Characterization of SSeCKS, a Src-suppressed C kinase substrate, involved in tumor suppression, growth arrest and scaffolding.
Item
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Title
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Characterization of SSeCKS, a Src-suppressed C kinase substrate, involved in tumor suppression, growth arrest and scaffolding.
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Identifier
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AAI9917673
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identifier
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9917673
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Creator
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Lin, Xueying.
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Contributor
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Adviser: Irwin H. Gelman
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Date
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1999
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Biology, Molecular | Health Sciences, Oncology
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Abstract
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Identification and characterization of novel genes involved in the regulation of mitogenesis and suppression of oncogene-induced tumor formation is of particular interest. Using a modified PCR-based subtractive hybridization technique developed previously, clone 322 was isolated whose transcripts were down-regulated in v-src transformed NIH3T3 cells. Later, the 322 product was identified as a PKC substrate/binding protein. Thus, the 322 gene product was named SSeCKS (S&barbelow;rc S&barbelow;uppre&barbelow;ssed C&barbelow; K&barbelow;inase S&barbelow;ubstrate).;SSeCKS transcript levels are suppressed in v-src, v- ras, but not in v-raf transformed rodent fibroblasts. The down-regulation of SSeCKS is dependent on Src kinase activity and is not a non-specific consequence of cell transformation. The finding that the down-regulation of SSeCKS correlates with anchorage-independent growth, and that SSeCKS decreases v-src-induced colony formation in soft agar, suggests that SSeCKS may encode a potential tumor suppressor. To study the putative tumor suppressive function of SSeCKS, conditionally transformed NIH3T3 cell lines (expressing ts72v-src) with tetracycline-regulated SSeCKS expression were developed.;Forced expression of SSeCKS suppresses parameters of v-src-induced malignant transformation, such as increased refractility, increased saturation density, growth factor- and anchorage-independent growth. The tumor suppressive effects of SSeCKS are not mediated by inhibition of Src kinase activity and MAPK (ERK2 and JNK) activity, nor by inhibition of Src and ERK2 expression. Interestingly, ERK2 activity is enhanced in v-src transformed cells upon SSeCKS overexpression. Forced expression of SSeCKS also restores cytoskeletal architecture such as formation of stress fibers and focal adhesion plaques, which are disrupted in transformed cells. These data suggest that SSeCKS functions as a tumor suppressor, likely by its ability to control cytoskeletal architecture and regulate cell signaling.;Forced expression of SSeCKS in untransformed NIH3T3 cells induces GI arrest, which correlates with suppression of cyclin D1. The decreased expression of cyclin D1 likely results from a suppression of serum-inducible ERK2 activity. Interestingly, ectopic expression of cyclin D1 fails to rescue SSeCKS-induced growth arrest in G1. SSeCKS sequence contains two potential cyclin-binding (CY) motifs which mediate association of cyclin D1 and SSeCKS in vitro . Overexpression of SSeCKS results in the sequestration of cyclin D1 in the cytoplasm, very likely via the CY motif-mediated binding. These data strongly support the notion that SSeCKS controls G1-S progression by inhibiting cyclin D1 expression and/or sequestering cyclin D1 in the cytoplasm.;Along with the progressive understanding of SSeCKS function, other groups showed that SSeCKS is highly homologous to AKAP250 (gravin), a mammalian scaffolding protein. Scaffolding proteins interact with more than one enzyme simultaneously and direct their associated molecules to specific subcellular compartments. By doing so, scaffolding proteins coordinate different signals to tightly regulate physiological functions spatially and temporally. SSeCKS has been shown to function as an anchoring protein for PKA. SSeCKS is a PKC substrate/binding protein, a calmodulin-binding protein, and a cyclin D1-interacting protein in vitro. Taken together, it is proposed that SSeCKS controls transformation and cell cycle progression by acting as a scaffolding protein.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
