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First published online 7 October 2008
doi: 10.1242/jcs.036798

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A Dictyostelium homologue of the metazoan Cbl proteins regulates STAT signalling

University of Dundee, School of Life Sciences, Dow Street, Dundee DD1 5EH, UK

View larger version (30K): [in this window] [in a new window]   Fig. 1. Comparison of CblA to metazoan Cbl protein family members. The top part of the figure is the output from a BLAST search, showing the predicted RING finger, EF-hand and SH2 domains. It also shows the positions of simple repeat sequence (blue) and the positions of four alpha helical segments (black arrowheads) and their length, as predicted by the program JPRED (Cuff and Barton, 2000). The lower three panels show alignments of CblA with Cbl family EF-hand domains, with a Cbl type and two orthodox SH2 domains and with Cbl family RING fingers. The red lines over the SH2 domains indicate the universally conserved R residue and the divergent adjacent residues that initially confused identification of the Cbl SH2 domain (Meng et al., 1999).

View larger version (41K): [in this window] [in a new window]   Fig. 2. Developmental time course of CblA expression. Cells were allowed to develop for the indicated times (in hours) and stages (s, slug; MC, mid-culminants; C, culminants) on filters. The times in B are equivalent to the following stages: 6 hours, streaming; 10 hours, tight aggregates; 12 hours, tipped aggregates and first fingers; and 14 hours, slugs. Cells were analysed for (A) CblA RNA by RT-PCR and (B) CblA protein by western transfer. The loading control in A is Ig7, a constitutively expressed RNA. Loading in the western transfer was monitored by total protein staining and was approximately equal in all lanes.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Intracellular localisation of CblA. Cells were starved for 4 hours then separated into cytosolic, nuclear (Strmecki et al., 2007) and crude membrane fractions (Theibert and Devreotes, 1986). Aliquots of the fractions were analysed by western transfer using the CblA antibody.

View larger version (45K): [in this window] [in a new window]   Fig. 4. Validation of a cblA-null strain. (A) Individual clones were first checked for gene disruption by PCR of genomic DNA and candidate clones, such as this (clone no 31), were assayed for cblA transcripts using RT-PCR (primers fwd960 5'-AAA CTC AAA GAT ATT CAG TGG TTT CAT A-3' and rev1926 5'-TGA ACA TAA TGAACA ACA ACT TAA ATG A-3'). The positive control was a random integrant from the same screen and the RNA loadings were controlled using Ig7 as in Fig. 2A. (B) A cblA– clone and Ax2 control cells at the indicated developmental stages were lysed and subjected to western transfer using the CblA antibody.

View larger version (87K): [in this window] [in a new window]   Fig. 5. Developmental phenotype of the cblA– strain. (A) cblA– cells and control Ax2 cells were allowed to develop on water-agar to the slug stage (left) and late culmination stage (right) and photographed under phase-contrast optics. The arrows on the cblA– slugs indicate the break points that are a common feature of DIF signalling mutants and the arrows on the Ax2 culminants show the position of the basal disc. (B) cblA– cells and control Ax2 cells were transformed with an ecmAO-lacZ reporter construct. This reporter is expressed in all the major prestalk and stalk cell sub-types. The transformed cells were allowed to develop on nitrocellulose filters to the late culmination stage then fixed and stained with X-Gal.

View larger version (46K): [in this window] [in a new window]   Fig. 6. A comparison of ecmA induction and DimB nuclear import in Ax2 and cblA– cells. (A) Ax2 and cblA– cells, developed to the mound stage, were dissociated and induced with different concentrations of DIF in a monolayer assay. RNA was then extracted and ecmA concentration was evaluated by RT-PCR. (B) Mound cells were dissociated, suspended in KK2 buffer and treated with 100 nM DIF. At the indicated times, cells were fixed with 85% cold methanol and stained with anti-DimB antibody and then with Alexa-Fluor-488-conjugated secondary antibody (Molecular Probes). Samples were observed by confocal microscopy. This is a low affinity antibody. Hence the nuclear staining that is observed (white arrows) is relatively weak.

View larger version (38K): [in this window] [in a new window]   Fig. 7. A comparison of inducible STATc tyrosine phosphorylation in control and cblA– cells. (A) cblA– cells and control Ax2 cells were developed by starvation in shaken suspension for 4 hours. They were then treated with 100 nM DIF and samples were removed at the indicated times. They were analysed by western transfer with a STATc anti-phosphotyrosine antibody and, as a loading control, with a general STATc antibody. (B) Quantitative comparison of four experiments, as in A, was performed. In some experiments the cblA+ control strain was Ax2 whereas in others it was a random integrant. (C) cblA– and control Ax2 cells were developed in suspension as in A. They were then treated with sorbitol at 200 mM, samples were removed after 15 minutes and analysed as in A, above. This is a typical example of three such experiments.

View larger version (41K): [in this window] [in a new window]   Fig. 8. Western transfer analysis of the PTP3 tyrosine phosphatase. (A) Cells transformed with PTP3:myc or PTP3CS:myc and selected with 20 µg/ml G418 were developed as in Fig. 7A, lysed and analysed by western transfer using affinity purified PTP3 antibody. The loading control was GSK3. (B) Ax2 cells or Ax2 cells transformed with PTP3:myc were developed as in Fig. 7A and analysed by western transfer as in A. The loading control was GSK3. (C) Ax2 cells or cblA– cells were developed as in Fig. 7A and then treated with 100 nM DIF. Samples removed at the indicated times were analysed by western transfer as in A. The loading control was total STATc protein.

View larger version (16K): [in this window] [in a new window]   Fig. 9. CblA, PTP3 and cellular signalling. This scheme suggests that the apparent downregulation of PTP3 by CblA leads to a stimulation in the DIF-induced tyrosine phosphorylation of STATc. In the case of stress the strong negative effect engendered by osmotic stress, probably mediated by increased serine threonine phosphorylation, is not relieved by CblA. The cblA– has morphological phenotypes, similar to those of `DIF-less' mutants, such as the dimB-null strains. We suggest, therefore that both PTP3 and the DIF synthesis and response genes impinge at a common point to regulated morphogenesis. However, CblA is not necessary for DIF responsiveness of ecmA or for DimB activation, hence the proposed dichotomy.

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