FIGURE SUMMARY
Title

Alternative splicing generates an isoform of the human Sef gene with altered subcellular localization and specificity

Authors
Preger, E., Ziv, I., Shabtay, A., Sher, I., Tsang, M., Dawid, I.B., Altuvia, Y., and Ron, D.
Source
Full text @ Proc. Natl. Acad. Sci. USA

Schematic representation of hSef-a and hSef-b-(A) Residues unique to each Sef isoform are shown in bold letters. *, signal for secretion; arrows, potential N-linked glycosylation sites; black box, transmembrane domain; Y, putative tyrosine phosphorylation site; light and dark gray boxes, Ig-like domain and the IL-17R-like domain. (B) Identification of hSef products. HEK 293 cells were transiently transfected with control empty vector (lane 1) or Myc-tagged hSef-b or hSef-a vectors (lanes 2 and 3, respectively). Equal amounts of protein were subjected to gel electrophoresis, and hSef products were identified by immunoblotting with α-myc antibody. (C) In vitro translation of hSef-b. Lane 1, translation in the presence of hSef-b vector; lane 2, translation in the presence of an empty vector. The assay was performed as described in Materials and Methods. Products were analyzed by SDS/PAGE and visualized by phosphoimaging. (D) Subcellular localization of hSef isoforms. HEK 293 cells, transfected with myc-tagged hSef-a (a and b) or myc-tagged hSef-b (c and d) expression vectors, were fixed 48 h later and stained with α-myc (red; a and c) or α-hSef antibody (green; b and d). Nuclei were counterstained with bisbenzimide (blue).

Expression pattern of hSef isoforms. Expression was determined by RT-PCR by using total RNA from the indicated human tissues and human primary cells. Amplification was performed with primers derived from hSef common region (A) and with specific primers for hSef-a (B) or hSef-b (C). Amplification of GAPDH transcript (D) compares RNA levels in each sample. Adrenal m. and adrenal c. denote adrenal medulla and cortex, respectively. A.E. cells, primary aortic endothelial cells. HUVEC, human umbilical vein endothelial cells; F, fetal; A, adult; NC, negative control. Templates for positive controls (PC) are plasmids containing hSef-a (A and B), hSef-b (C), or GAPDH (D).

(A) hSef-b suppresses colony formation in NIH 3T3 cells. Cells were stably transfected with expression vector bearing hSef-b (pCDNA/hSef-b) or an empty vector (pCDNA). After 1 day, cells were diluted (1/25) and marker-selected for 2–3 weeks. Resistant clones were counted at the end of the selection process (five plates for each vector). The results are representative of three experiments. (B) Induced expression of hSef-b in the tet-off NIH 3T3 cells. Cells were grown in 10% serum in the presence and absence of tet. After 24 h, the cells were lysed, and hSef-b expression was analyzed by immunoblotting with hSef-specific antibodies. Control cultures denote parental cells transfected with an empty pTet-splice vector. (C) The effect of hSef-b on apoptosis. NIH 3T3/hSef-b cells were grown for 48 h in the presence or absence of tet or in the absence of tet and serum (–tet, –NBS). Cells were washed and fixed, and apoptosis was then evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. (D and E) hSef-b inhibits the mitogenic activity of FGF2. Confluent cultures of control cells (D) or hSef-b-expressing cells (E) were serum starved and grown in the presence or absence of tet for 24 h. FGF2 was added at the above-indicated concentrations. [3H]Thymidine incorporation assay was performed as described (24, 26).

hSef-b reduces cyclin D1 levels and inhibits the Ras/MAPK pathway in FGF2-stimulated cells. Control and hSef-b inducible NIH 3T3 cells were serum starved for 24 h in the presence and absence of tet and then stimulated with FGF2 (20 ng/ml) for the indicated time periods. (A) The effect of hSef-b on the levels of cyclin D1 protein. The levels of cyclin D1 were evaluated in total cell lysates over 20 h of stimulation. Cyclin D1 and cdk proteins were analyzed by immunoblotting with anti-D1 monoclonal antibody or rabbit anti-cdk4. (B) The effect of hSef-b on FGF2-induced signaling pathways. Equal amounts of total cell lysates were analyzed by immunoblotting. The membranes were successively incubated with the indicated antibodies. (C) Activation of ERK1/2 MAPK in the control cultures. In cells transfected with control vector, there was no effect of tet removal on ERK1/2 activation. Each experiment was repeated at least twice and by using two independent clones of hSef-b inducible cells. P-ERK1/2, P-Akt, P-p38, and P-MEK1/2 are antibodies directed against the phosphorylated (P) form of the kinases.

Coimmunoprecipitation of Sef isoforms and FGFR1. HEK 293 cells were transfected with the indicated constructs, and whole-cell lysates were either immunoblotted with α-FGFR1 (H76) or α-hSef antibodies or immunoprecipitated with α-myc antibody and immunoblotted with H76 or α-hSef antibodies.

The spectrum of inhibitory activity of hSef-b. Mitogenic assay in control cultures (A) or cells expressing hSef-b (B) was performed as described in the legend to Fig. 3. Fold increase (FI) in biological activity was calculated by dividing cpm values obtained in the presence of the indicated stimulators with those obtained in 0.2% serum alone. Percent [3H]thymidine incorporation is relative to FI obtained in cultures stimulated with 10% serum in the presence of tet that was set at 100%. The concentrations of FGFs, insulin, epidermal growth factor, and serum are those that gave rise to a maximal biological response. F, FGF; INS, insulin. (C) hSef-b inducible NIH 3T3 cells were serum starved for 24 h in the presence and absence of tet and then stimulated with PDGF (20 ng/ml) for the indicated time periods. Equal amounts of total cell lysates were analyzed by immunoblotting with anti-P-ERK1/2 and anti-ERK antibodies. These experiments were repeated at least three times and by using two independent hSef-b-expressing clones.

Acknowledgments
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