To assess whether MELK has a role in mammary carcinogenesis, we knocked down the expression of endogenous MELK in breast cancer cell lines using mammalian vector-based RNA interference. Furthermore, we identified a long isoform of Bcl-G (Bcl-GL), a pro-apoptotic member of the Bcl-2 family, as a possible substrate for MELK by pull-down assay with recombinant wild-type and kinase-dead MELK.
To investigate the biological functions of MELK in breast cancer cells, we searched for substrates of MELK in cancer cells by in vitro protein pull-down assays using wild-type MELK (WT-MELK) and kinase-dead MELK (D150A-MELK) recombinant proteins. Comparison of silver staining of SDS-PAGE gels containing the pulled-down proteins identified an approximately 30 kDa protein in the lane corresponding to proteins pulled-down with WT-MELK but not in that corresponding to proteins pulled-down with D150A-MELK (Figure 3a).
Furthermore, we demonstrated that His-tagged WT-MELK could pull-down with Bcl-GL but His-tagged D150A-MELK could not, indicating that, in vitro, Bcl-GL interacts directly with WT-MELK but not with D150A-MELK (Figure 3d).
Thus, to investigate the biological significance of MELK in breast cancer cells, we searched for a possible substrate(s) of MELK by means of in vitro pull-down assays with recombinant wild-type MELK (WT-MELK) and kinase-dead MELK (D150A-MELK).Flag-tagged protein was overexpressed in COS7 cells and the cell lysate was obtained as described above. The cell lysate obtained was incubated with anti-Flag M2 affinity gel (Sigma-Aldrich) at 4 °C overnight with rotation. The Flag-tagged protein was pulled down by the affinity gel, and the affinity gel was washed twice with lysis buffer (0.5% NP-40, 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM NaF [pH 7.5]) with various protease inhibitors (1 mM PMSF, 1 mM NaOV, 2 μg/ml antipain, 10 μg/ml leupeptin, 30 nM okadaic acid, 5 mM benzamidine, and 10 μg/ml aprotinin). One to two milligrams of proteins prepared from brain tissues was incubated with the affinity gel, and the Flag-tagged protein pulled down by the affinity gel for 1 h. The affinity gel was washed twice with lysis buffer supplemented with protease inhibitors. The proteins pulled down by the Flag-tagged protein were subjected to Western blot analysis.
(E) The membrane fraction of adult brain lysates was incubated with or without Flag-tagged Cdk5. Flag-tagged Cdk5 pulled down TrkB from the membrane fraction of adult brain lysates.
Quality of the purified GST and GST-fusion proteins used in the GST pull-down assay was verified by Coomassie blue staining.GST-pull down and co-immunoprecipitation assays were performed to further characterize this interaction.
GST-pull down assay to identify the region of BRCA1 interacting with PP1. (A) Fragments used in GST pull down assays (BR 2 to 5) are diagrammed. (B) Gel depicting co-precipitation of GST-bound BR-4 with PP1. Following incubation of GST-BRCA1 proteins with equal amounts of cell lysate, a western blot was performed and probed with an antibody to the catalytic region of PP1. (C) Analysis of the effect of mutations of the KVTF PP1 interacting domain on the BRCA1- PP1 interaction. GST-bound-BR4 V-A and GST-bound-BR4 F-A binds PP1 with decreased intensity, compared to WT GST-bound-BR4.To study APPL1−Rab5 interaction in solution, we performed glutathione S-transferase (GST)-mediated pull-down assays. The APPL1 BAR-PH domain (residues 5−385) and a longer fragment with a 40-residue extension downstream of the PH domain, APPL1 (5−419), were each effectively pulled down by GTP-bound GST−Rab5 fusion protein (Figure 4). The APPL1 protein was pulled down by either WT Rab5 preloaded with non-hydrolysable GTP analog (GppNHp) or Rab5-Q79L defective in GTP hydrolysis (with or without preloaded GTP analog), but could not be effectively pulled down by either the WT Rab5 preloaded with GDP or Rab5-S34N defective in GTP binding (Figure 4 and data not shown).
Therefore, we tested APPL1 binding specificity towards other members in the Rab5 subfamily, using GST−Rab21 (full length) and GST−Rab22 (2−192) to pull down APPL1 (5−419).
On the other hand, we were unable to detect any binding between APPL1 and Rab22 in the pull-down assay (Figure 4).
To further define the Rab5−APPL1 binding mode, we performed extensive pull-down analyses between variants of Rab5 and APPL1, looking for reversal mutants that could rescue the lost binding ability of others. We identified one such pair; APPL1-N308D abolished the binding to Rab5, while Rab5-L38R had no effect on APPL1 binding. However, Rab5-L38R was found to bind with APPL1-N308D, but not with the other tested APPL1 variants of similar hydrophobic-to-charged mutations, including V25D, A318D, and L321D (Supplementary Figure 6). This result suggests that Rab5-L38R restores binding for APPL1-N308D through complementary, electrostatic, yet specific interactions. It further implies that the position 308 in the β3 strand of APPL1 PH domain is in the vicinity of position 38 in the α1 helix of Rab5 in their complex.
