Structure and Chemical Inhibition of the Ret Tyrosine Kinase Domain.

The RET proto-oncogene encodes a receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family of ligands. Loss-of-function mutations in RET are implicated in Hirschsprung disease, whereas activating mutations in RET are found in human cancers, including familial medullar thyroid carcinoma and multiple endocrine neoplasias 2A and 2B. We report here the biochemical characterization of the human RET tyrosine kinase domain and the structure determination of the non-phosphorylated and phosphorylated forms. Both structures adopt the same active kinase conformation competent to bind ATP and substrate and have a pre-organized activation loop conformation that is independent of phosphorylation status. In agreement with the structural data, enzyme kinetic data show that autophosphorylation produces only a modest increase in activity. Longer forms of RET containing the juxtamembrane domain and C-terminal tail exhibited similar kinetic behavior, implying that there is no cis-inhibitory mechanism within the RET intracellular domain. Our results suggest the existence of alternative inhibitory mechanisms, possibly in trans, for the autoregulation of RET kinase activity. We also present the structures of the RET tyrosine kinase domain bound to two inhibitors, the pyrazolopyrimidine PP1 and the clinically relevant 4-anilinoquinazoline ZD6474. These structures explain why certain multiple endocrine neoplasia 2-associated RET mutants found in patients are resistant to inhibition and form the basis for design of more effective inhibitors

Podocyte-specific loss of cdc42 leads to congenital nephropathy

Rho family GTPases are molecular switches best known for their pivotal role in dynamic regulation of the actin cytoskeleton. The prototypic members of this family are Cdc42, Rac1, and RhoA; these GTPases contribute to the breakdown of glomerular filtration and the resultant proteinuria, but their functions in normal podocyte physiology remain poorly understood. Here, mice lacking Cdc42 in podocytes developed congenital nephropathy and died as a result of renal failure within 2 weeks after birth. In contrast, mice lacking Rac1 or RhoA in podocytes were overtly normal and lived to adulthood. Kidneys from Cdc42-mutant mice exhibited protein-filled microcysts with hallmarks of collapsing glomerulopathy, as well as extensive effacement of podocyte foot processes with abnormal junctional complexes. Furthermore, we observed aberrant expression of several podocyte markers and cell polarity proteins in the absence of Cdc42, indicating a disruption of the slit diaphragm. Kidneys from Rac1- and RhoA-mutant mice, however, had normal glomerular morphology and intact foot processes. A nephrin clustering assay suggested that Cdc42 deficiency, but not Rac1 or RhoA deficiency, impairs the polymerization of actin at sites of nephrin aggregates. Taken together, these data highlight the physiological importance of Cdc42, but not Rac1 or RhoA, in establishing podocyte architecture and glomerular function

Yap- and Cdc42-dependent nephrogenesis and morphogenesis during mouse kidney development.

Yap is a transcriptional co-activator that regulates cell proliferation and apoptosis downstream of the Hippo kinase pathway. We investigated Yap function during mouse kidney development using a conditional knockout strategy that specifically inactivated Yap within the nephrogenic lineage. We found that Yap is essential for nephron induction and morphogenesis, surprisingly, in a manner independent of regulation of cell proliferation and apoptosis. We used microarray analysis to identify a suite of novel Yap-dependent genes that function during nephron formation and have been implicated in morphogenesis. Previous in vitro studies have indicated that Yap can respond to mechanical stresses in cultured cells downstream of the small GTPases RhoA. We find that tissue-specific inactivation of the Rho GTPase Cdc42 causes a severe defect in nephrogenesis that strikingly phenocopies loss of Yap. Ablation of Cdc42 decreases nuclear localization of Yap, leading to a reduction of Yap-dependent gene expression. We propose that Yap responds to Cdc42-dependent signals in nephron progenitor cells to activate a genetic program required to shape the functioning nephron

<i>Yap</i> is required for kidney development.

<p>(A) Stages of nephrogenesis and their relationship to the UB (black) tips. Signals released from UB tips induce mesenchyme cells to condense around UB tips forming the CM (blue). Some of these CM cells aggregate forming the PA that converts into epithelial RV. The late RV fuses with UB tips and develops into comma (CSB) and S-shaped (SSB) body. (A′) Schematic diagram of the nephron components. (B) Confocal images for Yap, E-cadherin and DAPI staining in late RV at E14.5. Nuclear Yap is observed in the proximal segment of the RV (arrowheads), while Yap expression disappears in Six2:Cre expressing cells (D - arrows point to CM cells, arrowhead points to an early nephron). (C) Confocal images of p-Yap/E-cadherin/DAPI staining shows ubiquitous p-Yap expression. Individual channels images are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003380#pgen.1003380.s002" target="_blank">Figure S2</a>. (E) Immunohistochemistry using Yap/Taz antibody in RV and SSB shows a similar expression pattern observed with Yap antibody in previous panels (arrowheads). (F–F″) Confocal images for Yap/E-cadherin/DAPI staining in SSB at E14.5. Nuclear Yap is observed in proximal and distal segments of the SSB (arrowheads). (G,H) Macroscopic view of the urogenital system from wild-type and <i>Yap</i> mutant kidneys at P0. Note bilateral reduction in kidney size of mutant compared to control and empty bladder in mutant animals. (I,J) PAS staining of P0 kidneys from wild-type and <i>Yap^CM−/−</i> animals. Arrows point to the papilla. (K,L) Closer view of the cortical zone shows limited nephrogenesis in <i>Yap^CM−/−</i>. (M,N) Higher magnification shows abnormal glomeruli structure and tubules with barely discernable lumens (asterisk) in <i>Yap^CM−/−</i>. k: kidney; b: bladder; cd: collectiong duct; csb: comma-shaped body; d: distal; g: glomeruli; ic: inner cortex; ma: medulla; m: medial; nz: nephrogenic zone; p: proximal; pt: proximal tubule; ssb: S-shaped body. Scale bars represent 25 µm (B–F″; M–N), 1 mm (G–J), 200 µm (K,L).</p

Loss of <i>Cdc42</i> phenocopies <i>Yap^CM−/−</i> phenotype.

