1. Tyrosine Phosphorylation-Independent Nuclear Translocation of a Dictyostelium STAT in Response to DIF Signaling 
Masashi Fukuzawa, Tsuyoshi Araki, Iris Adrian, Jeffrey G. Williams 
Molecular Cell 
Volume 7, Issue 4, Pages (April 2001)
DOI: /S (01)

2. Figure 1
Analysis of the Dd-STATc Protein by Sequence Comparison and Phosphotyrosine Assignment
(A) Alignment of the C-terminal proximal regions of the Dd-STATa and Dd-STATc proteins. This alignment was made using the program Clustal W at the default settings, and the numbers at the start indicate the position in each protein at which the alignment is initiated. The asterisk marked over the alignment near the C terminus shows the predicted site of tyrosine phosphorylation (see Figure 1B). The asterisk in the region marked “putative DNA binding domain” (this is marked over the alignment) shows a highly conserved valine residue known to be necessary for nuclear translocation.
(B) Analysis of the Dd-STATc protein by Western transfer and alignment of the predicted site of tyrosine phosphorylation of Dd-STATc and the Drosophila STAT. The asterisk over the alignment (below the Western transfer) of Dd-STATc and the Drosophila STAT (Hou et al., 1996; Yan et al., 1996) shows the predicted site of tyrosine phosphorylation. In order to confirm this site, two peptides with the sequence of the C-terminal 15 amino acids of Dd-STATc were synthesized, one with Tyr-922 unmodified, the other with Tyr-922 phosphorylated. An affinity-purified polyclonal antibody directed against the unmodified peptide (termed the N:STATc antibody) was used to probe a Western blot of a developmental time course (lower panel). Dd-STATc is present at an approximately uniform concentration during most of development, and the apparent concentration drop at culmination most probably results from incomplete extraction of protein from the cellulose-ensheathed stalk cells. A polyclonal antibody was also prepared against the p-Tyr peptide, and this was sequentially affinity purified over the p-Tyr and Tyr forms of the peptide, to yield an antibody that was specific for the tyrosine-phosphorylated form (Araki et al., 1998). The specificity of the p-Tyr:STATc antibody for the tyrosine-phosphorylated form of Dd-STATc was confirmed using cells that were transformed with a mutant form of Dd-STATc, wherein Tyr-922 is mutated to phenylalanine. This showed no signal in Western blot analysis (data not shown). There is a small amount of tyrosine-phosphorylated Dd-STATc protein in growing cells but this amount is highly variable from experiment to experiment, and we suspect it to be due to stress-activated tyrosine phosphorylation caused by the centrifugation steps used to wash the cells free of growth medium. During the first 2 hr of development, the concentration of tyrosine-phosphorylated Dd-STATc falls to a very low level and then rises to reach a peak at the tight aggregate stage. The amount of tyrosine-phosphorylated Dd-STATc sometimes shows a slight drop during slug formation, followed by a second small peak at the start of culmination, but the magnitude of the drop and the second peak varies from experiment to experiment
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
The Pattern of Nuclear Localization of Dd-STATc at the Slug Stage
(A) Whole mount of a slug immunohistochemically stained to detect the Dd-STATc protein. Ax2 cells were developed on a water agar plate to the slug stage and the resultant structures were mounted, fixed, and stained with the p-Tyr:STATc antibody. They were analyzed using confocal microscopy and the Z series was used to make a 3D reconstruction (Araki et al., 1998). This is a side view generated from such a reconstruction.
(B) Nuclear localization of a GFP:Dd-STATc fusion protein. The GFP:Dd-STATc construct utilizes the semiconstitutive, actin 15 promoter and contains red-shifted GFP fused to the extreme N terminus of Dd-STATc. Stably transformed cells were set up for development and the resulting slugs were visualized directly, without fixation
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Analysis of Gene Expression in Dd-STATc Null and Parental Ax2 Cells by Northern Transfer
Total cellular RNA purified at the indicated stages of development was subjected to Northern transfer. The filter was then hybridized with the indicated probes (see Results for a description of these probes)
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
Analysis of Prestalk-Specific Gene Expression in the Dd-STATc Null Mutant Using lacZ Fusion Gene Reporters
The upper two panels show β-galactosidase-staining patterns of a construct containing the ecmAO promoter fused to a modified lacZ gene that encodes an unstable β-galactosidase protein wherein the N-terminal amino acid is replaced by an Ile residue (Detterbeck et al., 1994). Such a construct more closely monitors ongoing gene transcription rates than does a stable β-galactosidase fusion construct. In order to stay in the linear range of the assay, a short time of staining was used for both samples and, because of this, staining in the pstO region of the parental slugs is barely detectable. The two panels below show β-galactosidase staining patterns of parental and Dd-STATc null slugs transformed with ecmO:gal, a reporter that is specific for pstO cell differentiation. The ecmO:gal transformant in the null strain shows diffuse, low-level β-galactosidase expression from the start of development, as do null cells transformed with ecmAO:gal (see Results), and this accounts for the haziness of this image relative to the equivalent wild-type structure. The four panels at the bottom are structures derived from parental or Dd-STATc null cells transformed with ecmA:gal, a reporter construct that is specific for pstA cell differentiation (Early et al., 1995). Again, developing structures were fixed and stained for β-galactosidase at the indicated stages
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5 The Activation of Dd-STATc by DIF
(A) The induction of specific Dd-STATc tyrosine phosphorylation by DIF. Exponentially growing Ax2 cells were harvested and developed in shaking suspension in KK2 buffer. After 4 hr of starvation, cells were placed in fresh flasks and DIF was added to a concentration of 50 nM. Cells were collected at the indicated times and lysed in SDS sample buffer. After Western blotting, the level of tyrosine phosphorylation of Dd-STATc was analyzed using the p-Tyr:STATc antibody. In the panel at the right, Ax2 cells were developed for 4 hr as described above and then treated with the indicated concentrations of DIF. Samples were collected after 3 min of induction and analyzed as above. The presence of tyrosine-phosphorylated Dd-STATc protein in untreated cells (RHS) or cells at time 0 (LHS) is most likely to be the result of endogenously produced DIF.
