INTRODUCTION

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Research over the last 3 decades suggests that environmental cues related to cell shape and cell-cell contacts play an important role in regulating the nuclear processes of cell cycle, gene expression, and cell viability (17, 20). Efforts to decipher routes for transmission of this environmental information have identified a number of mechanisms, generally involving the sequential activation of kinase cascades that are coordinated initially by assembly of cascade constituents at a cytoskeletal membrane focal site. For communication of signals across the nuclear membrane, in some cases particular activated kinases undergo translocation from the cytoplasm into the nucleus in response to proliferative signals (12). In other cases, signaling substrates such as transcription factors are initially cell membrane associated or cytoplasmic and become nuclear following activation (32). Based on the results of this study, we propose that the HEF1 protein might function in a novel pathway transmitting growth control signals between focal adhesions at the cell periphery and the mitotic spindle.

We recently described HEF1 (34), a novel member of a newly defined family of docking adapter proteins that also includes p130^Cas (for Crk-associated substrate) (59) and Efs (33) (also designated as Sin [1]). The members of this family have a common domain structure, with an amino-terminal SH3 domain, a large central domain encompassing multiple SH2-binding sites, and a carboxy-terminal domain conserved among all members of the HEF1-p130^Cas-Efs protein family but not similar to any other protein in GenBank. Because of the multiplicity of interactive domains encompassed in each of these proteins, they appear likely to play a central role in the coordination of growth signal processing.

As p130^Cas was the first characterized member of the p130^Cas-HEF1-Efs group, its study is most advanced and has established a framework for evaluation of the other family members. p130^Cas was initially described as a protein phosphorylated in response to Crk transformation via Abl and Src kinases (34, 49, 53). p130^Cas has been shown by immunolocalization to reside primarily at focal adhesions and along adhesion-proximal regions of stress fibers (52), where it associates with focal adhesion kinase (FAK) (53). During integrin-mediated adhesion to extracellular matrix, p130^Cas is phosphorylated along with other cytoskeletal proteins including FAK, paxillin, tensin, and cortactin (45, 52, 69). A central p130^Cas function in transformation of some cell types has been suggested by findings that expression of antisense constructs of p130^Cas in Ras- or Src-transformed NIH3T3 cells is sufficient to revert cells morphologically to a flat cell phenotype (4). Cumulatively, these data support a model in which p130^Cas acts together with paxillin, tensin, and talin to coordinate stable connections between integrin receptors and the actin cytoskeleton: as such, altered regulation of p130^Cas function by oncogenic phosphorylation might play a central role in establishing the characteristically altered cell morphologies of transformed cells.

In our prior characterization of HEF1, we determined that the protein shares not only a domain structure but also a number of functional properties with p130^Cas. The HEF1 SH3 domain confers association with FAK, and immunofluorescence detects a substantial quantity of HEF1 at focal adhesions (34). HEF1 associates with and is phosphorylated by Abl (34). HEF1 phosphorylation occurs in response to integrin ligation, resulting in association with the CrkL adapter protein (38) and signaling to C3G (3). However, HEF1 differs from p130^Cas in several potentially important ways. While p130^Cas is abundant in many cell types, HEF1 mRNA levels vary extensively between tissues. Based on a limited initial analysis, we had previously noted that HEF1 expression might be particularly abundant in epithelial cell populations (34), while we and others have determined that HEF1 is also highly expressed and is an important signaling intermediate in differentiating B (38) and T (41) cells. Further, in contrast to p130^Cas, which localizes exclusively to focal adhesions and the cytoplasm, immunofluorescence localizes a pool of the HEF1 population in the nucleus. The previous studies seeking to compare HEF1 and p130^Cas expression were, however, limited by the lack of reagents capable of distinguishing the two proteins, given their high degree of similarity.