<p>(A,B) Macroscopic view of the urogenital system from wild-type and <i>Cdc42^CM−/−</i>kidneys at P0. Note the reduction in kidney bladder size in mutant animals. (C–F) PAS staining (P0) from wild-type and <i>Cdc42^CM−/−</i> animals showing smaller papilla (arrows), dramatic reduction of both CM-derived epithelial structures and glomeruli in the mutant. (G–J) Sections of P0 kidneys using late nephron-specific markers confirms the abnormal glomeruli and proximal tubules formation in <i>Cdc42</i> mutant kidneys. Glomeruli (Podocin, G,H). Proximal tubules (LTL, I,J). (K,L) NCAM staining (E15.5) reveals dramatic reduction in the number of CM-derived structures (arrowheads) in mutants compared to wild-type. k: kidney; b: bladder; g: glomeruli; pt: proximal tubule. Scale bars represent 1 mm (A–D), 200 µm (E–L).</p

Loss of CM-derived epithelial structures and abnormal morphogenesis in <i>Yap</i> mutants.

<p>(A–J) Sections of P0 kidneys stained using late nephron markers confirm abnormal nephron formation in <i>Yap^CM−/−</i> kidneys. Glomeruli (Podocin, A,B; Podocin-WT1-Tomato lectin, C–D′). Proximal tubules (LTL, E,F). Henle's loop (<i>Slc12a1</i>, G,H). Distal tubules (<i>Slc12a3</i>, I, J). (K,L) Overview of an E14.5 nephrogenic zone reveals the presence of CM cells (arrows) in both genotypes, but CM-derived epithelial structures (arrowheads) are greatly reduced in mutant when compared to control littermates. (M–N′) Higher magnification shows histological morphology defects of mutant SSB compared to wild-type controls at E13.5. Scale bars represent 500 µm (A,B), 50 µm (C–D′), 200 µm (E–J), 100 µm (K–L).</p

<i>Cdc42</i> is necessary for Yap to be normally localized and active.

<p>(A–B′″) Staining for Six2 and Yap shows reduce nuclear Yap staining in most of the Six2 positives cells (arrows) of <i>Cdc42^CM−/−</i> compared to wild-type at E12.5. Control (C–C′″) and Cre infected (D–D′″) <i>Cdc42^flox/flox</i> mouse embryonic fibroblasts (MEFs) stained with Yap antibody and doubly counterstained with phalloidin and Hoechst 33258. (E) Quantification from panels A–B′″ of Yap nuclear staining in CM and UB cells from controls (black columns) and <i>Cdc42^CM−/−</i> (white columns) kidneys at E12.5. Data represent mean fluorescence intensity per nucleus area (100 nuclei for each genotype - ***p<0.0001). (F–M) Expression of Cited1 (F), <i>Capn6</i> (H), <i>Traf1</i> (J), <i>Meox2</i> (L) in control E14.5 kidneys, demonstrating expression in nephron progenitor cells. <i>Cdc42</i> deletion results in loss of expression of these genes in CM cells (G, I, K, M), similar to what is seen in <i>Yap^CM−/−</i> mutant. (N,O) <i>IS</i>H reveals increase in levels of <i>Fgf10</i> expression specifically in CM cells of mutant kidneys compared to wild-type controls. Scale bars represent 25 µm (A–B′″), 10 µm (C–D′″), 100 µm (F,G), 200 µm (H–O).</p

<i>Yap</i> deletion impairs nephron induction, without affecting self-renewal of the CM population.

<p>(A,B) Immunostaining analysis for Six2 (E14.5) shows no change in expression pattern in both genotypes (arrows). E-cadherin was used to visualize the UB compartment. (C,D) Dramatic reduction in nephrogenesis visualized by loss of NCAM-expressing structures (arrowheads) in the nephrogenic zone of <i>Yap</i> mutant compared to wild-type (E16.5). Note the reduced NCAM expression in CM cells. Calbindin highlights the UB and CD. (E) Quantification of early nephron structures in E15.5 controls (black columns) and <i>Yap</i> mutants (white columns) based on NCAM staining. Total***: p<0.0001; PA***: p<0.0001; RV*: p = 0.0209; CSB*: p = 0.0018; SSB***: p<0.0001. (F,G) ISH analysis shows maintained <i>Gdnf</i> expression in CM of control and <i>Yap</i> mutants (E15.5). (H,I) WT1 staining (E18.5) reveals staining in CM cells (arrows) for both genotypes, and dramatic reduction in number of renal MET-derived structures in mutants compared to wild-type. (J,K) Immunostaining analysis for the CM marker Sall1 (E14.5) shows no change in expression pattern in both genotypes. E-cadherin was used to visualize the UB compartment. Scale bars represent 100 µm.</p