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Pseudo-phosphatase Sbf1 contains an N-terminal GEF homology domain that modulates its growth regulatory properties

Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA
^* Author for correspondence (e-mail: mcleary{at}stanford.edu )

View larger version (57K): [in a new window]   Fig. 1. Sbf1 is a unique member of the myotubularin family of phosphatases. (A) Schematic representation of Sbf1 and myotubularin-related proteins. A highly conserved region that spans the PTP consensus motif is indicated by black rectangles; PTP motifs containing germline mutations are indicated with half white/half black rectangles. Gray oval in Sbf1 indicates CRS domain conserved in various signaling proteins. Gray box at Sbf1 N terminus denotes 80 amino acids of additional similarity with the N terminus of Rab3GEF. Start and stop codons are indicated in proteins where they are known. (B) Sequence alignment of the N-terminal CRS domain in Sbf1 reveals similarity with several known and hypothetical proteins. Black boxes indicate identical residues; shaded boxes indicate conservative amino acid substitutions. AA481292, AL161790 and AI158686 are unique human or mouse ESTs. C12714 is a predicted C. elegans protein. Sequences for CRAG, ST5, Rab3GEF, Rab6BP have been reported previously (Xu et al., 1998; Janoueix-Lerosey et al., 1995; Wada et al., 1997). (C) Anti-Sbf1 western blot analysis of NIH 3T3 cells stably transfected with wild-type Sbf1 or vector alone.

View larger version (58K): [in a new window]   Fig. 2. Wild-type Sbf1 lacks oncogenic effects in NIH 3T3 cells. (A) Soft agar assays were performed using NIH 3T3 cell lines expressing the indicated constructs. Data are the average of three experiments performed in triplicate. (B) Representative colony growth in soft agar is shown for the cell lines tested. (C) Western blot analysis using an anti-Sbf1 monoclonal antibody demonstrates comparable expression of wild-type Sbf1 and Sbf1N750 in stably transfected NIH 3T3 cells.

View larger version (25K): [in a new window]   Fig. 3. Structure and expression of mutant Sbf1 proteins. (A) A schematic depiction of wild-type Sbf1 is shown at the top. Bars depict amino acid compositions of various Sbf1 deletion mutants. (B) Western blot analysis with an anti-Sbf1 monoclonal antibody demonstrates equivalent expression of Sbf1 deletion constructs in stably transfected NIH 3T3 cell lines.

View larger version (26K): [in a new window]   Fig. 4. Oncogenic activation of Sbf1 by N-terminal deletions. (A) Representative focus-forming assay shows that the N111 mutant, but not wild-type, Sbf1 induces foci in monolayers of stably transfected NIH 3T3 cells. (B) Bar graph indicates focus-forming activity of mutant Sbf1 proteins. Data are the average of three experiments performed in triplicate.

View larger version (65K): [in a new window]   Fig. 5. Forced expression of wild-type Sbf1 inhibits proliferation and induces morphological changes in NIH 3T3 cells. (A) Growth curves show the increase in cell numbers for NIH 3T3 cells expressing wild-type or mutant Sbf1 constructs. Data are shown for a representative experiment. (B) Proliferation rates of stably transfected NIH 3T3 cells were determined by BrdU incorporation. Data represent the average and standard deviations from three experiments performed in duplicate. (C) Cytoskeletal architecture of NIH 3T3 cell stably transduced with wild-type Sbf1 (left) or vector (right). Green fluorescence corresponds to tubulin staining, which was revealed using primary anti-tubulin and secondary fluorescein isothiocyanate-conjugated antibodies. DAPI staining is shown in blue.

View larger version (77K): [in a new window]   Fig. 6. Wild-type Sbf1 and myotubularin are cytoplasmic proteins. (A) Western blot analyses of Sbf1, G6PD and c-Myb performed on nuclear (N) and cytoplasmic (C) fractions prepared from Raji cells show Sbf1 predominantly in the cyoplasmic fraction. (B) Immunohistochemical analysis of Sbf1 in tissue sections using an anti-Sbf1 monoclonal antibody shows specific cytoplasmic staining for Sbf1 in the cerebellum from wild-type (left) but not Sbf1-/- (right) mice. (C) NIH 3T3 cells were examined by immunofluorescence 48 hours after co-transfection of constructs expressing FLAG-tagged Sbf1 or MTM1. Green fluorescence corresponds to FLAG-tagged Sbf1 or MTM1 staining, which was revealed using primary anti-FLAG and secondary fluorescein isothiocyanate-conjugated antibodies.

View larger version (60K): [in a new window]   Fig. 7. N-terminal truncation of Sbf1 results in its partial nuclear localization. Subcellular localization of wild-type and mutant Sbf1 proteins was evaluated in stably transfected NIH 3T3 cell lines by immunofluorescence. Sbf1 proteins were detected using primary anti-Sbf1 and secondary fluorescein isothiocyanate-conjugated antibodies.

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