In this study, we have sought to answer key questions about HEF1 expression and function. Using a panel of antibodies specific for HEF1, we have found that the full-length transfected HEF1 cDNA is processed into at least four specific protein products, p115^HEF1, p105^HEF1, p65^HEF1, and p55^HEF1. In contrast to earlier reports stating that HEF1 expression may be peculiar to hematopoietic cells, we demonstrate that the HEF1 protein is also abundant in cell lines and primary tissues derived from breast and lung epithelium. Expression and phosphorylation of the different HEF1 isoforms are cell growth and cell cycle regulated, with p105^HEF1 and p115^HEF1 appearing early following serum induction or release from the G₁/S boundary, while p55^HEF1 expression peaks at mitosis. In contrast, p130^Cas levels remain essentially constant throughout the cell cycle. Using phosphatase treatment, we demonstrate that p115^HEF1 arises from p105^HEF1 via phosphorylation; using targeted mutagenesis, we demonstrate that p55^HEF1 arises from full-length HEF1 as the result of cleavage at a DLVD candidate caspase motif. By cell fractionation and immunofluorescence, we show that, while p105^HEF1 and p115^HEF1 are predominantly cytoplasmic, p55^HEF1 associates with the mitotic spindle. Supporting this localization, a two-hybrid library screen with HEF1 resulted in the identification of the mitotic spindle regulatory protein Dim1p as a specific interactor with a region encompassed by p55^HEF1. These results suggest a novel model for HEF1 function, in which this protein integrates signals related to cell adhesion with those controlling progression of the cell cycle through mitosis.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Cell lines. MCF-7 and BT474 are human breast carcinoma cell lines. A549 and SKLU are human lung carcinoma cell lines. H9 is a human T-cell lymphoma cell line. Jurkat is a human acute T-cell leukemia cell line. Nalm-6 is a human pre-B-cell line. All cells were cultured under standard medium conditions prescribed by the American Type Culture Collection.

Plasmids. For overexpression of HEF1 in mammalian cells, the plasmid pcDNA3-HEF1 was used. To construct this plasmid, an assembled full-length HEF1 cDNA encoding the 834-amino-acid (aa) HEF1 protein (34) was inserted into the EcoRI-XhoI-cut vector pcDNA3 (Invitrogen), which expresses the protein from the cytomegalovirus promoter. pcDNA3-HEF1_DLVA was created by using oligonucleotide-directed PCR mutagenesis to create a DA change at aa 363 of full-length HEF1; this construct is otherwise identical to pCMV-HEF1, with the coding region of HEF1 completely sequenced.

Antibodies. The rabbit polyclonal antibody -HEF1-R1 (previously designated -HEF1-SB) has been described elsewhere (34). The antibody -HEF1-R2 was similarly raised with the same multiple-antigen peptide (MAP) (8) in a different rabbit. The affinity purification of the -HEF1 antibodies was performed as previously described (34). The rabbit polyclonal antibodies -p130^Cas-B (raised against p130^Cas aa 318 to 486) and -p130^Cas-F (raised against p130^Cas aa 670 to 896) were a generous gift of Amy Bouton and Thomas Parsons and have been described elsewhere (30). The mouse monoclonal antibody to p130^Cas (raised against aa 644 to 819) was purchased from Transduction Laboratories. aa 318 to 486 of p130^Cas (based on the original numbering of the 968-aa cDNA described in reference 59) maximally align with aa 163 to 318 of HEF1 (based on the original numbering of the 834-aa cDNA described in reference 34), aa 644 to 819 of p130^Cas maximally align with aa 489 to 686 of HEF1, and aa 670 to 896 of p130^Cas maximally align with aa 526 to 762 of HEF1. See Fig. 1C for a diagram of the above antibody epitopes.

Manipulation of cells. Transfection of cells with pcDNA3-HEF1 or pcDNA3-HEF1_DLVA was done with Lipofectamine (Gibco BRL), under conditions as described by the manufacturer.

Immunofluorescence and microscopy. Cells were plated on coverslips 24 h before being processed for immunofluorescence. All steps were carried out at room temperature. Coverslips were washed twice in 1× phosphate-buffered saline (PBS) and then fixed in 3.5% paraformaldehyde for 7 min. Cells were permeabilized for 5 min in buffer B (0.1 M Tris [pH 7.5], 1.5 M NaCl, 1% bovine serum albumin) with 0.2% Triton X-100. Coverslips were then washed for 5 min in buffer B. The primary antibody incubations were done for 1 h at room temperature. The antibody dilutions were made in buffer B and were identical to those used for immunoblotting. Coverslips were washed for 5 min in buffer B plus Triton X-100 followed by a 5-min wash in buffer B alone. Secondary antibody (either fluorescein isothiocyanate-conjugated anti-mouse [Jackson Laboratories] or biotinylated anti-rabbit [Vector Laboratories]) was diluted in buffer B and incubated with slides for 1 h. Cells were washed twice for 5 min in buffer B, and those that had biotin-conjugated secondary antibodies were incubated with Texas red streptavidin (Vector Laboratories) for 15 min. Following this, the cells were washed twice with buffer B and then mounted with antifade mounting medium from Vector Laboratories.

