(3,4), and two SH2-containing adapter proteins, Grb2 and Shc, have been implicated in its activation. Specifically, these proteins have been shown to bind directly to tyrosine-phosphorylated receptors (5-7) or SH2 docking proteins (such as the insulin receptor substrate 1) (8). Grb2, a 25-kDa protein with two SH3 domains flanking one SH2 domain, shuttles the Ras guanine nucleotide exchange factor, Sosl, to activated receptors (or to insulin receptor substrate 1) (5, 7-11) so that Sosl can activate Ras by catalyzing the exchange of GDP for GTP (5, 9-11). Shc, another widely expressed protein that contains an N-terminal phosphotyrosine binding (PTB) domain (12-16) and a C-terminal SH2 domain (17), can associate, in its tyrosine-phosphorylated form, with Grb2-Sosl complexes and may increase Grb2-Sosl interactions after growth factor stimulation (9,18,19 MATERIALS AND METHODS Reagents. COS-cell-derived murine IL-3 and GM-CSF were produced as described (29). Rabbit antiserum to the Shcassociated p145 protein was generated by immunizing rabbits with a 15-mer obtained from amino acid sequencing (VPAE-GVSSLNEMINP) and crosslinked to keyhole limpet hemocyanin with glutaraldehyde (30). The glutathione S-transferase (GST) fusion proteins, consisting of the 27-kDa N-terminal and of GST linked to the Grb2 N-terminal (amino acid residues 5-56) or C-terminal (residues 163-215) SH3 domains of Grb2, were expressed in Escherichia coli in pGEX-2T plasmids (Pharmacia/LKB) and the fusion proteins were recovered from clarified lysates with glutathione (GSH)-agarose beads as described (31 Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; GSH, glutathione; GST, glutathione S-transferase; IL-3, interleukin 3; NRS, normal rabbit serum; 5-ptase, inositol polyphosphate 5-phosphatase; Ptd1ns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; SH2, src homology 2 domain; PTB, phosphotyrosine binding; p145, the Shc-associated 145-kDa protein.

Lowe syndrome, also known as oculocerebrorenal syndrome, is caused by mutations in the X chromosomeencoded OCRL gene. The Lowe syndrome, also known as oculocerebrorenal syndrome, is an X chromosome-linked disorder that is characterized by severe mental retardation, areflexia, hypotonia, a variety of eye abnormalities, and a renal Fanconi syndrome with impaired reabsorption of glucose, amino acids, phosphate urate, and bicarbonate. Patients have renal tubular acidosis, metabolic bone disease, and growth failure. The eye abnormalities include cataracts, decreased acuity, and glaucoma (1, 2). The protean manifestations of the disorder suggest that the defect involves a gene that mediates a critical cellular function shared by many cell types and organs. In 1992 Nussbaum and coworkers (3) identified a candidate Lowe syndrome gene by positional cloning of X chromosome breakpoints in two affected females. The predicted protein, designated OCRL, was found to be 51% identical over a span of 744 aa to inositol polyphosphate 5-phosphatase type II (5-phosphatase II) (4), an enzyme of the inositol phosphate signaling pathway that was originally isolated from human platelets (5) inositol polyphosphate 5-phosphatase type I (5-phosphatase I) (6-9). These soluble inositol phosphates are involved in calcium signaling reactions, and thus the 5-phosphatase enzymes function in signal-terminating reactions (10). Recently it has been shown that 5-phosphatase II also hydrolyzes the 5-phosphate from the phospholipid phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2] (9, 11). We now report that OCRL, the protein encoded by the Lowe syndrome locus, also is a Ptdlns(4,5)P2 5-phosphatase. OCRL also hydrolyzes the other 5-phosphatase substrates, albeit less well than the other isoenzymes. Sph I site is located (5'-CCTCATTGGCATCAGGCATGC-3'). This PCR product encodes amino acids Met264 to Met506 after digestion with Sph I and BamHI. The second cDNA was constructed by PCR using the same antisense primer and a sense primer that is identical to that shown above with insertion of nucleotides encoding seven histidine residues following nt 806-808. (5'-CGGGATCCAAAATGCACCAC-CACCACCACCACCACCTTCCACGTGAAAAAGAAG-C-3'). The two PCR products were digested with BamHI/Sph I and subcloned into the same region of ps.OCRLB-1 to replace the original aa 1-506 of the OCRLB-1 cDNA. The resulting plasmids ps.OCRL4 and ps.OCRL5 (with histidine tag) were sequenced throughout the PCR region to exclude mutations (Sequenase 2.0; United States Biochemical). ps.OCRL4 and ps.OCRL5 were subsequently subcloned between the BamHI and Xba I sites of the baculovirus transfer vector pVL1393 (PharMingen). MATERIALS AND METHODS

These findings strongly implicate the inositol polyphosphate 5-phosphatases in Shc- and Grb2-mediated signal transduction. Furthermore, SIP-110, SIP-130 and SIP-145 prefer 3-phosphorylated substrates, suggesting a link to the phosphatidylinositol 3-kinase signaling pathway.

The COP9 signalosome (CSN) is a central component of the activation and remodelling cycle of cullin-RING E3 ubiquitin ligases (CRLs), the largest enzyme family of the ubiquitin–proteasome system in humans. CRLs are implicated in the regulation of numerous cellular processes, including cell cycle progression and apoptosis, and aberrant CRL activity is frequently associated with cancer. Remodelling of CRLs is initiated by CSN-catalysed cleavage of the ubiquitin-like activator NEDD8 from CRLs. Here we describe CSN5i-3, a potent, selective and orally available inhibitor of CSN5, the proteolytic subunit of CSN. The compound traps CRLs in the neddylated state, which leads to inactivation of a subset of CRLs by inducing degradation of their substrate recognition module. CSN5i-3 differentially affects the viability of tumour cell lines and suppresses growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has potential for anti-tumour therapy.