SUMMARY Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. Here we show that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP.

Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor β (TGF-β) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Smad Mothers against Decapentaplegic (Mad) as the readout, we carried out a whole-genome RNAi screening in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-β. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. We have thus identified new evolutionarily conserved proteins that are important in the signal transduction of TGF-β and BMP into the nucleus.

The level of TGF-β/bone morphogenetic protein (BMP) signaling through Smad is tightly regulated to ensure proper embryonic patterning and homeostasis. Here we show that Smad activation by TGF-β/BMP is blocked by a highly conserved phosphorylation event in the α-helix 1 region of Smad [T312 in Drosophila Smad1 (MAD)]. α-helix 1 phosphorylation reduces Smad interaction with TGF-β/BMP receptor kinase and affects all receptor-activated Smads except Smad3. Tissue culture and transgenic studies in Drosophila further demonstrate that the biological activity of MAD is repressed by T312 phosphorylation in vivo. Through RNAi screening of the kinome, we have identified Misshapen (Msn) and the mammalian orthologs TNIK, MINK1, and MAP4K4 as the kinases responsible for α-helix 1 phosphorylation. Targeted expression of an active form of Msn in the wing imaginal disk disrupted activation of endogenous MAD by Dpp and expression of the Dpp/MAD target gene. Msn kinases belong to the Ste20 kinase family that has been shown to act as MAP kinase kinase kinase kinase (MAP4K). Our findings thus reveal a function of Msn independent of its impact on MAP kinase cascades. This Smad inhibition mechanism by Msn likely has important implications for development and disease. myristoylation | TKV interaction

Trafficking of Smad proteins between the cytoplasm and nucleus is a critical component of transforming growth factor ␤ (TGF-␤) signal transduction. Smad4 translocates into the nucleus either in response to TGF-␤ stimulation or when its nuclear export is blocked by leptomycin B (LMB). We demonstrate that both TGF-␤-induced and basal state spontaneous nuclear import of Smad4 require importin 7 and 8 (Imp7,8). Our data suggest that in the nuclear import of Smad4, the role of Imp8 is irreplaceable by Imp7, and that Smads preferentially bind Imp8. Interestingly, in contrast to its mammalian counterpart Smad4, Drosophila Medea appears to utilize different mechanisms for TGF-␤-induced or basal state nuclear accumulation, with the latter independent of Msk (Drosophila Imp7/8) function. In addition, overexpression of Imp8 alone was sufficient to cause an increased concentration of Smad1,3 and 4 in the nucleus, but had very limited effects on Smad2. These observations suggest selective involvement of Imp8/Msk in nuclear import of different Smads under different conditions.

Cytoplasm-to-nucleus translocation of Smad is a fundamental step in transforming growth factor ␤ (TGF-␤) signal transduction. Here we identify a subset of nucleoporins that, in conjunction with Msk (Drosophila Imp7/8), specifically mediate activation-induced nuclear translocation of MAD (Drosophila Smad1) but not the constitutive import of proteins harboring a classic nuclear localization signal (cNLS) or the spontaneous nuclear import of Medea (Drosophila Smad4). Surprisingly, many of these nucleoporins, including Sec13, Nup75, Nup93, and Nup205, are scaffold nucleoporins considered important for the overall integrity of the nuclear pore complex (NPC) but not known to have cargo-specific functions. We demonstrate that the roles of these nucleoporins in supporting Smad nuclear import are separate from their previously assigned functions in NPC assembly. Furthermore, we uncovered novel pathway-specific functions of Sec13 and Nup93; both Sec13 and Nup93 are able to preferentially interact with the phosphorylated/activated form of MAD, and Nup93 acts to recruit the importin Msk to the nuclear periphery. These findings, together with the observation that Sec13 and Nup93 could interact directly with Msk, suggest their direct involvement in the nuclear import of MAD. Thus, we have delineated the nucleoporin requirement of MAD nuclear import, reflecting a unique trans-NPC mechanism.Transforming growth factor ␤ (TGF-␤) cytokines critically regulate a diverse array of cellular properties in development and homeostasis, through an evolutionarily conserved mechanism that centers around the Smad transcription factors (18, 34). TGF-␤ induces phosphorylation of the Smad proteins and consequently drives Smads into the nucleus, ensuring that changes in the gene transcription program are strictly signal dependent (24,26). Two critical elements in nuclear import are the transport receptors (i.e., karyopherins or importins) and the nuclear pore complex (NPC) (28, 30). We recently identified Imp7/8 as the importin for TGF-␤-activated Smads, but how the Imp7/8-Smad complex translocates through the NPC has yet to be elucidated (36). The NPC consists of more than 30 evolutionarily conserved nucleoporins, each with a particular localization within the NPC based on biochemical, biophysical, electron microscopy, and computational studies (2,3,25). Many nucleoporins contain repeats of phenylalanine-glycine (FG), and the highly hydrophobic and unstructured FG domains occupy the central tunnel of the NPC, constituting a gating mechanism that restricts the movement of macromolecules through the NPC (19). In vivo genetic studies have suggested redundancy among FG domains in maintaining the permeability barrier as well as nuclear import (29, 38). On the other hand, many of the non-FG nucleoporins assemble into subcomplexes within the NPC, most prominently the Nup107-160 (consisting of Nup107, Nup133, Nup75, Sec13, and Seh1, etc.) and Nup53-93 (containing Nup53, Nup93, and Nup205, etc.) complexes, and they are believed to serve mostly as scaffo...