Immunoprecipitations. Cell lysates were prepared as follows with all steps done at 4°C. The cells were washed twice in 1× PBS and then scraped in buffer A-PTY buffer (50 mM HEPES [pH 7.5], 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 50 mM NaF, 10 mM Na₄P₂O₇ plus 1 mM Na₃VO₄, 1 mM phenylmethylsulfonylfluoride, 0.01 mg of aprotinin per ml, 0.01 mg of leupeptin per ml). The lysates were then spun in a microcentrifuge for 10 min at ~12,000 × g. Following this, the supernatant was removed and used in the immunoprecipitation experiments. Ten microliters of anti-HEF1 antiserum was incubated with 0.2 mg of cell lysate and 10 µl of 50% protein A-Sepharose beads (Sigma) overnight at 4°C. Beads were washed four times in 500 µl of buffer A and analyzed by immunoblotting. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels were used to resolve the products of the immunoprecipitation protocol.

Phosphatase treatment. HeLa cells were transfected with pcDNA3-HEF1 under conditions previously noted. The transfectants were harvested in buffer A (lacking NaF and Na₃VO₄), and the soluble supernatant was used for the experiments. Fifteen micrograms of lysate was treated with 2,000 U of lambda phosphatase (New England Biolabs) in the provided buffer for 30 min at 30°C.

Cell fractionation. Cell fractionation was performed essentially as described previously for HeLa cells (21). Protein concentration was assayed by a bicinchoninic acid assay (Pierce).

Immunohistochemistry. Selected adult and fetal human tissues fixed in 10% phosphate-buffered formaldehyde embedded in paraffin were used to evaluate the immunohistochemical localization of HEF1. Antigen retrieval was accomplished by boiling deparaffinized 5-µm-thick paraffin sections for 10 min in distilled water with a 750-W microwave oven at a low setting. After preincubation in goat serum and peroxidase blocking, the sections were incubated overnight at 4°C with a polyclonal rabbit anti-HEF1 antiserum (-HEF1-R1 or -HEF1-R2) diluted 1:100. Negative controls were incubated overnight in PBS. After the sections were washed with PBS for 10 min, the immunohistochemical reaction was accomplished with a commercial avidin-biotin-peroxidase kit (Vectastain Elite; Vector) with diaminobenzidine as chromogen. Note that while -HEF1-R1 recognized HEF1 more strongly by Western blot analysis, it also cross-reacted with at least one additional cellular protein, p95: -HEF1-R2 was specific for HEF1 in Western blot analysis but generally of lower affinity. Therefore, only production of identical patterns of recognition by both antibodies was described as a positive result.

Two-hybrid library screening and analysis of specificity of Dim1p-HEF1 interaction. A two-hybrid library screening was performed by standard protocols (26) with LexA-HEF1_102-229 expressed from the plasmid pEG202 (26) as bait in the strain EGY48 containing lexAop-LacZ reporter construct pJK103 to screen a pJG4-5 HeLa cDNA library (28). Approximately 5 × 10⁵ yeast transformants were screened; positives were confirmed by retransformation into naive EGY48/pJK103/LexA-HEF1_102-229 yeast. To map the interaction of the positive hsDim1p with HEF1 and to analyze the specificity of interaction, the pJG4-5-hsDim1p activation domain fusion was independently tested against LexA-HEF1_1-154, LexA-HEF1_151-229, LexA-HEF1_1-105; the series of GUS334 (LexA-p85SH2), GUS370 (LexA-SHC), GUS365 (LexA-IRS1), and GUS307 (IR), which contain identical fragments of p85, SHC, IRS1, and IR as described for pJG4-5 fusions in references 27 and 48 (gifts of T. Gustafson); and pRFHMI (LexA-bicoid) (18), a gift of Russ Finley. Expression of all fusion proteins was assayed by Western blot analysis, and -galactosidase values were calculated by a standard assay. Data points shown in Table 1 reflect the averages of six independent transformants, in a typical experiment out of two to three repetitions.

