N-myristoylated c-Abl tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor

Abstract

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2-treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH(2)-terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH(2)-terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors.

FIGURE 1.

A, domain structure of Abl family members (5). The numbers indicate amino acid residues in c-Abl 1b, and the recombinant protein constructs used in this study encompass amino acids 65–534, 83–534, and 248–531. B, ribbon representation of the c-Abl kinase NH₂-terminal half residues, including the SH3, SH2, and kinase domains (Protein Data Bank code 1OPK) (7). The NH₂-terminal cap (amino acids 2–79) is indicated by dotted lines (8). The myristate-binding site and ATP binding pocket are indicated by arrows. C, ribbon representation of an enlarged view of GNF-2 (colored gold) bound to the c-Abl myristate binding site. The location of Ala³⁵⁶ is indicated.

FIGURE 2.

GNF-2 inhibits recombinant Abl kinase activity in vitro. A, recombinant Abl (amino acids 65–534) was diluted in kinase buffer containing 0.1% of the indicated detergents, incubated with either DMSO or 10 μm compounds for 30 min, and then its tyrosine kinase activity was measured by an ELISA-based assay. It is of note that Brij 35 interferes with recombinant Abl kinase activity as well as the ability of GNF-2 to inhibit recombinant Abl kinase activity. B, recombinant Abl (amino acids 65–534) was diluted in kinase assay buffer in the absence or presence of 0.1% Brij 35, incubated with either DMSO or compounds for 30 min, and then its tyrosine kinase activity was assessed by radioenzymatic assay. The phosphorylation of GST-Abltide was monitored by SDS-PAGE and autoradiography (top). The total substrate used (GST-Abltide) was visualized by Coomassie staining (bottom). M, molecular weight marker. C, a continuous spectrophotometric assay confirms the GNF-2 inhibition of recombinant Abl kinase activity. The recombinant Abl (amino acids 65–534) was diluted to a final concentration of 50 nm in kinase buffer (50 mm Tris-Cl (pH 7.4), 50 mm KCl, 10 mm MgCl₂) in the absence or presence of GNF-2. Kinase activity was measured with 300 μm substrate and 500 μm ATP for 20 min and expressed as pmol/pmol·min.

FIGURE 3.

A, indicated recombinant Abl proteins were incubated with Sepharose-immobilized GNF-2 or Me-GNF-2 (top), and the bound proteins were visualized by silver staining (bottom). B, the SH3 and/or SH2 domains in Abl are required for GNF-2 to exert its activity. Indicated recombinant Abl proteins were incubated with either DMSO or 10 μm of GNF-2, and then its tyrosine kinase activity was monitored by an ELISA-based assay. Data are expressed as a percentage of DMSO controls (mean ± S.D.; n = 3).

FIGURE 4.

GNF-2 targets c-Abl in tissue culture cells. A, GNF-2 binds to c-Abl in 3T3 fibroblasts. The lysates of 3T3 WT fibroblasts were incubated with either Sepharose-immobilized Me-GNF-2 or Sepharose-immobilized GNF-2 in the absence or presence of GNF-2, and the bound proteins were analyzed by Western blot with an anti-Abl antibody (8E9). B, GNF-2 inhibits the phosphorylation of CrkII. 3T3 WT fibroblasts were treated with either DMSO or various concentrations of compounds for 1 h, and then total lysates were analyzed by Western blot. C, GNF-2 inhibits c-Abl-induced phosphorylation of CrkII. The abl^−/−arg^−/− 3T3 cells and abl^−/−arg^−/− cells reconstituted with c-Abl were treated with either DMSO or 10 μm compounds for 1 h, and then total lysates were analyzed by Western blot. D, the point mutation in the myristate-binding site renders c-Abl resistant to inhibition by GNF-2. The indicated c-Abl proteins were immunoprecipitated following transfection into HEK293T cells, treated with either DMSO or 10 μm compounds, and then their kinase activities were assessed by ELISA-based assay. Data are expressed as a percentage of DMSO controls (mean ± S.D.; n = 3 for a representative experiment).

FIGURE 5.

