. Parental Ax2 cells and PTP3OE cells were starved for 4 hours and then left untreated, exposed to 100 nM DIF-1 (A) for 5 minutes or exposed to 200 mM sorbitol (B) for 5 minutes. One aliquot of cells was lysed, subjected to western transfer and the blot was analysed with an antibody specific to the tyrosine phosphorylated form of STATc. As a loading control, a parallel blot was probed with total STATc antibody. The second aliquot of cells was fixed and immunostained for STATc.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Analysis of the effects of PTP inhibition on STATc tyrosine phosphorylation. (A) Induction assay using dominant-negative forms of PTP3 cells transformed with myc:PTP3CS were starved in suspension for 4 hours and then exposed to DIF-1 at 100 nM. The activation of STATc was analysed by western transfer using an antibody specific to the tyrosine phosphorylated form of STATc. As a loading control, a parallel blot was probed with a non phospho-specific STATc antibody. (B) Pervanadate-mediated inhibition of PTP3 Ax-2 cells. Cells were starved in suspension for 4 hours, then exposed to DIF-1 at 100 nM or pervanadate at 2 mM (1 mM H2O2 and 2 mM NaVO4) for the indicated times and analysed as in A.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Interaction of PTP3 with STATc revealed by substrate-trapping. (A) In vitro binding of PTP3. STATc parental Ax2 cells and STATc-null cells were starved for 4 hours and then left untreated (control), exposed to DIF-1 (100 nM) or exposed to sorbitol (200 mM). After 5 minutes of treatment, extracts were prepared from the cells and subjected to affinity chromatography using the C to S substrate-trapping form of PTP3. The eluates from the columns were subjected to western transfer using a general phosphotyrosine-specific antibody (upper panel) or a tyrosine phosphorylation-specific STATc antibody (lower panel). The strongest DIF-1 and stress-induced signal detected by the phosphotyrosine-specific antibody is a 130 kDa species that corresponds to a previously described protein (Gamper et al., 1999). This protein co-migrates with the strongest signal detected by the STATc antibody and both species are absent from the STATc null. (B) In vivo interaction of PTP3 and STATc parental Ax2 cells or cells expressing the myc tagged C to S substrate-trapping form of PTP3 (myc:PTP3CS, labelled as PTP3CS) were starved for 4 hours and then left untreated (control), exposed to DIF-1 at 100 nM or exposed to sorbitol at 200 mM. After 5 minutes of treatment, extracts were prepared from the cells and immunoprecipitated using a myc antibody. After western transfer, the blot was stained using a general STATc antibody and a tyrosine phosphorylation-specific STATc antibody. We estimate that 5% of STATc protein in the cell is recovered by the co-IP procedure.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Osmotic stress and DIF-1 treatment decrease cellular PTP3 activity. Cells at 4 hours of development in suspension and expressing myc:PTP3 were left untreated or exposed to DIF-1 (100 nM), sorbitol (200 mM) or pervanadate (1 mM H2O2 and 2 mM Na3VO4) for 5 and 15 minutes. They were lysed in a non-ionic detergent and immunoprecipitated using a myc antibody. The precipitates were dissolved and assayed for tyrosine phosphatase activity using the general substrate pNPP. As controls, parental Ax2 cell extracts or cells transformed with myc:PTP3CS were immunoprecipitated with myc antibody. Neither precipitation yielded significant activity (data not shown). For purposes of comparison, the western blot at the bottom shows the level of the STATc phosphorylation in parental Ax2 cells after the treatments described above.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Electrophoretic mobility shifts of myc-PTP3CS after DIF-1 treatment. Cells transformed with myc:PTP3CS were starved for 4 hours, exposed to DIF-1 at 100 nM for 3 minutes or left untreated, where indicated the samples were treated with lambda phosphatase. All samples were analysed by 2D gel electrophoresis (pH 6-10 for first dimension and 4-12% gradient gel for second dimension). After western transfer, the resultant blots were stained using a myc antibody.

View larger version (28K): [in this window] [in a new window]   Fig. 7. A model for DIF-1 and stress-induced activation of STATc. The model proposes regulation of STATc at the level of de-phosphorylation. In untreated cells, PTP3 activity is relatively high and STATc is, therefore, minimally tyrosine phosphorylated. Upon stress or DIF-1 treatment, PTP3 is serine-threonine phosphorylated. This reduces PTP3 activity and the level of tyrosine phosphorylation of STATc increases.

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