Sequence analysis. Sequence alignments were done with the GCG package of programs (15). PEST motifs were identified with the program PESTfind (55), available at http://www.at.embnet.org/embnet/tools/bio/PESTfind/.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

A single HEF1 cDNA is processed to p115, p105, p55, and p65. HEF1 and p130^Cas have similar electrophoretic mobilities, with each protein detected endogenously as multiple species migrating between ~105 and ~130 kDa. As a first step to characterizing HEF1 expression, we transfected HeLa cells with a plasmid in which the cytomegalovirus promoter expressed a full-length HEF1 cDNA. We then used two HEF1-directed affinity-purified polyclonal antisera (details in Materials and Methods) to determine the sizes of the resulting HEF1 protein products (Fig. 1A, R1 and R2).

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Transfection of full-length HEF1 cDNA into HeLa cells produces p105HEF1, p115HEF1, p65HEF1, and p55HEF1. (A) HEF1 isoforms detected by -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 -HEF1-R1 (R1) or -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: -p130Cas-B, -p130Cas-F, -p130Cas-TL, and -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 -p130Cas-B (B), -p130Cas-F (F), -p130Cas-TL (TL), and -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.

Abundant HEF1 expression in epithelial cells in vitro and in vivo. We had previously performed a limited analysis showing that the HEF1 mRNA was particularly abundant in tissues including human lung and placenta (34). However, one research group has claimed that expression of HEF1 protein is specific to lymphocytes (e.g., description of Cas-L [41]), and we and others have shown that HEF1 protein function is particularly required for signaling in B and T cells (2, 38, 60). To resolve the issue of the HEF1 expression pattern prior to analysis of the endogenous protein's regulation, we used -HEF1 antibody to directly examine cell lines and primary tissues for HEF1 protein (Fig. 2).

View larger version (125K): [in this window] [in a new window]   FIG. 2.   HEF1 protein isoforms are abundant in breast, lung, and lymphoid cell lines and in primary bronchial tissue. (A) Antibody -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 -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.

Serum stimulation and cell cycle progression regulate expression of p115^HEF1 and p105^HEF1 in G₁/S. We had hypothesized that HEF1 proteins might coordinate signaling between multiple cellular compartments (34). Other proteins with similar properties have been shown to be regulated in response to cell growth stimulation and during the cell cycle (5, 51). We therefore characterized HEF1 protein expression during serum stimulation and throughout cell cycle progression in synchronized cells. MCF-7 breast carcinoma cells were chosen for this analysis, based on our identification of abundant HEF1 expression in this cell line and the tractability of this line for cell cycle and morphological studies.

View larger version (29K): [in this window] [in a new window]   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 -HEF1; cross-reactive p95 confirms the equal loads of lanes. Numbers at right and left indicate molecular mass in kilodaltons.

View larger version (12K): [in this window] [in a new window]   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 (---), -HEF1 (H), or the -p130Cas-TL antibodies (C). Lysates were resolved by SDS-PAGE and probed in Western blot analysis with -HEF1 antibodies (A); the blot was then stripped and reprobed with -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.

p115^HEF1 is a phosphorylated modification of p105^HEF1. Both HEF1 and p130^Cas are known to be phosphorylated on tyrosines during integrin engagement and following oncogenic transformation (reviewed in reference 29). To determine whether p115^HEF1 might represent a tyrosine-phosphorylated form of p105^HEF1 and to determine whether phosphorylation of HEF1 was regulated during the cell cycle, we assessed HEF1 phosphotyrosine content in MCF-7 cells synchronized by thymidine block. HEF1 was immunoprecipitated at 0, 1, 3, 6, 9, 12, or 24 h following release from block, and the phosphotyrosine (Fig. 5A) and HEF1 (Fig. 5B) content of immunoprecipitates was determined by visualization with the antiphosphotyrosine antibody RC20 or -HEF1-R1, respectively. By this means, the p105^HEF1 and p115^HEF1 species appeared to be tyrosine phosphorylated to comparable levels and with similar time courses as the proteins accumulated.

View larger version (48K): [in this window] [in a new window]   FIG. 5.   (A and B) p105HEF1 and p115HEF1 are 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 -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 -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 -HEF1. Numbers in the margins of each panel represent molecular mass in kilodaltons.

