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CELL GROWTH AND DEVELOPMENT

Cell Cycle-Regulated Processing of HEF1 to Multiple Protein Forms Differentially Targeted to Multiple Subcellular Compartments

Susan F. Law, Yu-Zhu Zhang, Andres J. P. Klein-Szanto, Erica A. Golemis
Susan F. Law
Division of Basic Science and
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Yu-Zhu Zhang
Division of Basic Science and
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Andres J. P. Klein-Szanto
Division of Medical Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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Erica A. Golemis
Division of Basic Science and
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DOI: 10.1128/MCB.18.6.3540
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  • Fig. 1.
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    Fig. 1.

    Transfection of full-length HEF1 cDNA into HeLa cells produces p105HEF1, p115HEF1, p65HEF1, and p55HEF1. (A) HEF1 isoforms detected by α-HEF1-SB antisera. HeLa cells were either mock transfected (M) or transfected with pCMV-HEF1 (HEF1), and crude lysates were analyzed by SDS-PAGE and Western blot analysis, with either α-HEF1-R1 (R1) or α-HEF1-R2 (R2) antiserum for visualization of HEF1 isoforms. Numbers outside the lanes indicate molecular mass in kilodaltons. (B) Epitope mapping detects p55HEF1 and p65HEF1. HeLa cells were either mock transfected (m) or transfected with pCMV-HEF1 (HEF1). Cell lysates were resolved by SDS-PAGE and probed with four antibodies reacting with HEF1: α-p130Cas-B, α-p130Cas-F, α-p130Cas-TL, and α-HEF1-R1 (described in Materials and Methods). (C) Locations of epitopes for HEF1-reactive antisera and predicted p55HEF1-p65HEF1 boundaries. Shown are the locations of epitopes for the antibodies α-p130Cas-B (B), α-p130Cas-F (F), α-p130Cas-TL (TL), and α-HEF1 (SB) on the full-length 834-aa HEF1 coding sequence; details are in Materials and Methods. Assignment of endpoints for p55HEF1 and p65HEF1 is approximate, based on patterns of reactivity demonstrated in panel B.

  • Fig. 2.
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    Fig. 2.

    HEF1 protein isoforms are abundant in breast, lung, and lymphoid cell lines and in primary bronchial tissue. (A) Antibody α-HEF1 was used to visualize HEF1 protein in cell lysates prepared from multiple cell lines: MCF-7 (lane 1), BT474 (lane 2), A549 (lane 3), SKLU (lane 4), H9 (lane 5), Jurkat (lane 6), Nalm-6 (lane 7), mock-transfected HeLa (lane M), and HeLa transfected with pcDNA3-HEF1 (lane HEF1). For the M and HEF1 lanes, significantly less lysate was added. Numbers at right indicate molecular mass in kilodaltons. (B) Immunohistochemical detection of HEF1 in the human bronchiolar epithelium with antibody α-HEF1. Note that the immunostain is localized in the cytoplasm of the epithelial cells lining the lumen of the bronchiole. The stained nuclei seen in the epithelium and wall of this pulmonary structure are stained with hematoxylin and are HEF1 negative. The panel shows immunoperoxidase and hematoxylin stain of a paraffin section. Magnification, ca. × 52.

  • Fig. 3.
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    Fig. 3.

    Induction of p105HEF1 and p115HEF1 following serum stimulation in MCF-7 cells. MCF-7 cells were brought to quiescence by starvation for serum (St.), then the medium was changed to DMEM–10% calf serum, and lysates were made at the times indicated after medium addition (15 or 30 min and 1, 2, 4, 6, or 24 h). Crude cell lysates were resolved by SDS-PAGE, and HEF1 species were visualized by α-HEF1; cross-reactive p95 confirms the equal loads of lanes. Numbers at right and left indicate molecular mass in kilodaltons.

  • Fig. 4.
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    Fig. 4.

