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.The RET (rearranged during transfection) gene was originally isolated as an oncogenic fusion protein in cell transformation assays (1). The RET proto-oncogene (2), on human chromosome 10q11.2, encodes a receptor tyrosine kinase (RTK) 4 (3-5) activated by members of the glial cell line-derived neurotrophic factor (GDNF) ligand family (GDNF, neurturin, artemin, and persephin) (6) in conjunction with a ligand-specific coreceptor (GFR␣1-4) (7). RET signaling is essential for development, survival, and regeneration of many neuronal populations such as those in the enteric and sympathetic nervous systems (6) and the kidney (8, 9). The domain organization of RET is shown in Fig. 1A; orthologs exist from Drosophila to human and share a high degree of sequence similarity (90% in vertebrates) throughout the cytoplasmic domain and, to a lesser extent, within the extracellular region.GDNF family ligands do not interact directly with RET; instead, signaling via RET depends on formation of a tripartite complex of RET, a GDNF family ligand, and its cognate glycosylphosphatidylinositol-linked GFR␣ (10, 11). In addition, ligand binding requires Ca 2ϩ ions chelated to the RET extracellular domain (12, 13). According to the classical RTK paradigm, formation of the complex promotes RET dimerization, leading to trans-autophosphorylation within the RET intracellular domain (RET-ICD). There are two tyrosine residues (Tyr 900 and Tyr 905 ) in the RET tyrosine kinase domain (RET-KD) activation loop (...

The Josephin domain plays an important role in the cellular functions of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We have determined the solution structure of Josephin and shown that it belongs to the family of papain-like cysteine proteases, sharing the highest degree of structural similarity with bacterial staphopain. A currently unique structural feature of Josephin is a flexible helical hairpin formed by a 32-residue insertion, which could determine substrate specificity. By using the Josephin structure and the availability of NMR chemical shift assignments, we have mapped the enzyme active site by using the typical cysteine protease inhibitors, transepoxysuccinyl-L-eucylamido-4-guanidino-butane (E-64) and [L-3-trans-(propylcarbamyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline (CA-074). We also demonstrate that the specific interaction of Josephin with the ubiquitin-like domain of the ubiquitin-and proteasome-binding factor HHR23B involves complementary exposed hydrophobic surfaces. The structural similarity with other deubiquitinating enzymes suggests a model for the proteolytic enzymatic activity of ataxin-3.cysteine protease ͉ NMR ͉ polyglutamine disease ͉ SCA3 ͉ ubiquitin

The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH) 2 domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3 0 flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes.

Securin inhibits the proteolytic activity of separase in a 2-fold manner. While inhibiting separase, securin is able to promote nuclear accumulation of separase and help separase to become fully activated after securin's own destruction at anaphase onset.