(B) The induction of Dd-STATc dimerization by DIF. Whole-cell extracts, prepared as in the Experimental Procedures, were fractionated by glycerol gradient (10%–40%) centrifugation. Each gradient fraction was analyzed by 7.5% SDS-PAGE followed by immunoblotting with either the N:STATc (upper panel) or p-Tyr:STATc, tyrosine phosphate–specific antibody (lower panel).
(C) The induction of nuclear translocation of Dd-STATc by DIF. Ax2 cells were allowed to develop in shaking culture for 4 hr in KK2 buffer and then treated with 50 nM DIF. Samples were collected over a 30 min incubation at the times indicated. The samples were analyzed for nuclear localization of Dd-STATc by immunohistochemical staining using the N:STATc antibody
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6 Nuclear Translocation of Dd-STATc Mutant Proteins
All constructs utilize the semiconstitutive, actin 15 promoter and have red-shifted GFP fused to the extreme N terminus of Dd-STATc. The Dd-STATc-derived constructs were transformed into Dictyostelium using a blasticidin resistance cassette because preliminary experiments using a G418 cassette yielded transformants where the fusion protein was non-DIF inducible. We believe that the relevant difference between the two selectable markers is the much higher copy number, and hence expression level, obtained with G418. However, we have not proven this directly. Dd-STATa:GFP is, by contrast, fully regulatable by cAMP in a multicopy vector (Dormann et al., 2001), and so a G418 cassette was used for the construct shown in (D). The constructs in (A) contain the entire Dd-STATc protein (WT) or a mutant (Y to F) bearing a point mutation that converts the site of tyrosine phosphorylation into a phenylalanine residue. The symbol R denotes an invariant arginine residue that is needed for SH2 domain function. The letters VI show the core of a conserved hydrophobic region known to be necessary for DNA binding and nuclear translocation (Herrington et al., 1999). The construct in (B) contains a deletion derivative of Dd-STATc that lacks the site of tyrosine phosphorylation and most of the SH2 domain. The construct in (C) is a chimera between Dd-STATc and Dd-STATa that contains N-terminal proximal sequences from Dd-STATc and C-terminal proximal sequences from Dd-STATa. The construct in (D) is a control construct that contains the C-terminal proximal sequences from Dd-STATa used in construct (C). In all cases, pooled transformant populations were used for the DIF induction. Note that the presence of cells with multiple nuclei is a common occurrence when Dictyostelium cells are grown axenically
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7 A Model for the Regulation of pstA Cell Differentiation
This is a schematic representation of the predicted protein occupancy and transcriptional activity of the pstA-specific and pstO-specific elements within the promoter of the ecmA gene in either pstA or pstO cells. The model hypothesizes an as yet unidentified activator of pstO-specific transcription (the O activator) that is activated by DIF. The differentiation of pstA cells is inducible in monolayer assay by DIF (Early et al., 1995) but, surprisingly, pstA differentiation still takes place in mutant slugs where DIF levels are greatly reduced (Thompson and Kay, 2000). Hence, it is an open question whether the A activator is DIF regulated during normal development. In pstA cells, the A activator functions to direct pstA-specific gene expression, but in pstO cells the Dd-STATc protein acts to prevent the A activator from functioning. Hence, the pstA-specific elements are not utilized in pstO cells because Dd-STATc acts as a specific repressor of the A activator. Thus, we suggest that DIF functions through a negative feedback loop; DIF induces the differentiation of pstO cells but also activates the Dd-STATc protein, and this serves to limit the domain of pstA-specific gene expression
Molecular Cell 2001 7, DOI: ( /S (01) )