    Induction of HEF1 but not p130Cas during reentry into cell cycle. Crude lysates were made from either exponentially growing MCF-7 cells (Expo.) or MCF-7 cells synchronized by thymidine block and released for the number of hours noted (0, 1, 3, 6, 9, 12, or 24). Lysates were immunoprecipitated by either control (---), α-HEF1 (H), or the α-p130Cas-TL antibodies (C). Lysates were resolved by SDS-PAGE and probed in Western blot analysis with α-HEF1 antibodies (A); the blot was then stripped and reprobed with α-p130Cas-TL antibody to p130Cas (B). Note that, although antibody to p130Cas is additionally cross-reactive with HEF1, because of the different electrophoretic mobilities of the two proteins, HEF1- or p130Cas-derived species can be readily discriminated by superimposing enhanced chemiluminescence-visualized Western blots sequentially probed with the two antibodies. IP, immunoprecipitation. Numbers at left of each panel indicate molecular mass in kilodaltons.

  • Fig. 5.
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    Fig. 5.

    (A and B) p105HEF1 and p115HEF1are tyrosine phosphorylated to comparable extents. Crude lysates were made from MCF-7 cells synchronized by thymidine block and released for the number of hours noted (0, 1, 3, 6, 9, 12, or 24). Lysates were immunoprecipitated either by control (---) or α-HEF1 (H) antibody. These lysates were resolved by SDS-PAGE and probed in Western blot analysis with the RC20 antibody to phosphotyrosine (A); following stripping, the blot was reprobed with α-HEF1 antibody (B). (C) p115HEF1 levels are reduced by treatment with lambda phosphatase. Lysates from HeLa cells transfected with pCMV-HEF1 were treated either with (+) or without (--) lambda phosphatase, resolved by SDS-PAGE, and visualized with α-HEF1. Numbers in the margins of each panel represent molecular mass in kilodaltons.

  • Fig. 6.
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    Fig. 6.

    (A) Specific appearance of p55HEF1 in mitotic shakeoff. Crude lysates were made from either exponentially growing cells (Ex) or cells synchronized by thymidine block and released for the number of hours notes (0, 1, 3, 6, 9, 12, or 24). At 9 h following release, a mitotic shakeoff was prepared (M). As a control to indicate the position of the p105HEF1, p115HEF1 and p55HEF1 species, HeLa cell lysates (known to express relatively low levels of endogenous HEF1) either mock transfected (--) or transfected with pcDNA3-HEF1 (HEF1) were also analyzed. The lysates were resolved by SDS-PAGE and probed in Western blot analysis with α-HEF1 antibodies. (B) Endogenous p55HEF1 can be immunoprecipitated by antibody to HEF1. Five hundred micrograms of whole-cell lysate prepared from mitotic shakeoffs of MCF-7 cells 9 h after release from thymidine block was used for immunoprecipitation with either control (--) or α-HEF1 antibodies (H), followed by visualization with α-HEF1. Note that the prominent diffuse band migrating at ∼59 to 64 kDa represents the immunoglobulin blob generally detected in immunoprecipitations. (C) p55HEF1 is abundant in nocodazole-blocked cells and is replaced by p105HEF1 and p115HEF1 following release. MCF-7 cells were blocked in mitosis by incubation in 1 μM nocodazole for 14 h and released. Cell lysates were prepared from cells at 1, 4, 8, 12, and 24 h after release. As before, as a control for sizes, an aliquot of mock-transfected (M) or HEF1-transfected (HEF1) HeLa cells was included. Lysates were resolved by SDS-PAGE and probed in Western blot analysis with α-HEF1. (D) Production of p55HEF1 results from a cleavage of the full-length HEF1 protein at a DLVD motif located at aa 360 to 363. PCR-based mutagenesis was used to alter DLVD360–363 to DLVA in the context of the full-length 834-aa HEF1 coding sequence, and the mutant was cloned into the pCMV expression vector. Whole-cell lysates from HeLa cells mock transfected (M), transfected with pcDNA3-HEF1 (H), or transfected with pcDNA3-HEF1DLVA (DLVA) were visualized with α-HEF1 antibodies. Numbers in the margins of each panel indicate molecular mass in kilodaltons.

