The Interactive Fly
Zygotically transcribed genes
Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila
Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila
The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, a genome-wide RNA interference (RNAi) screen was carried out in cultured Drosophila cells. One hundred and twenty-one genes were identified whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, it was found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, a key negative regulator of Drosophila JAK/STAT signaling was identified, protein tyrosine phosphatase PTP61F; it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. This study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway (Baeg, 2005).
To more clearly elucidate the roles of positive regulators, their requirement for the phosphorylation of STAT92E was assayed. Tyrosine phosphorylation is a key step in STAT activation upon cytokine/receptor stimulation. Thus, monitoring steady-state levels of phosphorylated STAT in dsRNA-treated cells would provide insight into the molecular functions of the candidate genes. As expected, Upd stimulation of S2-NP cells leads to a dramatic increase in tyrosine-phosphorylated STAT92E, as shown by Western blot analysis. The effect was measured of dsRNAs against the 29 positive regulators on Upd-induced STAT92E phosphorylation. Thirteen genes (besides STAT92E) were found to be required for Upd-induced STAT92E phosphorylation. As expected, these genes included the canonical components Dome and hop. In contrast to the initial assay in the primary screen, exogenous Upd was used to activate STAT92E phosphorylation, and thus it was not possible to identify genes that act upstream of the receptor, such as Upd2. Notably, two of the 13 genes (CG16790 and CG4329) that regulate STAT92E phosphorylation have no predicted function, yet clearly have human orthologs; further investigation of their molecular functions in JAK/STAT signaling in Drosophila may advance understanding of the mammalian pathway (Baeg, 2005).
Interestingly, this assay revealed that RNAi knockdown of the cyclin-dependent kinase 2 gene (cdc2) resulted in a decrease in STAT92E tyrosine phosphorylation, suggesting that cdc2 modulates JAK/STAT signaling by affecting tyrosine phosphorylation of STAT92E. Consistent with this observation, Warts/Lats, which has been shown both biochemically and genetically to interact with cdc2 and to negatively regulate its kinase activity, was identified in the screen as a potential negative regulator of JAK/STAT signaling. These results suggest that STAT92E plays an important role in Warts/Lats-mediated inhibition of cell proliferation (Baeg, 2005).
echinoid (ed) was identified as a positive regulator required for Upd-dependent STAT92E tyrosine phosphorylation. ed encodes a cell adhesion molecule and has been shown to be a negative regulator of the EGFR signaling pathway during Drosophila eye development. Previous experiments have shown both positive and negative interactions between the JAK/STAT pathway and the EGFR pathway. For example, STAT92E mutants phenocopy mutants in the EGFR pathway. Furthermore, studies using mammalian tissue culture systems have demonstrated that EGFR signaling activates both JAK1 and STAT1. In addition, EGFR-induced cell migration is mediated predominantly by the JAK/STAT pathway in primary esophageal keratinocytes. Similarly, ed has been shown to be responsible for defective cell migration in Caenorhabditis elegans. Therefore studying the role of ed in JAK/STAT signaling in different contexts may facilitate understanding of the genetic and biochemical mode of STAT activation by EGFR signaling, and provide insights into the mechanisms governing cancer cell metastasis in humans (Baeg, 2005).
Another step in the activation of the JAK/STAT signaling pathway is the translocation of STATs into the nucleus. In resting cells, STATs reside mainly in the cytoplasm. Upon cytokine stimulation, they are phosphorylated on key tyrosine residues and rapidly translocate to the nucleus, where they trans-activate target genes. Previous studies have shown that Importin alpha5 and Ran are required for the nuclear import of phosphorylated (activated) STATs. To reset the cells after stimulation, STATs are exported out of the nucleus into the cytoplasm in preparation for the next round of signaling using an Exportin-1/CRM-1-dependent mechanism. These observations suggest that defective nucleocytoplasmic shuttling of STATs can disrupt steady-state distribution of STATs and induce aberrant biological responses. Among all 121 candidates, seven genes were identified that are potentially involved in protein trafficking based on their predicted molecular functions and protein domains. These include Rab26, Ran, CG10225, which encodes the Drosophila homolog of Ran-binding protein 3 (RanBP3), CG11763, which encodes the Drosophila homolog of Ran-binding protein 10 (RanBP10), and the Drosophila homolog of Cellular Apoptosis Susceptibility gene product (CAS) that was initially identified as a Ran-binding protein. In addition, Drosophila homologs of Transportin 1 and Nucleoporin 196, which have been implicated in protein import and/or export in mammals, were identifed. The subcellular localization of phosphorylated STAT92E was examined under conditions where each of the seven candidates was depleted by RNAi except Rad26. As a control it was found that under resting conditions tyrosine phosphorylated STAT92E is detected predominantly in the cytoplasm. Moreover, a significant reduction was observed in phosphorylated STAT92E levels in the cytoplasm when cells were treated with dsRNA against the receptor dome. Upon stimulation with Upd, STAT92E accumulated in the nuclei of 27% of cells. These results illustrate the specificity and sensitivity of the assay. Interestingly, it was found that cells treated with dsRNAs against CG11763 or CG10225 displayed a significant increase in phospho-STAT92E nuclear accumulation upon Upd stimulation. This was not due to changes in the total phosphorylation levels of STAT92E. No significant effects of dsRNA-mediated knockdown of Cas or Trn on STAT92E translocation was detected. In contrast, the role of Ran and Nup98 in STAT92E translocation could not be assessed in this assay due to difficulties in introducing the Upd expression vector into cells upon RNAi knockdown of these two genes. Taken together, these results strongly suggest that the Drosophila homologs of RanBP3 and RanBP10 are novel regulators of JAK/STAT signaling that affect signal-dependent STAT92E nuclear transport (Baeg, 2005).
