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We describe a protein kinase, Shkl, from the fission yeast Schizosaccharomyces pombe, which is structurally related to the Saccharomyces cerevisiae Ste2O and mammalian p65PAK protein kinases. We provide genetic evidence for physical and functional interaction between Shkl and the Cdc42 GTP-binding protein required for normal cell morphology and mating in S. pombe. We further show that expression of the STE20 gene complements the shki null mutation and that Shkl is capable of signaling to the pheromone-responsive mitogen-activated protein kinase cascade in S. cerevisiae. Our results lead us to propose that signaling modules composed of small GTP-binding proteins and protein kinases related to Shkl, Ste2O, and p65PAK, are highly conserved in evolution and participate in both cytoskeletal functions and mitogen-activated protein kinase signaling pathways.ras genes are highly conserved in evolution and encode small GTP-binding proteins that regulate cell growth and differentiation in a broad spectrum of eukaryotic organisms (1). The fission yeast Schizosaccharomyces pombe possesses a single known ras homolog, rasl, the product of which is required for at least two distinct cellular functions. First, Rasl is required for sexual differentiation-namely, conjugation and sporulation-which is induced by starvation and by peptide mating pheromones that bind to serpentine receptors (2-4). Rasl functions upstream of a mitogen-activated protein (MAP) kinase (MAPK) In this report, we describe a protein kinase, Shk,1, and provide evidence that it mediates functions of the Rasl/Cdc42 signaling complex in S. pombe. Shkl is highly related in structure to the Ste2O kinase, which is required for sexual response in Saccharomyces cerevisiae (19,20), and to the mammalian Cdc42/Racl-binding kinase, p65PAK (Pak) (21).Our results lead us to propose that signaling pathways mediated by small GTP-binding proteins and protein kinases related to Shkl are conserved in evolution and participate in regulation of the cytoskeleton and MAPK modules.MATERIALS AND METHODS Microbial Manipulation and Analysis. S. pombe strains SP870 (h9O ade6-210 leul-32 ura4-D18) and SP66 (h9o ade6-216 leul-32) were provided by D. Beach (Cold Spring Harbor Laboratory). SP870D (h90 ade6-210/ade6-210 leul-32/leul-32 ura4-D18/ura4-D18) is a spontaneous diploid derived from SP870 (V. Jung, personal communication). SP206U (h90 ade6-210/ade6-210 leul-32/leul-32 ura4-D18/ura4-D18 shk1::ura4/shk1) was constructed by transformation of SP870D with an Ec1136II-Msc I fragment of shkl::ura4 from plasmid pBSSHK1::URA4. SP206UA (h90 ade6-210/ade6-210 leul-32/leul-32 ura4-D18/ ura4-D18 shk1::ura4:.ADE2/shk1+) was constructed by transforming SP206U with a Not I fragment of ura4:.ADE2 obtained from pVIN (22). SP42N17 (h90 ade6-210 leul-32 ura4::adh1-cdc42[T17N]-ADE2) was constructed by transforming the S.Abbreviations: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; GST, glutathione Stransferase.

Mammalian cells redirect their movement in response to changes in the physical properties of their extracellular matrix (ECM) adhesive scaffolds, including changes in available substrate area, shape, or flexibility. Yet, little is known about the cell's ability to discriminate between different types of spatial signals. Here we utilize a soft-lithography-based, microcontact printing technology in combination with automated computerized image analysis to explore the relationship between ECM geometry and directional motility. When fibroblast cells were cultured on fibronectin-coated adhesive islands with the same area (900 micrometers2) but different geometric forms (square, triangle, pentagon, hexagon, trapezoid, various parallelograms) and aspect ratios, cells preferentially extended new lamellipodia from their corners. In addition, by imposing these simple geometric constraints through ECM, cells were directed to deposit new fibronectin fibrils in these same corner regions. These data indicate that mammalian cells can sense edges within ECM patterns that exhibit a wide range of angularity and that they use these spatial cues to guide where they will deposit ECM and extend new motile processes during the process of directional migration.

Characterizing the genetic alterations leading to the more aggressive forms of estrogen receptor positive (ER+) breast cancers are of critical significance in breast cancer management. Here we identify recurrent rearrangements between estrogen receptor gene ESR1 and its neighbor CCDC170, which are enriched in the more aggressive and endocrine-resistant luminal-B tumors, through large-scale analyses of breast cancer transcriptome and copy number alterations. Further screening of 200 ER+ breast cancers identifies eight ESR1-CCDC170 positive tumors. These fusions encode N-terminally truncated CCDC170 proteins (ΔCCDC170). When introduced into ER+ breast cancer cells, ΔCCDC170 leads to markedly increased cell motility and anchorage-independent growth, reduced endocrine sensitivity, and enhanced xenograft tumor formation. Mechanistic studies suggest that ΔCCDC170 engages Gab1 signalosome to potentiate growth factor signaling and enhance cell motility. Together, this study identifies neoplastic ESR1-CCDC170 fusions in a more aggressive subset of ER+ breast cancer, which suggests a new concept of ER pathobiology in breast cancer.

Summary eIF3 promotes translation initiation, but relatively little is known about its full range of activities in the cell. Here, we employed affinity purification and highly sensitive LC-MS/MS to decipher the fission yeast eIF3 interactome, which was found to contain 230 proteins. eIF3 assembles into a large supercomplex, the translasome, which contains elongation factors, tRNA-synthetases, 40S and 60S ribosomal proteins, chaperones, and the proteasome. eIF3 also associates with ribosome biogenesis factors and the importins-β Kap123p and Sal3p. Our genetic data indicated that the binding to both importins-β is essential for cell growth, and photobleaching experiments revealed a critical role for Sal3p in the nuclear import of one of the translasome constituents, the proteasome. Our data reveal the breadth of the eIF3 interactome and suggest that factors involved in translation initiation, ribosome biogenesis, translation elongation, quality control, and transport are physically linked to facilitate efficient protein synthesis.