  • Fig. 7.
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    Fig. 7.

    The p105HEF1 and p115HEF1species are predominantly cytoplasmic. MCF-7 cells were brought to quiescence by serum deprivation and then refed with DMEM-10% serum, and cell lysates were made at the times indicated (0, 12, and 24 h). Lysates were separated into nuclear (N), cytoplasmic (C), and combined membrane (M) fractions as described in Materials and Methods. Fractions were resolved by SDS-PAGE and probed in Western blot analysis with α-HEF1. Numbers at right indicate molecular mass in kilodaltons.

  • Fig. 8.
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    Fig. 8.

    p55HEF1 associates with the mitotic spindle. Cells in prophase (A to C), metaphase (D to F), late anaphase (G to I), and cytokinesis (J to L) were stained with α-HEF1-R2 antiserum to HEF1 and visualized with rhodamine (A, D, G, and J) or stained with α-tubulin and visualized with fluorescein isothiocyanate (B, E, H, and K); a merged image is shown in panels C, F, I, and L, with HEF1-tubulin colocalization shown in yellow. Note punctate staining of α-HEF1-R2, which does not colocalize with microtubules in nonmitotic cells. Focal adhesion staining is not visible in this optical section, although it is clearly present in nonmitotic cells. Identical results were obtained with α-HEF1-R2 in a single stain, excluding bleedover from tubulin as a source of HEF1 staining (data not shown).

Tables

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  • Table 1.

    HEF1 associates specifically with the mitotic regulatory protein hsDim1pa

    LexA fusionValue for construct
    hsDim1p-activation domainActivation domain vector
    Specificity of interaction
     HEF1102–229 572 ± 32421 ± 9
     p85ΔSH25 ± 24 ± 1
     SHC12 ± 39 ± 3
     IRS13 ± 14 ± 4
     Insulin receptor3 ± 14 ± 1
     Bicoid7 ± 46 ± 3
    Mapping of site on HEF1
     HEF11–105 5 ± 16 ± 1
     HEF11–124 153 ± 328 ± 1
     HEF11–154 109 ± 2213 ± 2
     HEF1102–175 20 ± 45 ± 2
     HEF1102–229 640 ± 4529 ± 2
     HEF1125–229 50 ± 518 ± 2
     HEF1151–229 45 ± 822 ± 4
     Bicoid4 ± 14 ± 0
     Alone4 ± 16 ± 0
    • ↵a EGY48 yeast cells were transformed with the pJK103 LexA operator-LacZ reporter and indicated combinations of LexA-fused and activation-domain-fused (pJG4-5) proteins. β-Galactosidase activities were calculated as the average values for six separate colonies for each data point. Numbers shown are the β-galactosidase values (± standard deviations) obtained for different constructs.

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Cell Cycle-Regulated Processing of HEF1 to Multiple Protein Forms Differentially Targeted to Multiple Subcellular Compartments
Susan F. Law, Yu-Zhu Zhang, Andres J. P. Klein-Szanto, Erica A. Golemis
Molecular and Cellular Biology Jun 1998, 18 (6) 3540-3551; DOI: 10.1128/MCB.18.6.3540

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Cell Cycle-Regulated Processing of HEF1 to Multiple Protein Forms Differentially Targeted to Multiple Subcellular Compartments
Susan F. Law, Yu-Zhu Zhang, Andres J. P. Klein-Szanto, Erica A. Golemis
Molecular and Cellular Biology Jun 1998, 18 (6) 3540-3551; DOI: 10.1128/MCB.18.6.3540
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KEYWORDS

Cell Compartmentation
cell cycle
Phosphoproteins

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