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

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.

Induction of p105^HEF1 and p115^HEF1 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.

Induction of HEF1 but not p130^Cas 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-p130^Cas-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-p130^Cas-TL antibody to p130^Cas (B). Note that, although antibody to p130^Cas is additionally cross-reactive with HEF1, because of the different electrophoretic mobilities of the two proteins, HEF1- or p130^Cas-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.

(A and B) p105^HEF1 and p115^HEF1are 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) p115^HEF1 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.

(A) Specific appearance of p55^HEF1 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 p105^HEF1, p115^HEF1 and p55^HEF1 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 p55^HEF1 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) p55^HEF1 is abundant in nocodazole-blocked cells and is replaced by p105^HEF1 and p115^HEF1 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 p55^HEF1 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 DLVD_360–363 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-HEF1_DLVA (DLVA) were visualized with α-HEF1 antibodies. Numbers in the margins of each panel indicate molecular mass in kilodaltons.

The p105^HEF1 and p115^HEF1species 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.