Huntingtin-interacting protein 1 phosphorylation by receptor tyrosine kinases

Abstract

Huntingtin-interacting protein 1 (HIP1) binds inositol lipids, clathrin, actin, and receptor tyrosine kinases (RTKs). HIP1 is elevated in many tumors, and its expression is prognostic in prostate cancer. HIP1 overexpression increases levels of the RTK epidermal growth factor receptor (EGFR) and transforms fibroblasts. Here we report that HIP1 is tyrosine phosphorylated in the presence of EGFR and platelet-derived growth factor β receptor (PDGFβR) as well as the oncogenic derivatives EGFRvIII, HIP1/PDGFβR (H/P), and TEL/PDGFβR (T/P). We identified a four-tyrosine "HIP1 phosphorylation motif" (HPM) in the N-terminal region of HIP1 that is required for phosphorylation mediated by both EGFR and PDGFβR but not by the oncoproteins H/P and T/P. We also identified a tyrosine residue (Y152) within the HPM motif of HIP1 that inhibits HIP1 tyrosine phosphorylation. The HPM tyrosines are conserved in HIP1's only known mammalian relative, HIP1-related protein (HIP1r), and are also required for HIP1r phosphorylation. Tyrosine-to-phenylalanine point mutations in the HPM of HIP1 result in proapoptotic activity, indicating that an intact HPM may be necessary for HIP1's role in cellular survival. These data suggest that phosphorylation of HIP1 by RTKs in an N-terminal region contributes to the promotion of cellular survival.

Fig 1

HIP1 association with and phosphorylation mediated by EGFR and EGFRvIII. (A) HIP1 was immunoprecipitated (IP) from mouse lung tissue by use of anti-HIP1 polyclonal antibodies UM410 and UM323. Western blotting with antiphosphotyrosine monoclonal antibody 4G10 (Millipore) showed a HIP1-sized phosphotyrosine band. (B) Fibroblasts were serum starved for 24 h and then stimulated with EGF (100 ng/ml) for 15 min prior to extract preparation. One set of cells was treated with 1 μM AG1478 (also known as tyrphostin) for 30 min prior to addition of EGF. HIP1 was immunoprecipitated from 2 mg of HIP1-transformed fibroblast WCL (23) by use of anti-HIP1 polyclonal antibody UM410. Antiphosphotyrosine monoclonal antibody 4G10 was used to detect phosphorylated HIP1 in the immunoprecipitates. Anti-HIP1 monoclonal antibody 4B10 was used to detect HIP1. (C) Cells were treated with sodium orthovanadate (NaVa) at a final concentration of 2 mM for different periods, and EGF was added to the NaVa-containing medium for the last 15 min of each period, prior to harvest. HIP1 was immunoprecipitated and analyzed as described for panel B. The protein that migrated slower than (above) HIP1 did not plateau at 60 min. The identity of this band is unknown, as it did not comigrate with HIP1 (bottom panel). (D) HEK293T cells were cotransfected with HIP1-Myc and EGFR-V5 or EGFRvIII-V5 and lysed 24 h after transfection. HIP1-Myc was immunoprecipitated from 1 mg of lysate by use of anti-Myc antibody-conjugated agarose beads (Sigma). Anti-V5 antibodies (Invitrogen) were used to detect EGFR. (E) Diagram illustrating cold-load stimulation, a strategy for precise temporal analysis of endocytic processes. (F) HIP1 tyrosine phosphorylation was assayed at several time points for up to 60 min after initiation of endocytosis. Cells were starved, and then EGFR was “loaded” with 100 nM EGF for 1 h at 4°C. The cold medium was then exchanged for medium prewarmed to 37°C. This temperature change allowed for receptor internalization to proceed. (G) HIP1-Myc-transfected HeLa cells were treated with 1 μM EGF-555 in the “cold-load stimulation” experimental paradigm. Cells were then fixed, stained for HIP1 (anti-Myc; Cell Signaling) and clathrin (X22; AbCAM) to mark clathrin-coated vesicles, and imaged using a 1-μm slice thickness on a Zeiss confocal microscope. (H) Quantification of EGF colocalization with HIP1 and clathrin over time. ***, P < 0.0001; **, P < 0.001 (n = 37 cells per time point over 3 experiments).