Combined results from our mutagenesis pull-down experiments (Figures 4, 5 and 6), crystal structures of the BAR-PH domain of APPL1 (Figure 1), and structures of GTPase domain of human Rab5 in different nucleotide binding modes (Zhu et al, 2003, 2004) clearly explain the requirement of GTP-bound Rab5 for APPL1 binding. Based on available information, we have modeled the interaction between the two proteins. With the assumption that both proteins remain rigid bodies, our complex model satisfies constraints imposed by the mutagenesis pull-down results (Figure 7).
In the Rab5−APPL1 pull-down experiment, 30 μg GST−Rab fusion protein (52 kDa) was incubated with 60 μl of 30% slurry of GSH–Sepharose 4B (GE Healthcare) at 22°C for 30 min. Nucleotide loading reaction was performed on the GSH beads in an exchange buffer of (1 × PBS, 2 mM DTT, 1 mM MgCl2, 4 mM EDTA, and 400 μM GppNHp or GDP) at 22°C for 30 min. Increasing the magnesium ion concentration to 20 mM terminated the loading reaction.
Pull-down analysis of APPL1-Rab interaction. GST fusion proteins of Rab5, Rab21, and Rab22 were used to pull down His-tagged APPL1 (5−385) and (5−419) fragments in the presence of GDP or GTP analog (GppNHp).
(A) Pull-down assay on Rab5 variants. Point mutations in switch regions were introduced in the full-length Rab5-Q79L background of the GST fusion construct. Each mutant was expressed in E. coli, purified, and equal amounts of each Rab5 sample was used to pull down recombinant proteins of His-tagged WT APPL1 (5−419) (top panel) and His-tagged rabaptin5 (551−862) (bottom panel). The results were visualized by Coomassie blue stain.Consistent with prior reports [23,24], we failed to observe stable direct interaction between purified ST and PP2A C subunit using a glutathione S-transferase (GST) pull-down assay (unpublished data).Next, we used the recombinant, purified MRN complex in peptide pull-down experiments. These showed that MRN bound to the phosphorylated but not the unphosphorylated form of the SDTD peptide—confirming the direct nature of the interaction—yet did not bind to either version of the H2AX C-terminal peptide (Fig 3A). This indicates the specificity of MRN for the phosphorylated MDC1 SDTD motif and also shows that if, as previously reported, NBS1 binds to γH2AX directly (Kobayashi et al, 2002), this interaction must be less stable than MRN binding to the phosphorylated SDTD motif.
(B) Silver-stained SDS–polyacrylamide gel of an SDTD peptide pull-down. PP, S329 T331 doubly phosphorylated peptide and −, its non-phosphorylated equivalent; B, bead-interacting proteins removed from extracts in a pre-clearing step; M, molecular weight markers.
(B) A 200 ng portion of CK2-phosphorylated or mock-phosphorylated GST-SDTD6 was incubated in pull-down reactions with 25 μl of in vitro-translated (IVT) HA fusions corresponding to amino-acid residues 1–348 of NBS1 (HA-fNBS1), or analogous proteins bearing point mutations predicted to abolish either FHA (R28A/H45A) or BRCT2 (K160M) phosphorylation-dependent interactions.For the glutathione S-transferase (GST) pull-down experiments, plasmids pGEX-RNP-K (kindly provided by Dr. Levens) and pGEX-4T (GE Healthcare) were used to express GST-hnRNP-K fusion protein or GST alone in Escherichia coli.
GST-hnRNP-K and GST proteins were produced in E. coli BL21 cells, previously transformed with vector pGEX-RNP-K or pGEX-4T. Cells were induced with 0.1 mM IPTG for 2 h at 37 °C. Bacteria were harvested and suspended in lysis buffer (PBS, 1% Triton X-100, 1 mM PMSF, 5 mM DTT, and anti-proteases), and sonicated on ice. GST-hnRNP-K and GST alone were purified from cleared lysates by mixing with glutathione-sepharose 4B beads (GE HealthCare), 5 ml of cleared lysate/400 μl of beads, for 1 h at 4 °C. After extensive washing, GST-hnRNP-K or GST beads were incubated in binding buffer (50 mM HEPES, ph 7.5, 50 mM NaCl, 0.1% Nonidet P-40 with protease inhibitor mixture (Roche Molecular Biochemical)) at 4 °C for 1 h with insect cell extracts containing either p30 or p54 ASFV proteins overexpressed in a baculovirus system [8]. Equal amounts of GST, GST-hnRNP-K and ASFV proteins p30 and p54 were used as judged by Coomasie Blue staining.
The interaction was further confirmed by in vitro binding assays using a GST-hnRNP-K fusion protein bound to glutathione-sepharose 4B beads. GST pull-down experiments were carried out followed by Western blot with specific antibodies. First, p30 and another unrelated ASFV protein (in this case, p54 as negative control) were used to bind GST fusion protein or GST alone. Protein p30, and not p54, was retained in the presence of GST-hnRNP K, showing a band of appropriate size (30 kDa) for p30 in Western blotting. This band did not appear in the presence of GST alone, indicating specific interaction of p30 with hnRNP K and not with GST (Fig. 1A). Identical results were obtained in subsequent experiments using BA71V infected or mock-infected cells extracts instead of baculovirus infected cell extracts in the pull-down assay (Fig. 1B).
This interaction was further confirmed by an in vitro GST-fusion pull-down assay, using either p30 obtained from baculovirus system or ASFV infected cell extracts.