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S J Fashena

Publications and source records attributed to S J Fashena.

11 recordsLinked to original sources
PubMed2000

The continued evolution of two-hybrid screening approaches in yeast: how to outwit different preys with different baits.

The original two-hybrid system, an experimental approach designed to detect protein interactions, exploited the modular nature of many transcription factors. It has provided the intellectual and technical seed for the evolution of an array of innovative approaches, the application of which broadens the scope of experimentally feasible questions to include the interaction of proteins with diverse binding partners. The available array of modified and alternative approaches facilitates the analysis of complex cellular machinery and signaling networks that rely on multiple protein interactions. Such advances have facilitated the functional analysis of proteins on the genome level, a feat considered untenable a decade ago.

Animals↗
PubMed2000

Integrin signalling: a new Cas(t) of characters enters the stage.

Cellular morphology is determined by the organization of the intracellular actin cytoskeleton, which is influenced by external and internal cues. Focal adhesions are sites at which the actin cytoskeleton is linked to the extracellular matrix by integrin receptor complexes. In addition to providing structural tethering points for cells, integrin receptor complexes transduce signals that influence a broad range of cellular processes, including migration, proliferation, transformation and apoptosis. The Cas proteins (p130Cas, HEF1/Cas-L and Efs/Sin), a family of docking proteins containing multiple interaction domains, are important components of integrin receptor signalling and have been implicated in all of these processes.

Actins↗
PubMed2000

Signalling by adhesion receptors.

Over the past twenty years, intensive research has enabled us to identify components of specific signalling pathways downstream of an array of adhesion and growth-factor receptors. The first Gordon Research Conference on 'Signalling by Adhesion Receptors', which took place in Newport, Rhode Island, USA (23-28 July 2000), focused on these findings.

Animals↗
PubMed2000

The docking protein HEF1 is an apoptotic mediator at focal adhesion sites.

HEF1 (human enhancer of filamentation 1) is a member of a docking protein family that includes p130(Cas) and Efs. Through assembly of multiple protein interactions at focal adhesion sites, these proteins activate signaling cascades in response to integrin receptor binding of the extracellular matrix. The HEF1 protein is cell cycle regulated, with full-length forms cleaved in mitosis at a caspase consensus site to generate an amino-terminal 55-kDa form that localizes to the mitotic spindle. The identification of a caspase cleavage site in HEF1 led us to investigate whether HEF1 belongs to a select group of caspase substrates cleaved in apoptosis to promote the morphological changes characteristic of programmed cell death. Significantly, inducing expression of HEF1 in MCF-7 or HeLa cells causes extensive apoptosis, as assessed by multiple criteria. Endogenous HEF1 is cleaved into 65- and 55-kDa fragments and a newly detected 28-kDa form in response to the induction of apoptosis, paralleling cleavage of poly(ADP-ribose) polymerase and focal adhesion kinase (FAK); the death-promoting activity of over-expressed HEF1 is associated with production of the 28-kDa form. While the generation of the cleaved HEF1 forms is caspase dependent, the accumulation of HEF1 forms is further regulated by the proteasome, as the proteasome inhibitors N-acetyl-L-leucinyl-L-leucinyl-L-norleucinyl and lactacystin enhance their stability. Finally, the induction of HEF1 expression also increases Jun N-terminal protein kinase (JNK) activation, and activated JNK colocalizes with HEF1, implicating this pathway in HEF1 action. Based on these results, we propose that dysregulation of HEF1 and its family members along with FAK may signal the destruction of focal adhesion sites and regulate the onset of apoptosis.

Acetylcysteine↗
PubMed1999

Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain.

HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells. While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition. In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif. This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47. We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth. Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins. These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Transmembrane glycoprotein gp150 is a substrate for receptor tyrosine phosphatase DPTP10D in Drosophila cells.

