The PKC1-MPK1 pathway in yeast functions in the maintenance of cell wall integrity and in the stress response. We have identified a family of genes that are putative regulators of this pathway. WSC1, WSC2, and WSC3 encode predicted integral membrane proteins with a conserved cysteine motif and a WSC1-green f luorescence protein fusion protein localizes to the plasma membrane. Deletion of WSC results in phenotypes similar to mutants in the PKC1-MPK1 pathway and an increase in the activity of MPK1 upon a mild heat treatment is impaired in a wsc⌬ mutant. Genetic analysis places the function of WSC upstream of PKC1, suggesting that they play a role in its activation. We also find a genetic interaction between WSC and the RAS-cAMP pathway. The RAS-cAMP pathway is required for cell cycle progression and for the heat shock response. Overexpression of WSC suppresses the heat shock sensitivity of a strain in which RAS is hyperactivated and the heat shock sensitivity of a wsc⌬ strain is rescued by deletion of RAS2. The functional characteristics and cellular localization of WSC suggest that they may mediate intracellular responses to environmental stress in yeast.

A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.

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Phosphorylation regulates the function of ligand-gated ion channels such as the N-methyl D-aspartate (NMDA) receptor. Here we report a mechanism for modulation of the phosphorylation state and function of the NMDA receptor via an inhibitory scaffolding protein, RACK1. We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices. Peptides that disrupt the interactions between RACK1, NR2B, and Fyn induce phosphorylation and potentiate NMDA receptor-mediated currents. Therefore, RACK1 is a regulator of NMDA receptor function and may play a role in synaptic plasticity, addiction, learning, and memory.

Protein kinase C (PKC) isozymes move upon activation from one intracellular site to another. PKC-binding proteins, such as receptors for activated C kinase (RACKs), play an important role in regulating the localization and diverse functions of PKC isozymes. RACK1, the receptor for activated ␤IIPKC, determines the localization and functional activity of ␤IIPKC. However, the mechanism by which RACK1 localizes activated ␤IIPKC is not known. Here, we provide evidence that the intracellular localization of RACK1 changes in response to PKC activation. In Chinese hamster ovary cells transfected with the dopamine D2L receptor and in NG108-15 cells, PKC activation by either phorbol ester or a dopamine D2 receptor agonist caused the movement of RACK1. Moreover, PKC activation resulted in the in situ association and movement of RACK1 and ␤IIPKC to the same intracellular sites. Time course studies indicate that PKC activation induces the association of the two proteins prior to their co-movement. We further show that association of RACK1 and ␤IIPKC is required for the movement of both proteins. Our results suggest that RACK1 is a PKC shuttling protein that moves ␤IIPKC from one intracellular site to another.Specific intracellular localization of signaling proteins such as PKC 1 is important for the regulation of complex signal transduction cascades (1). PKC is a family of 10 isozymes that are localized to specific intracellular sites in unstimulated cells. Upon activation, each PKC isozyme moves to a different intracellular site (2). Localization of inactive or activated PKC isozymes is mediated, at least in part, by interaction with anchoring proteins (3, 4). For example, inactive PKC isozymes appear to be localized by binding to the scaffolding proteins AKAP-79 and gravin (5, 6). In contrast, activated PKC isozymes are localized by binding to receptors for activated C kinase (RACKs). RACK1 specifically binds the active form of ␤IIPKC (7, 8) thereby regulating PKC function (8 -12). In vitro, RACK1 binds PKC only in the presence of PKC activators and increases PKC kinase activity, presumably by stabilizing its active conformation (13). The RACK1 binding site on PKC is within the C2 region of the regulatory domain providing a direct protein-protein interaction (8). Indeed, RACK1 belongs to the WD40 family of proteins, and the WD40 motif is implicated in mediating protein-protein interactions (14). Furthermore, peptides derived from either PKC and/or RACK1 can alter PKC activity in vitro and in vivo (8,12,15,16).Although RACK1 binds activated PKC and is clearly important for PKC function, the mechanism by which RACK1 localizes ␤IIPKC to its site after activation is not understood. One prediction is that the anchoring protein RACK1 should always be localized to the same site that accepts ␤IIPKC after translocation. We therefore used confocal microscopy to determine whether RACK1 is co-localized with activated ␤IIPKC, whether RACK1 is localized to a specific organelle, and whether the intracellular localization of RACK1 changes...