Fig 2

The HIP1 ANTH domain and amino acids 753 to 799 are required for HIP1 phosphorylation mediated by EGFR. (A) Schematic of HIP1 deletion mutations as they relate to known HIP1 domains. Domains: ANTH, AP180 N-terminal homology; CHC, clathrin heavy chain binding; AP2, clathrin adaptor protein 2 binding; CC, coiled coil; CLC, clathrin light chain binding; LZ, leucine zipper; USH, upstream helix; TALIN, TALIN homology. (B to E) HEK293T cells were cotransfected with EGFR-V5 and either wild-type HIP1 or the deletion mutants, all of which were Myc tagged. Immunoprecipitation was performed with 1 mg lysate and anti-Myc beads, and phosphorylation was detected with antiphosphotyrosine antibody 4G10. (B) HIP1Δ184-400 and HIP1Δ401-599 were phosphorylated by EGFR, whereas the ΔANTH mutant was not. (C) HIP1Δ600-709 and HIP1Δ690-752 were phosphorylated by EGFR, and HIP1Δ753-799 was not. All mutants interacted with EGFR, as evidenced by their coimmunoprecipitation with EGFR. (D) Phosphorylation of HIP1 by EGFR does not require HIP1 binding to clathrin. The top panel shows phosphorylation of HIP1L486A (lane 3). Lane 4 contained the wild-type HIP1 control. Although the binding mutation is in the clathrin light chain binding site, we consistently observed a loss of binding to the entire clathrin triskelion, as represented by the blot for the clathrin heavy chain in this case. (E) The HIP1ΔANTH mutant is not phosphorylated by EGFR. Data are representative of three independent experiments.

Fig 3

Identification of tyrosine residues in HIP1 required for EGFR-mediated phosphorylation of HIP1. (A) Schematic of HIP1 Y-to-F mutations. The diagram also displays the locations of all 17 tyrosine residues (diamond-headed stalks) within the domains of HIP1. The key mutants that were not phosphorylated by wild-type RTKs are illustrated in the magnified area (HIP1 4xYF and 7xYF). Abbreviations are as described in the legend to Fig. 2. HPM, HIP1 phosphorylation motif. (B and C) HEK293T cells were cotransfected with EGFR-V5 and either Myc-tagged wild-type HIP1 or Myc-tagged HIP1 Y-to-F point mutants. Immunoprecipitation was performed with anti-Myc beads, and phosphorylation was detected with antiphosphotyrosine antibody 4G10. (B) The USH-deficient mutant (HIP1Δ753-799) and the HPM Y-to-F mutants (HIP1 4xYF) were not phosphorylated by EGFR or EGFRvIII. (C) Y152 of HIP1 inhibits EGFR phosphorylation. Phosphorylation-resistant mutants (lanes 5 and 7) were phosphorylation sensitive when the Y152F point mutation was included (lanes 6 and 8). The levels of EGFR were increased in the presence of wild-type HIP1 but not in the presence of phosphorylation-resistant mutants (bottom panel). (D) HEK293T cells were transfected with Myc-tagged HIP1 or the HIP1/HPM(4xYF) mutant and assayed for HIP1 levels by Western blotting with anti-Myc antibody over a 96-hour period. (E) Low GFP levels that were associated with cotransfected HIP1/HPM(4xYF) (lanes 7 to 9) were rescued by cotransfection of HIP1/HPM(4xYF)-ires-GFP with wild-type HIP1 (lanes 10 to 12). Wild-type HIP1 also increased GFP levels (lanes 4 to 6) compared to those in cells transfected with wild-type HIP1-ires-GFP (lanes 1 to 3). (F) Cos-7 cells were transfected with Myc-tagged wild-type HIP1, HIP1/HPM(4xYF), and HIP1/ΔANTH DNA constructs. Cells were stained with mouse monoclonal anti-Myc antibody (cytoplasmic staining) to allow for analysis of transfected cells and with DAPI to show nuclear morphology. Cells were scored at 24 h posttransfection and deemed apoptotic if nuclear condensation or fragmentation was observed (arrowheads). These images are representative of the overall results, where the HIP1-transfected cells were found more frequently with smooth, nonblebbed nuclei and contained large regions of HIP1-expressing cytoplasm. In comparison, the mutant cells displayed more condensed cytoplasm, and the nuclei were less frequently intact. (G) Apoptotic cells in the three different transfections described for panel F were quantitated. The experiment was performed on three separate occasions, and data were averaged. Error bars denote standard deviations. At least 100 cells from each sample in each experiment were scored for apoptosis by two blinded investigators (A.A.W. and A.C.), according to nuclear morphology. The percentages of apoptotic cells were compared to the baseline cell death frequency (15%) of surrounding HIP1-negative cells in each experiment.