Another important step in the JAK/STAT signal transduction pathway is the dephosphorylation of the signaling molecules JAKs and STATs. In mammals, several PTPs have been implicated in the dephosphorylation of JAK and/or STAT proteins both in the cytoplasm and in the nucleus. In contrast, no PTPs have been identified that regulate JAK/STAT signaling in Drosophila. PTP61F was identified as a strong negative regulator in the screen. Knockdown of PTP61F by RNAi resulted in a more than fourfold increase in STAT92E-dependent reporter activity. PTP61F encodes the Drosophila homolog of mammalian PTP-1B, which has been shown to attenuate insulin, PDGF, EGF, and IGF-I signaling by dephosphorylating tyrosine residues of JAKs and/or STATs in mammalian tissue culture. Therefore the hypothesis was tested that PTP61F might serve as the tyrosine phosphatase for Hop. A dramatic increase was observed in tyrosine phosphorylation of Hop upon RNAi knockdown of PTP61F, suggesting that Hop may be a substrate of PTP61F. A significant increase was detected in STAT92E phosphorylation in cells treated with dsRNA against PTP61F. This is consistent with the notion that STAT92E is a downstream target of Hop, although the possibility that both Hop and STAT92E may be targets of PTP61F cannot be ruled out (Baeg, 2005).
In both mammals and Drosophila, SOCS, a negative regulator of the JAK/STAT pathway, has been shown to be transcriptionally activated by JAK/STAT signaling, thus generating a negative feedback loop. This prompted an examination of the expression pattern of PTP61F and whether its expression is responsive to JAK/STAT signaling in vivo. It was found PTP61F is expressed in a striped pattern, reminiscent of the STAT92E expression pattern. In addition, overexpression of Upd under the control of prd-Gal4 resulted in a dramatic increase in PTP61F transcript levels in the paired domain. Furthermore, levels of the PTP61F transcript were greatly reduced in embryos lacking Hop activity, suggesting that PTP61F transcription is dependent on active JAK/STAT signaling. Taken together, these results demonstrate that PTP61F expression responds to JAK/STAT signaling in vivo (Baeg, 2005).
These data suggested that loss of PTP61F would result in an increase in JAK/STAT signaling. Thus, the genetic interaction between PTP61F and canonical components of the JAK/STAT pathway was examined, using Df(3)ED4238, a deficiency uncovering the PTP61F gene. The interaction was tested in the Drosophila eye following overexpression of Upd using GMR-Gal4 driver, which causes a dramatic overgrowth and deformation of the adult eye. The severity of this phenotype is proportional to the strength of the JAK/STAT-mediated signal, because removing one copy of STAT92E significantly suppresses the GMR-Upd eye phenotype. Consistent with PTP61F being a negative regulator of the JAK/STAT signaling pathway, flies heterozygous for Df(3)ED4238 showed an enhanced deformed eye phenotype. A PTP61F transgene rescues this enhanced deformed eye phenotype in flies heterozygous for Df(3)ED4238. In addition, the PTP61F transgene also rescues lethality in flies carrying UAS-Upd GMR-Gal4/+; Df(3)ED4238/+, presumably caused by leaky expression of UAS-Upd in conjunction with PTP61F deficiency (Baeg, 2005).
The genetic interaction between PTP61F and Hop was examined. Flies carrying a dominant hyperactive Hop allele (HopTum-l) display decreased viability and the formation of melanotic tumors. This tumor formation phenotype is sensitive to gene dosage. Previous studies have shown that reducing the levels of positive regulators, such as STAT92E, Cdk4, and CycE, increases the viability and/or decreases tumor formation. Therefore both viability and melanotic tumor formation were monitored in females heterozygous for HopTum-l and these results were compared to females heterozygous for both HopTum-l and Df(3)ED4238. Removing one copy of PTP61F in HopTum-l heterozygous females leads to a significant decrease in survival rate and a dramatic enhancement in the formation of melanotic tumors. Altogether, these results demonstrate that PTP61F is a bona fide negative regulator of the JAK/STAT pathway in Drosophila (Baeg, 2005).
Reference
Baeg, G. H., Zhou, R. and Perrimon, N. (2005). Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila. Genes Dev. 19: 1861-1870. 16055650
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.