QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online December 30, 2003
doi: 10.1242/10.1242/dev.00927

This Article Summary Full Text Full Text (PDF) Supplemental Data Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhukovskaya,, N. V. Articles by Williams, J. G. Search for Related Content PubMed PubMed Citation Articles by Zhukovskaya,, N. V. Articles by Williams, J. G. Social Bookmarking                         What's this?

Dd-STATb, a Dictyostelium STAT protein with a highly aberrant SH2 domain, functions as a regulator of gene expression during growth and early development

Natasha V. Zhukovskaya,^1,*, Masashi Fukuzawa,^1,*, Masatsune Tsujioka¹, Keith A. Jermyn¹, Takefumi Kawata¹, Tomoaki Abe¹, Marketa Zvelebil² and Jeffrey G. Williams

¹ School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK
² University College London, Ludwig Institute for Cancer Research, The Cruciform Building, Gower Street, London WC1E 6BT, UK

View larger version (72K): [in a new window]   Fig. 1. Alignment of the Dd-STATa, b and c sequences after removal of their simple sequence component. The Accession Number of the complete predicted sequence of Dd-STATb is AJ581661 but here a truncated form of the sequence is presented. The N-terminal halves of Dd-STATa, b and c contain tracts of glutamine, asparagines and threonine. These are encoded by CAA repeats, a feature common to many Dictyostelium genes. In this alignment, Q, N and T tracts equal to or longer than three residues were omitted, to give Dd-STATa' (633 of 707 residues), Dd-STATb' (978 of 1147 residues)and Dd-STATc' (819 of 929 residues). The three STATs display only scattered regions of short homology in their N-terminal-proximal regions. No functions have thus far been mapped to the N-terminal-proximal regions of Dd-STATa or Dd-STATc and a BLAST search using the N-terminal-proximal region of Dd-STATb also yielded no hits (the search was run at NCBI with amino acids 1 to 505 and using blastP with an `E' value of 10). The predicted approximate positions of the DNA binding domains (closely spaced broken line), the SH2 domains (widely spaced broken line) and the site of the insertion in the Dd-STATb sequence (broad unbroken line) are indicated by double-headed arrows. The positions of the arginine to leucine substitution is indicated by a triangle, and the predicted site of tyrosine phosphorylation is indicated by an asterisk. (B) Alignment between the SH2 domains of Dd-STATs a to c, human STATs 1 to 6 and Src. The alignment was generated using ClustalW and then modified by hand to align the known secondary structure elements from STAT1 and the Src SH2 domain. This alignment was used in modelling the Dd-STATb SH2 domain (STATc). The dominant inserts are highlighted in yellow; red indicates identical residues; grey indicates similar residues. The inset shows a phylogenetic tree generated using the Nearest Neighbour joining method; blue boxes indicate proteins where a crystal structure is known.

View larger version (38K): [in a new window]   Fig. 2. Structural analysis of Dd-STATb. (A) The C trace of Dd-STATb (blue), superimposed on STAT1 (magenta), was used as a template for modelling Dd-STATb. The invariant arginine at position ßB5 is shown in red (in STAT1) and its equivalent residue in Dd-STATb, a leucine, is shown in blue. The two main inserts are highlighted. (B) Dd-STATb is superimposed on the Src SH2 domain, with the arginine/leucine variation highlighted and the Src phosphopeptide also shown. (C) A ribbon diagram of Dd-STATb (in blue) superimposed on the whole structure of STAT1, showing that the position of the large insert (insert1) would not disrupt a similar dimerisation as that seen for STAT1. (D) The C trace for Dd-STATb (red), STAT1 (blue) and STAT3 (green). The phosphorylated Serine residues are shown in STAT1 and STAT3. It is apparent that the positions of insert 1 and insert 2 are regions of dissimilarity in both STAT1 and STAT3.

View larger version (57K): [in a new window]   Fig. 3. (A) Western transfer analysis of Dd-STATb. Cells were allowed to develop on water agar, aliquots were harvested at the indicated times and they were subjected to western transfer analysis using the C:STATb antibody. This result, combined with other gel analyses using high molecular weight markers (B), show that Dd-STATb migrates with the expected approximate molecular weight of 130 kDa. (B) Western transfer analysis of two Dd-STATb disruptant clones and two random integrant clones. The four clones were grown to 2x106/ml and subjected to western transfer using the C:STATb antibody. Clones B15 and B16 are random integrant (Dd-STATb+) clones and B8 and B9 are disruptant (Dd-STATb-) clones. These assignments were further confirmed by immunohistochemical staining (data not shown). (C) Immunohistochemical analysis of the intracellular distribution of Dd-STATb in growing cells. Cells growing at 2x106/ml in HL5 medium were fixed and stained with the C:STATb antibody. Scale bar: 10 µm.

