The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD-95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD-95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K þ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage-dependent K þ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.

Apoptotic stimuli activate and oligomerize the proapoptotic proteins Bak and Bax, resulting in mitochondrial outer-membrane permeabilization and subsequent cell death. This activation can occur when certain BH3-only proteins interact directly with Bak and Bax. Recently published crystal structures reveal that Bax separates into core and latch domains in response to BH3 peptides. The distinguishing characteristics of BH3 peptides capable of directly activating Bax were also elucidated. Here we identify specific BH3 peptides capable of "unlatching" Bak and describe structural insights into Bak activation and oligomerization. Crystal structures and crosslinking experiments demonstrate that Bak undergoes a conformational change similar to that of Bax upon activation. A structure of the Bak core domain dimer provides a high-resolution image of this key intermediate in the pore-forming oligomer. Our results confirm an analogous mechanism for activation and dimerization of Bak and Bax in response to certain BH3 peptides.

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH)signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling. To determine whether a similar enhancement of signaling is observed in vivo or alternatively whether increased SOCS-2 levels repress growth in vivo, we generated and analyzed transgenic mice that overexpress SOCS-2 from a human ubiquitin C promoter. These mice are not growth-deficient and are, in fact, significantly larger than wild-type mice. The overexpressed SOCS-2 was found to bind to endogenous GH receptors in a number of mouse organs, while phosphopeptide binding studies with recombinant SOCS-2 defined phosphorylated tyrosine 595 on the GH receptor as the site of interaction. Together, the data implicate SOCS-2 as having dual effects on GH signaling in vivo.The suppressor of cytokine signaling (SOCS) 1 proteins are a family of eight SH2 domain-containing proteins, comprising cytokine-inducible SH2 domain-containing protein (CIS) and SOCS-1-7. Studies in many laboratories have implicated SOCS proteins in the attenuation of cytokine action through inhibition of the Janus kinase (JAK)/signal transducer and activators of transcription (STAT) signal transduction pathway. SOCS proteins operate as part of a classical negative feedback loop, in which activation of cytokine signaling leads to their expression. Once produced, SOCS proteins bind to key components of the signaling apparatus to prevent further signal transduction and possibly target them for degradation via a conserved C-terminal motif, called the SOCS Box, that recruits ubiquitin ligases (reviewed in Refs. 1-3).While in vitro studies have suggested that SOCS proteins may be promiscuous in their activity, gene deletion studies in mice have highlighted their importance in a limited number of signaling pathways. SOCS-1 is a key regulator of interferon ␥ signaling, T-cell homeostasis, and lactation (4 -6), while SOCS-3 is thought to play a crucial role in placental function (7). CIS-deficient mice are reported to have no phenotype, although CIS transgenic mice display growth retardation and defects in mammary development which are accompanied by reductions in STAT5 phosphorylation (8). Interestingly, this phenotype has similarities to those observed in STAT5a-and STAT5b-deficient mice (9 -11).SOCS-2-deficient animals exhibit accelerated post-natal growth resulting in a 30 -50% increase in body weight by 12 weeks of age, significant increases in bone and body lengths, thickening of the skin due to collagen deposition, and increases in internal organ size (12). This phenotype has striking similarities to those of insulin-like growth factor (IGF)-I and growth hormone (GH) transgenic mic...