Endogenous p55^HEF1 specifically appears at mitosis and arises through cleavage of the full-length HEF1 protein at a candidate caspase site. During assay of cell-synchronized MCF-7 cultures, we had noted the endogenous p55^HEF1 as a relatively minor species that appeared to increase in abundance at later time points in the cell cycle than the p105^HEF1 and p115^HEF1 species. We considered that the relative paucity of this species in contrast to our initial results with transfected HEF1 cDNA (Fig. 1) might reflect an actual difference in steady-state synthesis of p55^HEF1 from endogenous versus transfected full-length HEF1. Alternatively, if p55^HEF1 were specifically abundant in mitotic cells, this might also explain the underrepresentation, as the initial wash steps in the lysis procedure clearly removed many tenuously attached mitotic cells, and as mitosis is of such short duration (~30 min) that only a subpopulation of cells are in this phase of the cell cycle at any single time point. To test this second hypothesis, we directly analyzed lysates prepared from MCF-7 cells synchronized by thymidine block, released, and allowed to grow between 1 and 24 h, and in addition from cells prepared as mitotic shakeoffs from plates at 9 h after release from thymidine block (Fig. 6A), a time at which maximal numbers of cells should be entering mitosis (14). Results of this analysis were dramatic. Specifically, in cells prepared from mitotic shakeoff, p55^HEF1 was strongly induced, while p105^HEF1 and p115^HEF1 were diminished, supporting the idea that the endogenous p55^HEF1 protein might have a function specific to mitosis.

View larger version (21K): [in this window] [in a new window]   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 -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 -HEF1 antibodies (H), followed by visualization with -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 -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 -HEF1 antibodies. Numbers in the margins of each panel indicate molecular mass in kilodaltons.

p105^HEF1 and p115^HEF1 are predominantly cytoplasmic and associated with focal adhesions, whereas p55^HEF1 associates with the mitotic spindle. p130^Cas localizes to focal adhesions and the cytoplasm (52), whereas we had previously used immunofluorescence to detect -HEF1-reactive species at both the cell periphery and the nucleus (34). Based on our identification of multiple forms of HEF1 protein, with differential timing of expression, one possibility was that p55^HEF1, p105^HEF1, and p115^HEF1 might localize to different intracellular compartments. Accordingly, we performed cell fractionation followed by Western blot analysis with -HEF1-R1 to assay distribution of endogenous HEF1 proteins in MCF-7 cells (Fig. 7). By this means, p105^HEF1 and p115^HEF1 were found to localize predominantly to cytoplasmic fractions (which generally contain focal adhesion-associated proteins), with a minor population being detected in nuclear and membrane (combined Golgi and endoplasmic reticulum) fractions (visible on a longer exposure [data not shown]). This basic distribution did not change following growth induction.

View larger version (15K): [in this window] [in a new window]   FIG. 7.   The p105HEF1 and p115HEF1 species 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 -HEF1. Numbers at right indicate molecular mass in kilodaltons.

View larger version (141K): [in this window] [in a new window]   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 -HEF1-R2 antiserum to HEF1 and visualized with rhodamine (A, D, G, and J) or stained with -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 -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 -HEF1-R2 in a single stain, excluding bleedover from tubulin as a source of HEF1 staining (data not shown).

Association of HEF1 with Dim1p supports a p55^HEF1 function at the mitotic spindle. If the HEF1 protein possessed a function relevant to the mitotic spindle, this might reasonably be reflected in physical interaction between the region of HEF1 encompassed by p55^HEF1 and other proteins with spindle-associated functions. To investigate this possibility, we performed a two-hybrid library screen with LexA-fused HEF1_102-229, which excluded the HEF1 amino-terminal SH3 domain previously used for screening (34) but incorporated part of the previously defined SH2-binding site-rich substrate domain present in p55^HEF1. As analysis with the -phosphotyrosine antibody RC20 indicates that the p55^HEF1 species possesses little if any tyrosine phosphorylation (35) while the p105^HEF1 and p115^HEF1 species are tyrosine phosphorylated (Fig. 5), it seemed reasonable that, in the absence of tyrosine phosphorylation expected in yeast, p55^HEF1-interacting proteins might be enriched by this approach.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this study, we have identified a number of properties of the HEF1 gene that are likely to be important for understanding the activity of its protein products. First, we have shown that a single cDNA encoding an 834-aa protein species can be processed in vivo to produce at least four distinct protein species, p115^HEF1, p105^HEF1, p65^HEF1, and p55^HEF1. Second, we have demonstrated that at least three of these species (p115^HEF1, p105^HEF1, and p55^HEF1) are abundant endogenously and that their expression is differentially regulated during cell cycle progression. Third, we have established the mechanism of production of these species, with p115^HEF1 being a phosphorylated modification of p105^HEF1 and p55^HEF1 being cleaved at a DLVD caspase consensus from full-length HEF1. Fourth, we have shown that the different HEF1 species localize to different compartments of the cell, with p115^HEF1 and p105^HEF1 being predominantly cytoplasmic and associated with focal adhesions, while p55^HEF1 is associated with the mitotic spindle. Fifth, we have identified the human homolog of S. pombe Dim1p as a HEF1-interacting protein, providing a second link to spindle function. These results indicate that HEF1 may connect control of cell attachment to substrate with regulation of mitotic spindle in G₂/M, thus acting as a regulatory protein in the cellular decision to divide or initiate apoptosis.

