In and dominant temperature-sensitive (DTS) mutations affect the 2 2 and 6 proteasome subunit genes, respectively (3, 4). merge of images from two focal planes: the socket cells Rabbit polyclonal to JAK1.Janus kinase 1 (JAK1), is a member of a new class of protein-tyrosine kinases (PTK) characterized by the presence of a second phosphotransferase-related domain immediately N-terminal to the PTK domain.The second phosphotransferase domain bears all the hallmarks of a protein kinase, although its structure differs significantly from that of the PTK and threonine/serine kinase family members. (in green: Suppressor of Hairless) were found in the epithelial plane, whereas the sheath cells (in red: Prospero) were subepithelial. These Prospero-positive cells cannot correspond to glial cells for several reasons: first, at this late stage, no glial cells accumulate Prospero; furthermore glial cells migrate away from microchaetes around 23C24 hr APF and are no longer associated with microchaetes at 28 hr APF; finally, glial cell nuclei are distinctly smaller than sheath cell nuclei (7). In all panels anterior is usually up. Here, I show that decreased proteasome activity resulted in shaft-to-socket cell fate transformations in and heterozygous pupae, and that overexpression of the 6 dominant-negative mutant subunit led to neuron-to-sheath and shaft-to-socket cell fate changes. Such fate transformations usually result from increased Notch signaling. Consistently, the and mutations were shown to strongly enhance the double-socket phenotype associated with increased Notch signaling activity. Finally, an activated form of Notch was found to be stabilized in cells that expressed the 6 dominant-negative mutant subunit. This observation indicates that this form of Notch is usually a target of the proteasome. These data demonstrate that a wild-type level of proteasome activity is required for the proper regulation of at least two alternative cell fate decisions during sense organ development, and that proteasome-mediated protein degradation antagonizes Notch signaling activity in this process. These findings raise the possibility that this proteasome participates in limiting Notch signaling by targeting an active form of Notch for degradation. MATERIALS AND METHODS Flies. The and mutations are described in refs. 3 and 4 (kind gifts from J. Belote, Syracuse Univ., Syracuse, NY). The A101 P[that specifically expresses nuclear -galactosidase in pI and its progeny cells (11). The to generate the P[allele of the 6 proteasome catalytic subunit gene (ref. 3; plasmid p1B176; gift from J. Belote) was subcloned into pUAS-T between the and heterozygous pupae were heat-pulsed at the purchase Dexamethasone restrictive heat between 7 and 23 hr APF. Under these conditions, pupae developed to give pharate adults that did not emerge but showed specific bristle defects. A small number of double-socket sense organs were observed around the notum [Fig. purchase Dexamethasone 1 and and flies showed 8% (= 50/621 from six nota) and 2% (= 8/432 from five nota) of double-socket microchaetes around the notum], as well as around the stomach and on the wing margin (data not shown). Wild-type flies subjected to the same regimen showed no defects (Fig. ?(Fig.11 and or mutant pupae for a longer period of time, pupal development was blocked and no pharate adults were recovered (not shown). The double-socket phenotype seen in and adult flies suggested that this shaft cell was occasionally transformed into a second socket cell. This cell fate change was confirmed by using anti-Su(H) antibodies to specifically mark the socket cells (14): a few sense organs with two Su(H)-positive cells were observed in nota from and pupae that were dissected at 24 hr APF and that had developed at the restrictive heat as described above (Fig. ?(Fig.11 and and mutant pupae, Prospero was used as a sheath cell marker. Prospero, a divergent homeodomain protein, is usually detected in the pIIb, pIIIb, and sheath cells (7, 15, 16). purchase Dexamethasone It also accumulates transiently in the glial cell and in the neuron. At 28 hr APF, however, Prospero could be detected in only a single cell, the sheath cell (Fig. ?(Fig.11and mutant pupae, microchaetes with two cells accumulating Prospero were occasionally observed (Fig. ?(Fig.11 and phenotype resulted from a defect in proteasome activity, the cDNA encoding the DTS5 mutant 6 proteasome subunit was expressed in wild-type flies by using the UAS-system (17). When a homozygous viable driver inserted at the locus (and one copy of the UAS-transgene grew normally at 25C, but died at 29C as.