Compartment-specific Ras signaling is an emerging paradigm that may explain the multiplex outputs from a single GTPase. The fission yeast, Schizosaccharomyces pombe, affords a simple system in which to study Ras signaling because it has a single Ras protein, Ras1, that regulates two distinct pathways: one that controls mating through a Byr2-mitogen-activated protein kinase cascade and one that signals through Scd1-Cdc42 to maintain elongated cell morphology. We generated Ras1 mutants that are restricted to either the endomembrane or the plasma membrane. Protein binding studies showed that each could interact with the effectors of both pathways. However, when examined in ras1 null cells, endomembrane-restricted Ras1 supported morphology but not mating, and, conversely, plasma membrane-restricted Ras1 supported mating but did not signal to Scd1-Cdc42. These observations provide a striking demonstration of compartment-specific Ras signaling and indicate that spatial specificity in the Ras pathway is evolutionarily conserved.cancer ͉ Cdc42 ͉ Int6 ͉ mitogen-activated protein kinase ͉ eIF3

Quiescent, full-grown Xenopus oocytes, which are arrested at the G2/M border of meiosis, contain an inactive 42-kDa mitogen-activated protein kinase (p42MAPK) that is activated when oocytes are stimulated to resume the meiotic cell cycle. MATERIALS AND METHODSConstruction of Escherichia coli Expression Vectors. PCRs (Taq polymerase, Perkin-Elmer) (25) were performed by Vincent Jung to generate Ha-ras genes with the desired mutations and BamHI as the cloning site (underlined). The 5' oligonucleotide used was 5'-CCCTGAGGjATCCATGAC-GAATATAAG-3' which created a BamHI site (underlined) upstream of the initiation codon (in bold); the 3' oligonucleotide was 5'-CCTCAGGATCCTCAGGAGAGCACA-CACTT-3'. The 3' oligonucleotide used to mutate the membrane localization site was 5'-CCTCAGGATCCTCAG-GAGAGCACACGCCTT-3' with a point mutation (in bold) to change Cys'86 (CAC) to Arg (CGC). The PCR templates were derived from [Gly12]Ha-ras (wild type) and [Vall2]Ha-ras cDNAs (26). These PCR products were first cloned into ADHI promoter-driven expression vectors for Saccharomyces cerevisiae (27) to confirm their biological activity (28). Expression of [Gly12]Ha-ras in yeast suppressed the temperature sensitivity of a strain containing the temperaturesensitive mutation ras2ts. Expression of [Val'2]Ha-ras conferred heat shock sensitivity on a wild-type strain and suppressed the lethality of a strain containing cdc25ts. Finally, expression of [Val"2,Arg186]Ha-ras suppressed the heat shock sensitivity of a strain containing [Val19]RAS2 (29). The fragments were cloned into pTrcHis (Invitrogen, San Diego) and expressed as fusion proteins with N-terminal oligohistidine (30,31).Purification of Intact and Histidine-Tagged Fusion Proteins. Ha-Ras proteins were purified as described (32). His-tagged proteins were prepared according to ref. 31 and manufacturer's instructions (Invitrogen). The His6-Ras proteins were solubilized in sonication buffer [10 mM Tris HCI, pH 7.5/10 mM MgCl2/50 mM NaCI/1 ,M phenylmethanesulfonyl fluoride/20 AM leupeptin/aprotinin (2 ,tg/ml)/2 AM pepstatin/ 0.5% (wt/vol) dodecyl f3-D-maltoside (Boehringer Mannheim)]. The protein was bound to Pro-Bond resin (Invitrogen) overnight at 4°C and eluted with increasing imidazole concentrations. Fractions containing His-tagged proteins, identified by immunoblotting, were dialyzed against microinjection buffer (20 mM Hepes, pH 7.4/20 mM NaCl/4 mM MgCl2/0.5 mM 2-mercaptoethanol). His6-Ras preparations (at least 70% pure) were concentrated using Centriprep-10 concentrators (Amicon) and stored at -70°C in microinjecAbbreviations: MAP kinase, mitogen-activated protein kinase; p42MAPK, 42-kDa MAP kinase; MPF, maturation-promoting factor; GVBD, germinal vesicle breakdown; MBP, myelin basic protein; CHX, cycloheximide. 9831The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Yin6 is a yeast homolog of Int6, which is implicated in tumorigenesis. We show that Yin6 binds to and regulates proteasome activity. Overexpression of Yin6 strengthens proteasome function while inactivation weakens and causes the accumulation of polyubiquitinated proteins including securin/Cut2 and cyclin/Cdc13. Yin6 regulates the proteasome by preferentially interacting with Rpn5, a conserved proteasome subunit, and affecting its localization/assembly. We showed previously that Yin6 cooperates with Ras1 to mediate chromosome segregation; here, we demonstrate that Ras1 similarly regulates the proteasome via Rpn5. In yeast, human Int6 binds Rpn5 and regulates its localization. We propose that human Int6, either alone or cooperatively with Ras, influences proteasome activities via Rpn5. Inactivating Int6 can lead to accumulation of mitotic regulators affecting cell division and mitotic fidelity.