Evidence for a functional link between Dd-STATa and Dd-PIAS, a Dictyostelium PIAS homologue

Abstract

Several mammalian protein families inhibit the activity of signal transducer and activator of transcription (STAT) proteins. The protein inhibitor of activated STAT (PIAS) was initially identified through its ability to interact with human STAT proteins. We isolated a gene (pisA) encoding a Dictyostelium orthologue of PIAS, Dd-PIAS, which possesses almost all the representative motifs and domains of mammalian PIAS proteins. A Dd-PIAS null mutant strain displays a normal terminal morphology but with accelerated development once cells are aggregated. In contrast, Dd-PIAS overexpressor strains demonstrate delayed aggregation, almost no slug phototaxis, impaired slug motility, and a prolonged slug migration period. This strain is a near phenocopy of the Dd-STATa null mutant, although it eventually forms a fruiting body, albeit inefficiently. The expression of several Dd-STATa-activated genes is upregulated in the Dd-PIAS null mutant and there is ectopic expression of pstAB makers. The concentration of a PIAS-green fluorescent protein (GFP) fusion protein, expressed under the PIAS promoter, is greatest in the pstO cells and gradually decreases with proximity to the tip of the slug and culminant: a pattern diametrically opposite to that of Dd-STATa. Our results suggest a functional interrelationship between Dd-PIAS and Dd-STATa that influences gene expression and development.

Analysis of DrkA kinase’s role in STATa activation

“…The upper top panel: phosphorylated STATa (pSTATa); the upper middle panel: DrkA‐Myc; the lower panel: a beeswarm boxplot of quantification of band intensities of pSTATa. STATa phosphorylation was detected by Western analysis with anti‐phospho‐STATa antibody, pSC9 (Kawata et al, ). The intensities of pSTATa bands of five independent experiments were quantified by normalizing against that of the most intense band detected with Ponceau staining (Ponceau) using ImageJ software.…”

“…One day after DrkA induction, when the DrkA‐Myc protein level peaks (Figure a), cells were treated with 5 mM cAMP following prior starvation in buffer for 6 hr in shaken suspension. Activation of STATa by cAMP was analyzed by monitoring phosphorylation on Tyr702, using a purified polyclonal anti‐phospho‐STATa antibody, pSC9 (Kawata et al, ). In DrkA induced cells treated for 10 min with cAMP, the phosphorylation level of STATa was increased approximately 2.5‐fold relative to that observed in DrkA uninduced cells following 10 min of cAMP treatment (Figure b).…”

“…The primary antibodies used in the experiments were as follows: anti‐phospho‐STATa antibody pSC9 (Kawata et al, ) for STATa phosphorylated on Tyr702, antiphosphotyrosine antibody 4G10 (Merck Millipore) for general pTyr modification, antiphosphothreonine antibody RM102 (Merck Millipore) for phosphothreonine modification, anti‐GFP monoclonal antibody (No. 11 814 460 001; Roche Diagnostics) for GFP‐tagged proteins, anti‐Myc antibody 9E10 (Wako Pure Chemical) for Myc‐tagged proteins, antipolyhistidine antibody M136‐3 (MBL Co. Ltd.) for His‐tagged proteins, anti‐GST antibody 5A7 (Wako) for GST‐tagged proteins and anti‐actin antibodies C‐11 or C4 (Santa Cruz Biotechnology, Inc.) for monitoring total actin as a loading control.…”

“…The null mutant exhibits delayed aggregation due to inefficient chemotaxis to pulsatile cAMP. Although the null cells are capable of forming slightly short slugs, they do not display phototaxis (Kawata, Hirano, Ogasawara, Aoshima, & Yachi, ). In addition, the mutant shows abnormal morphogenetic cell movement (T. Kawata, N. Shimada, & M. Maeda, unpublished), which results in the formation of aberrant terminal structures (Mohanty et al, ).…”

Aberrant adhesion impacts early development in aDictyosteliummodel for juvenile neuronal ceroid lipofuscinosis

“…68 Knockout of grlJ (glutamate receptor-like protein) or pisA (protein inhibitor of STAT) results in precocious development following aggregation. 69,70 Finally, knockout of iplA (inositol 1,4,5-trisphosphate receptor-like protein) delays aggregation by 1-2 hours. 71 The observations that grlJ ¡ and iplA ¡ cells, which are linked to calcium homeostasis, display similar developmental timing as cln3 ¡ cells was interesting given that altered calcium homeostasis has been observed in mammalian cell models that lack functional CLN3.…”