N-Myristoyl group in c-Abl affects the ability of GNF-2 to inhibit c-Abl kinase activity. A, reconstitution of c-Abl^G2A in abl^−/−arg^−/− 3T3 cells increases the phosphorylation of CrkII, and GNF-2 inhibits c-Abl^G2A-induced phosphorylation of CrkII. The abl^−/−arg^−/− 3T3 cells and abl^−/−arg^−/− cells reconstituted with either c-Abl^WT or c-Abl^G2A were treated with either DMSO or 5 μm compounds for 1 h, and then total lysates were analyzed by Western blot with an anti-phospho-CrkII antibody (top) or anti-c-Abl antibody (8E9) (bottom). The blot was reprobed with an anti-CrkII antibody to show equal loading of proteins (middle). B, GNF-2 is more potent than STI-571 with respect to inhibition of c-Abl^G2A. The abl^−/−arg^−/− cells reconstituted with c-Abl^G2A were treated with either DMSO or various concentrations of compounds for 1 h, and then total lysates were analyzed by Western blot with an anti-phospho CrkII antibody. The data from three independent experiments were averaged for each condition and presented in the form of bar graphs (top). Data are expressed as a percentage of DMSO controls (mean ± S.D.; n = 3). One representative Western blot is shown (bottom). C, substitution of the myristoylation site (Gly²) in c-Abl with alanine renders c-Abl highly susceptible to inhibition by GNF-2. The abl^−/−arg^−/− cells reconstituted with either c-Abl^WT or c-Abl^G2A were treated with either DMSO or various concentrations of compounds for 1 h, and then total lysates were analyzed by Western blot.

FIGURE 6.

GNF-2 induces translocation of the myristoylated c-Abl to the ER. A and B, the indicated cells were plated on coverslips and grown in DMEM containing 10% fetal bovine serum. The cells were treated with either DMSO or 10 μm compound for 1 h, and c-Abl proteins were visualized by indirect immunofluorescence using an anti-c-Abl antibody (8E9) and Alexa Fluor 488 goat anti-mouse IgG. 4′,6-Diamidino-2-phenylindole was used to stain the nucleus. C, wide field fluorescence images. The cells were double-stained for c-Abl (green) and protein-disulfide isomerase (red) following treatment with either DMSO or 10 μm GNF-2 for 1 h. D, confocal fluorescence images. The cells were double-stained for c-Abl (green) and Sec61β (red) following treatment with either DMSO or 10 μm GNF-2 for 1 h. It is evident that the intense green fluorescence around the nucleus in GNF-2-treated cells colocalizes with the red fluorescence.

FIGURE 7.

GNF-2 inhibits Arg kinase activity in vitro and in tissue culture cells. A, GNF-2 inhibits tyrosine kinase activity of recombinant Arg. Recombinant Arg (amino acids 110–581) and Abl (amino acids 83–534) were incubated with various concentrations of compounds, and then their tyrosine kinase activity was monitored by radioenzymatic kinase assay with [γ-³²P]ATP and GST-Abltide as a substrate. The phosphorylation of GST-Abltide was visualized (top) and quantified (bottom) by phosphorimaging analysis. B, Western blot analyses of total lysates of the indicated 3T3 cells. It is evident that the anti-Arg antibody does not cross-react with c-Abl. C, GNF-2 interacts with Arg in abl^−/− 3T3 cells. The lysates of abl^−/− 3T3 fibroblasts were incubated with either Sepharose-immobilized Me-GNF-2 or Sepharose-immobilized GNF-2 in the absence or presence of GNF-2, and the bound proteins were analyzed by Western blot with an anti-Arg antibody. D, GNF-2 inhibits the phosphorylation of CrkII in abl^−/− 3T3 cells. The abl^−/− 3T3 cells were treated with either DMSO or various concentrations of compounds for 1 h, and then total lysates were analyzed by Western blot. E, The arg^−/− 3T3 cells reconstituted with Arg-yellow fluorescent protein (mouse 1b) were treated with either DMSO or GNF-2 (10 μm). The cells were imaged live using a Nikon TE2000U microscope, Hamamatsu ORCA camera, and Metamorph software (version 7.6.0) in the Nikon Imaging Center at Harvard Medical School.