p105^HEF1 and p115^HEF1 have recently become the topic of much study because of their implication as potentially key transducers of signaling related to integrin engagement during differentiation of B and T cells (2, 3, 38, 41, 60, 65). This study for the first time demonstrates that the abundance of these forms of HEF1 is cell cycle regulated, with expression being particularly high following initiation of cell division (Fig. 3 to 5). Our data indicate that p105 appears earlier in the cell cycle than does p115^HEF1, raising the possibility that a stage-specific kinase may phosphorylate p105^HEF1. However, this cannot be a complete explanation, as inspection of a panel of cell lines (Fig. 2) indicates that the relative abundance of the two forms varies between different lines of asynchronously growing cells. At this time, functional differences between the p105^HEF1 and p115^HEF1 forms of HEF1 remain unclear. Studies of the comparable p130^Cas doublet indicate that the slower-migrating, more phosphorylated form of this protein is more tightly associated with the cytoskeleton (54), suggesting that p115^HEF1 may function primarily at focal adhesions while the p105^HEF1 form may additionally function in other cellular compartments. Elsewhere, we present evidence that the HEF1 protein encompasses a carboxy-terminal helix-loop-helix motif and that the p105^HEF1, but not p115^HEF1, species of HEF1 forms endogenous complexes with helix-loop-helix proteins (36), raising the intriguing possibility that this form of the protein may bridge cell adhesion and transcriptional control related to cell differentiation in a manner similar to that demonstrated for -catenin (5).

Several points relative to the production of p55^HEF1 and p65^HEF1 are worthy of particular note. First, our data indicate that these species contain little if any tyrosine phosphorylation, despite the fact that p55^HEF1 encompasses the SH2-binding site-rich substrate domain and is derived from the p105^HEF1-p115^HEF1 species. This difference suggests that processing by cellular phosphatases (e.g., reference 24) may be enhanced either accompanying or subsequent to cleavage from p105^HEF1-115^HEF1, perhaps as a result of removal from the concentration of tyrosine kinases residing at focal complexes (43). Alternatively, part of the p105^HEF1-p115^HEF1 population may exist in a non-tyrosine-phosphorylated form, and this may serve as the precursor to p55^HEF1-p65^HEF1. Second, a particularly intriguing aspect of the production of p55^HEF1 is the implied involvement of caspases in the proteolytic cleavage of p105^HEF1 and/or p115^HEF1. The number of signaling molecules known to be targeted by caspase family members in apoptosis is rapidly increasing; at least some such targets include proteins either associated with HEF1, such as FAK (70), or in related signaling pathways, such as MEKK-1 (10). A much more limited set of molecules has been shown to be regulated by caspases in normally growing cells (e.g., reference 25), and the generality of this process has yet to be defined. The DLVD motif shown to be required for HEF1 cleavage is not conserved with p130^Cas, emphasizing that this processing and release of p55^HEF1 is likely to be a HEF1-specific phenomenon. Finally, it appears likely that HEF1 protein expression may be subject to additional forms of regulation; inspection for motifs (55) identifies a strongly predicted PEST sequence in the carboxy-terminal half of the protein (perhaps accounting for the very limited detection of endogenous p65^HEF1 [35]), while in vivo labelling experiments with ³⁵S have suggested that the protein possesses a short half-life (35), as has been observed for many proteins associated with cell cycle regulation processes. One as yet unresolved issue is that of why p55^HEF1 is abundant following transfection of cDNA, whereas this species is restricted to mitotic cells endogenously; it may be that the transient overexpression obtained following transfection overwhelms intracellular mechanisms that negatively regulate posttranslational cleavage.