View larger version (42K): [in a new window]   Fig. 4. (A) Determination of the relative growth rates of Dd-STATb- and Dd-STATb+ cells. A co-cultivation experiment, with three cycles, was performed using a mixture of Dd-STATb- and Dd-STATb+ cells that were derived from a single transformation with the Dd-STATb disruption construct (see text). The cells were allowed to grow to saturation (2x107/ml) and then diluted 1 in 100 for regrowth. (B) A compilation of five co-cultivation experiments. The first (top) experiment is that described in A and the other four experiments were performed in the same way. Although the rate of loss of the null cells was variable, the outcome was reproducible; the random integrant cells always came to dominate the population.

View larger version (105K): [in a new window]   Fig. 5. (A) Confirmation of the micro-array results for four selected ESTs. In a micro-array screen of 1700 ESTs, using RNAs from growing Dd-STATb+ and Dd-STATb-cells to make the labelled cDNAs, 38 ESTs showed a reproducible difference in hybridisation (see Table 1). Several of the characterised ESTs (i.e. those where the Dictyostelium gene had previously been described or, in the case of HGPRT, where a function could be inferred) were employed as probes in northern transfer, using RNAs extracted from cells growing in HL5 medium and at the indicated densities. In some cases, different northern blots were used for different analyses. A loading control was performed for each blot, using the constitutively expressed gene Ig7, and in each case the control confirmed the changes visualised here (data not shown). (B) Comparison of the levels of discoidin 1 gene expression in multiple Dd-STAb+ and Dd-STATb-clone. Independent clones from the same transformation, using the Dd-STATb disruption construct, were screened for Dd-STATb expression by immunostaining. Five `random integrant' clones (clones B12-16), where the blasticidin resistance cassette inserted non-homologously into the genome, and five Dd-STATb disruptant clones (B5, B6, B8, B9 and B10) were analysed by northern transfer using a discoidin 1 probe. The loading control shown was performed on the same blot, by melting off the discoidin 1 hybridisation signals and then using, as a probe, the constitutively expressed gene Ig7.

View larger version (13K): [in a new window]   Fig. 6. Comparison of developmental changes in the levels of discoidin 1 gene expression in Dd-STAb+ and Dd-STATb-clones. The kinetics and extent of discoidin 1 mRNA accumulation were determined for two random integrant, Dd-STATb+ clones (B15 and B16, Fig. 3B) and two disruptant, Dd-STATb-clones (B8 and B9, Fig. 3B). Cells were grown to a density of 2x106 cells/ml and subjected to development in shaken suspension in KK2 for the indicated time periods.

View larger version (20K): [in a new window]   Fig. 7. Size analysis of endogenous Dd-STATb protein on a glycerol gradient. Cells at 4 hours of development in shaken suspension were treated with 100mM sorbitol for 30 minutes (Araki et al., 2003). A whole cell protein extract was then centrifuged through a 10%-40% glycerol gradient (Fukuzawa et al., 2001). We have previously calibrated this system using commercial size markers but additionally, in this experiment, the activated (dimeric) form of Dd-STATc was generated by the sorbitol treatment and this was used as an internal marker (see text).

View larger version (63K): [in a new window]   Fig. 8. Biochemical analysis of potential interactions between Dd-STATb and other STATs. (A) Growing cells were lysed and subjected to immunoprecipitation using the C:STATb antibody and the pellet was assayed for the three STATs using the analysis protocol described on the figure. (B) Genetic analysis of potential interactions between Dd-STATb and other STATs. Slugs were generated using either Ax-2 cells or cells that are null for both the Dd-STATa and the Dd-STATc genes. The latter strain was created by sequential inactivation, using ura and blasticidin disruption cassettes (Kawata et al., 1996; Kalpaxis et al., 1991). Absence of both STAT proteins in the selected strain was confirmed immunochemically. Whole-mount slugs were fixed and stained using the C:STATb antibody and visualised by confocal microscopy. Only the front approximate half of each slug is shown and their tips are facing left.

View larger version (26K): [in a new window]   Fig. 9. Glycerol gradient analysis of unmutated and mutated forms of Dd-STATb. Two mutant versions of Dd-STATb, and the unmutated Dd-STATb, were cloned downstream of the constitutive actin 15 promoter; in LR residue L1025 is converted to arginine and in YF residue Y1143 is converted to phenylalanine. These three DNAs were stably transformed into Dictyostelium using G418 as the selective agent. This yields multiple copies of the transforming DNA, the number varying from cell. Clones with a high expression level were selected and analysed as described in the legend to Fig. 7. Scale bar: 10 µm.

View larger version (127K): [in a new window]   Fig. 10. Immunohistochemical analysis of cells expressing unmutated and mutated forms of Dd-STATb. Growing cells expressing the unmutated and mutated versions of Dd-STATb, described in Fig. 8 were subjected to immunohistochemical analysis exactly as described in Fig. 3. Scale bar: 100 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter