The exact mechanism of Ptp61F remains unclear but potentially calls for the dephosphorylation of Stat92E. SOCS proteins and PTPases trigger international downregulation of the JAK STAT pathway by inhibition of the receptor/JAK complicated while in the cytoplasm or phosphorylated STATs in the nucleus, respectively. Not too long ago, a JAK STAT inhibitor was uncovered in Drosophila that didn’t act within this worldwide trend. The ken & barbie gene was originally recognized in a P element mutagenesis screen for male sterility, and mutants of this gene lacked external genitalia. ken was later implicated to be a novel interactor from the JAK STAT pathway. In a genetic screen designed to uncover modifiers of the adult eye overgrowth phenotype caused by Upd overexpression during the developing eye imaginal disc, ken enhanced the eye overgrowth phenotype suggesting that, on this tissue, it normally inhibited the JAK STAT signaling pathway. Ken is characterized by an N terminal Broad complex, tramtrack, brica brac domain and C terminal zinc finger motifs, a domain structure shared by known transcriptional repressors.
Ken was discovered to bind the sequence GAAA, which overlaps with a subset of Stat92E consensus binding sites. Furthermore, ectopic expression of Ken while in the embryo inhibits the expression of known JAK STAT target genes ventral veins lacking, trachealess, and knirps. In contrast, misexpression of selleckchem Ken does not affect the expression of the JAK STAT target Socs36E. Therefore, Ken behaves as a selective inhibitor of a subset of JAK STAT targets that contain DNA binding sites that accommodate both Stat92E and Ken binding sites. Here, we investigate the role of Ken while in the Drosophila testis niche. Although ken is expressed throughout the testis apex, it is cell autonomously required in CySCs but not GSCs for their maintenance.
Furthermore, expression of Ken while in the CySC lineage is sufficient to lead to CySCs as well as GSCs to self renew outside of their normal niche. Materials and methods Fly stocks and culture Flies were raised on standard yeast/molasses medium at 25 C unless inhibitor GSK256066 otherwise stated. The following stocks were used: y w, ken alleles : ken1, ken 02970, ken k11035, UAS ken, UAS zfh1, UAS hop TumL, UAS stat92E RNAi, UAS zfh1 RNAi GAL4, nanos GAL4, hs upd, and hs ken. Induction of ectopic ken, zfh1, hopTumL, upd, and RNAi constructs Ectopic Ken, Zfh1, or HopTumL was induced in c587 GAL4/Y;; UAS ken/tub GAL80 ts, c587 GAL4/Y; UAS zfh1/, UAS/tub GAL80 ts /, or c587 GAL4/Y; UAS hop TumL, UAS/tub GAL80 ts / males by setting up crosses at 18 C to permit survival until adulthood. Newly eclosed males were then shifted to 31 C or 29 C for two weeks before dissection.
Stat92E RNAi and Zfh1 RNAi were induced in c587 GAL4/Y; UAS stat92ERNAi/, tub GAL80 ts / or c587 GAL4/Y; UAS zfh1 RNAi/, tub GAL80 ts / males by shifting newly eclosed males raised at 18 C to 31 C for one week before dissection.