One of the most surprising findings of this study is the colocalization of p55^HEF1 with the mitotic spindle and the subsequent establishment of HEF1 interaction with Dim1p. This spindle association does not reflect a generic association of p55^HEF1 with tubulin, as analysis of cells transfected with the full-length HEF1 cDNA and thus overexpressing p55^HEF1 during interphase shows no indication of association with microtubule networks (35). Therefore, localization of p55^HEF1 to the mitotic spindle seems most likely to indicate an affinity of the p55^HEF1 species for either a bundled form of tubulin specific to mitosis or a spindle-associated protein(s). We believe the latter possibility is more likely, because scrutiny at high magnification of HEF1 immunofluorescence in mitotic cells (35) indicates that the protein is arrayed in a striated or punctate pattern along the spindle, similar to that previously reported for MAPs such as mitotic-specific motors and spindle assembly factors (13). Thus, HEF1 may dock with the spindle via association with MAPs or potentially hsDim1p (whose localization has not yet been determined); alternatively, a number of signaling and cell cycle control-related proteins have been reported to associate with the mitotic spindle, including among others p34^cdc2 (57), MAP kinase (42), and the CAS protein (involved in regulation of apoptosis and cyclin B degradation) (62).

Following the early observations that morphological and adhesive properties of cells govern their viability and proliferative capacity (17, 20), multiple groups have focused on elucidating specific signaling pathways involved in such regulation. Cell adhesion, and in particular stimulation of the integrin receptor, is required in some cell lines for DNA synthesis (47); for appropriate differentiation of a cell type, as reflected by the induction of cell-type-specific gene expression (16, 64); and more recently, for prevention of anoikis, defined as programmed cell death induced by loss of attachment (22, 40, 58). Perhaps significantly, proteins known to associate with HEF1 or p130^Cas have been implicated in some of these pathways. For example, disruption of contacts between the HEF1-p130^Cas-Efs partner protein FAK and the integrin receptor results in induction of apoptosis (31), while expression of constitutively active FAK is protective against anoikis (23). HEF1 may function in activation of the MAP kinase pathway required for cell proliferation and gene expression, either through interaction with FAK (11, 37, 61) or through further association of the HEF1 partner Crk (3, 38, 41) with C3G (67), promoting activation of Ras, or by some other means. The data in this study allow us to propose a model in which p105^HEF1 and p115^HEF1 coordinate signaling complexes at focal adhesions in response to adhesion or growth factor signals initiating cell proliferation: cleavage of HEF1 at G₂/M removes p105^HEF1-p115^HEF1 from focal adhesions, potentially disrupting these complexes; as cell attachment to substrate diminishes and cells round up for mitosis, addition of p55^HEF1 to the mitotic spindle contributes to signals favoring cell cycle progression, potentially via interaction with the recently defined mitotic regulator hsDim1p. Based on the implication of caspases as HEF1-cleaving enzymes and the known association of HEF1 with FAK, aberrant HEF1 cleavage may additionally play a role in the progression of anoikis. Finally, the identification of HEF1 as a helix-loop-helix-containing protein with the capacity to associate with other helix-loop-helix factors, described elsewhere, indicates that HEF1 may couple integrin-related cell proliferation signals with regulation of cellular differentiation status. These connections to cell division, apoptosis, and differentiation position HEF1 to be an important central regulator of cell growth controls.

In a final summary, since the initial description of p130^Cas, a number of studies have established mechanisms by which this protein complexes with signaling partners at focal adhesions to presumably regulate cell shape and motility during normal cell adhesion and oncogenesis (4, 9, 19, 24, 30, 39, 44-46, 50, 52-54, 56, 59, 68, 69). This current study departs from the preexisting literature on the p130^Cas-HEF1-Efs family to suggest that, via regulated expression of synthesis, HEF1 in particular may function in part as a cell cycle sensor, whereas p130^Cas does not. We further demonstrate that cleaved HEF1 is associated with the mitotic spindle, whereas our studies to date indicate that p130^Cas is unlikely to be cleaved and is exclusively cytoplasmic. While p130^Cas is abundant in many cell types, including fibroblasts, HEF1 is the predominant species in an alternative group of cell types, including some epithelial and hematopoietic lineages. Cumulatively, these results suggest that, for the HEF1-p130^Cas-Efs family, sequence homology and conserved domain structure may nevertheless be adapted to diverse cellular functions.