We have begun to explore the downstream signaling pathways of receptor protein tyrosine phosphatases (RPTPs) that control axon guidance decisions in the Drosophila central nervous system. We have focused our studies on the adhesion molecule-like gp150 protein, which binds directly to and is an in vitro substrate for the RPTP DPTP10D. Here we show that gp150 and DPTP10D form stable complexes in Drosophila Schneider 2 (S2) cells and in wild-type larval tissue. We also demonstrate that the DPTP10D cytoplasmic domain is sufficient to confer binding to gp150. gp150 has a short cytoplasmic domain containing four tyrosines, all found within sequences similar to immunoreceptor family tyrosine-based activation motifs (ITAMs). We demonstrate that gp150 is tyrosine phosphorylated in wild-type larvae. In S2 cells, gp150 becomes tyrosine phosphorylated following incubation with PTP inhibitors or upon coexpression of the Dsrc tyrosine kinase. Phosphorylated Dsrc and an unknown 40-kDa phosphoprotein form stable complexes with gp150, thereby implicating them in a putative gp150 signaling pathway. When coexpressed with gp150, either full-length DPTP10D or its cytoplasmic domain mediates gp150 dephosphorylation whereas a catalytically inactive DPTP10D cytoplasmic domain does not. The neural RPTP DPTP99A can also induce gp150 dephosphorylation but does not coimmunoprecipitate with gp150. Taken together, the results suggest that gp150 transduces signals via phosphorylation of its ITAM-like elements. Phosphotyrosines on gp150 might function as binding sites for downstream signaling molecules, thereby initiating a signaling cascade that could be modulated in vivo by RPTPs such as DPTP10D.

Animals↗
PubMed1992

A poly(dA-dT) upstream activating sequence binds high-mobility group I protein and contributes to lymphotoxin (tumor necrosis factor-beta) gene regulation.

Lymphotoxin (LT; also known as tumor necrosis factor-beta) is a pleiotropic cytokine whose expression is tightly regulated in most cells and is repressed prior to activation signals. In some early B cells and Abelson murine leukemia virus-transformed pre-B-cell lines, LT mRNA is constitutively expressed. To examine the molecular regulation of the LT gene in a constitutively expressing cell line, we studied the Abelson murine leukemia virus-transformed lines PD and PD31. As demonstrated by primer extension analysis, constitutively expressed pre-B-cell-derived and inducibly expressed T-cell-derived LT mRNA were initiated at the same cap sites and predominant cap site utilization was conserved. Furthermore, we delineated an upstream activating sequence that was an important functional component of lymphotoxin transcriptional activation in PD and PD31 cells. The upstream activating sequence was localized to an essentially homopolymeric A + T-rich region (LT-612/-580), which was bound specifically by recombinant human high-mobility group I protein (HMG-I) and a PD/PD31 nuclear extract HMG-I (HMG-I-like) protein. The nuclear extract-derived HMG-I-like protein was recognized by anti-HMG-I antibody and bound to LT DNA to effect an electrophoretic mobility shift identical to that of bound recombinant human HMG-I. These findings implicate HMG-I in the regulation of constitutive lymphotoxin gene expression in PD and PD31 cells. HMG-I and HMG-I-like proteins could facilitate the formation of active initiation complexes by altering chromatin structure and/or by creating recognition sites for other activator DNA-binding proteins, some of which may be unique to or uniquely modified in these constitutive LT mRNA producers.

Base Sequence↗
PubMed1990

The murine lymphotoxin gene promoter. Characterization and negative regulation.

Murine lymphotoxin (LT; TNF-beta) gene upstream regulatory elements were identified by linking fragments of 5' DNA to the chloramphenicol acetyl transferase gene. Fragment LT1 (-293 to +77 in relation to the proximal cap site) exhibited promoter activity which drove CAT expression in transfected murine fibroblasts and T lymphomas. Primer extension analysis of endogenous LT message confirmed that LT1 contained the necessary elements required for promoter function. Promoter activity was not observed when LT2 (-662 to +77), LT3 (-1186 to +77), or LT3 delta AX (-1186 to +77 (delta-662/-269)) were ligated to the chloramphenicol acetyl transferase gene and transfected into fibroblasts or T lymphomas. At least one upstream repressor element is postulated to account for this promoter inhibition. In contrast to the results obtained with fibroblast and T cell transfectants, LT1 was inactive in the B cell transfectants A20 and P3X63. This suggests that some B cells express a repressor factor that inhibits the LT promoter and/or they lack the necessary positive regulatory factors.

Animals↗