Fig 4

HIP1r requires the HPM for phosphorylation mediated by EGFR. (A) Amino acid alignment of HIP1 and HIP1r N termini. Amino acids in gray are completely conserved. The tyrosines in black are the HPM tyrosines. The tyrosine at position 152 of HIP1 is not conserved in HIP1r, and the amino acid at this position (residue 143) in HIP1r is a phenylalanine. (B and C) HEK293T cells were cotransfected with EGFR-V5 and either wild-type HIP1r or HIP1r Y-to-F point mutants. Immunoprecipitation was performed with anti-HIP1r UM374 polyclonal antibody, and phosphorylation was detected with antiphosphotyrosine monoclonal antibody 4G10. (B) The HIP1r HPM Y-to-F mutants [HIP1r Y(135,142)F and Y(191,236)F] were not phosphorylated by EGFR. (C) The F143Y mutant of HIP1r inhibited phosphorylation of HIP1r. The phosphorylated protein migrating at 120 kDa in the first lane is endogenous HIP1r. Two separate repetitions of these experiments are presented.

Fig 5

HIP1 association with and phosphorylation mediated by PDGFβR and its oncogenic derivatives. (A to D) HEK293T cells were cotransfected with HIP1-Myc and RTKs. HIP1-Myc was immunoprecipitated from 1 mg of HEK293T whole-cell lysate by use of anti-Myc beads. Phosphorylation was detected with antiphosphotyrosine antibody 4G10, and clathrin association was detected with anticlathrin antibody TD.1 (Sigma). Anti-PDGFβR (BD Pharmingen) was used to detect PDGFβR, H/P, and T/P. (A) Cells were cotransfected with HIP1-Myc and either PDGFβR, H/P, or T/P. HIP1 phosphorylation was observed in these cotransfections, and the phosphorylation was enhanced when NaVa was added to cells 1 h before lysis. (B) The HPM Y-F mutants (HIP1 4xYF and 7xYF) were not phosphorylated by PDGFβR but were phosphorylated by H/P and T/P. Lanes 10 and 11 were spliced and rearranged from the same gel for presentation purposes. (C) T/P requires amino acids 281 and 475 in HIP1 for HIP1 phosphorylation, as the HIP1 15xYF mutant had only these two tyrosines intact. (D) In contrast to HIP1, H/P readily interacted with clathrin when H/P was coexpressed with HIP1. Extracts were immunoprecipitated with anti-PDGFβR (lanes 1 to 3 and 7 to 9) to directly precipitate H/P rather than HIP1. Extracts were immunoprecipitated with anti-Myc (lanes 4, 5, 10, and 11) to directly precipitate Myc-HIP1 rather than H/P. The extracts in the panels on the right were derived from imatinib-treated cells to inhibit H/P phosphorylation, as demonstrated in the lower panels.

Fig 6

Identification and phosphorylation of two alternative HIP1 isoforms. (A) Diagram of alternative human HIP1 transcripts and their amino acid sequences, designated hHIP1a and hHIP1b. These isoforms differ only in their initial exon, resulting in alternative N-terminal amino acid sequences. (B to E) HEK293T cells were cotransfected with various RTKs and either HIP1a or HIP1b, both of which were tagged with Myc. Immunoprecipitation was performed with anti-Myc beads, and HIP1 phosphorylation was detected with antiphosphotyrosine antibody 4G10. (B) EGFR phosphorylates HIP1b less than HIP1a in the presence or absence of NaVa. (C) EGFRvIII phosphorylates HIP1b to a slightly lesser degree than that for HIP1a in both the presence and absence of NaVa. (D) PDGFβR phosphorylates both HIP1b and HIP1a. (E) H/P and T/P phosphorylate HIP1a and HIP1b equally in both the presence and absence of NaVa.