RACK1 was originally identified as an anchoring protein for protein kinase C (4), but in the past decade, its role has been greatly expanded through structure prediction, localization, and identification of novel binding partners (reviewed in Ref. 5). RACK1 is a 36-kDa protein containing seven WD40 repeats that mediate its protein-protein interaction properties (6). Homology with the G protein subunit G␤ has led to the prediction of the structure of RACK1 as being a seven-bladed ␤-sheet propeller (7,8), suggesting the availability of multiple protein interaction surfaces. RACK1 acts as a scaffolding/anchoring protein for a number of binding partners, ranging from signaling proteins such as kinases (1, 9, 10), a phosphodiesterase (11), and a phosphatase (12) to numerous intracellular tails of receptors (1,10, 13,14). In addition, recent data suggest that RACK1 binding may play a role in gene expression, translation, and ribosome assembly and activation (3, 15-17). Therefore, RACK1 is a multi-purpose protein that regulates various biological functions.We recently demonstrated that RACK1 acts as a novel inhibitory scaffolding protein associating in a tri-molecular complex with the tyrosine kinase Fyn and its substrate, the NR2B subunit of the glutamate-gated ion channel, NMDAR (1).1 We found that this association prevents the phosphorylation of NR2B by Fyn and inhibits NMDAR-mediated activity (1). However, removal of RACK1 from the NMDAR complex via the activation of the cAMP/PKA pathway by the adenylate cyclase activator forskolin or PACAP(1-38), enables NR2B phosphorylation by Fyn and enhances NMDAR channel function (1, 3).It is interesting that Fyn and NR2B share a region of sequence homology that mediates their binding at a single RACK1 binding site located in the first WD40 repeat at amino acids 35-48 (1). Our previous in vitro data suggest that Fyn and NR2B interact with RACK1 simultaneously and that RACK1 serves as a physical bridge between the kinase and its

Protein kinase C (PKC) is involved in many neuroadaptive responses to ethanol in the nervous system. PKC activation results in translocation of the enzyme from one intracellular site to another. Compartmentalization of PKC isozymes is regulated by targeting proteins such as receptors for activated C kinase (RACKs). It is possible, therefore, that ethanol-induced changes in the function and compartmentalization of PKC isozymes could be due to changes in PKC targeting proteins. Here we study the response of the targeting protein RACK1 and its corresponding kinase betaIIPKC to ethanol, and propose a novel mechanism to explain how ethanol modulates signaling cascades. In cultured cells, ethanol induces movement of RACK1 to the nucleus without affecting the compartmentalization of betaIIPKC. Ethanol also inhibits betaIIPKC translocation in response to activation. These results suggest that ethanol inhibition of betaIIPKC translocation is due to miscompartmentalization of the targeting protein RACK1. Similar events occurred in mouse brain. In vivo exposure to ethanol caused RACK1 to localize to nuclei in specific brain regions, but did not affect the compartmentalization of betaIIPKC. Thus, some of the cellular and neuroadaptive responses to ethanol may be related to ethanol-induced movement of RACK1 to the nucleus, thereby preventing the translocation and corresponding function of betaIIPKC.

Scaffolding proteins such as receptor for activated C kinase (RACK) 1 are involved in the targeting of signaling proteins and play an important role in the regulation of signal transduction cascades. Recently, we found that in cultured cells and in vivo, acute ethanol exposure induces the nuclear compartmentalization of RACK1. To elucidate a physiological role for nuclear RACK1, the Tat protein transduction system was used to transduce RACK1 and RACK1-derived fragments into C6 glioma cells. We found that nuclear RACK1 is mediating the induction of the immediate early gene c-fos expression induced by ethanol. First, transduction of full-length RACK1 (Tat-RACK1) resulted in the induction of c-fos expression and enhancement of ethanol activities. Second, we determined that the C terminus of RACK1 (Tat-RACK1⌬N) is mediating transcription. Third, we identified a dominant negative fragment of RACK1 that inhibited the nuclear compartmentalization of endogenous RACK1 and inhibited ethanol-induction of c-fos mRNA and protein expression. Last, acute exposure to ethanol or transduction of full-length Tat-RACK1 resulted in an increase in mRNA levels of an activator protein 1 site-containing gene, PAC1 (pituitary adenylate cyclase-activating polypeptide receptor type I), suggesting that nuclear RACK1 is involved in the regulation of the expression of genes that are altered upon acute ethanol treatment. These results may therefore have important implications for the study of alcohol addiction.