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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.