File S1 - A Protoberberine Derivative HWY336 Selectively Inhibits MKK4 and MKK7 in Mammalian Cells: The Importance of Activation Loop on Selectivity

Supporting information figures. Figure S1, HWY336 inhibits MKK4 and MKK7 selectively but does not inhibit other mammalian MEKs and MAPKs. MKK4 and MKK7 were immunoprecipitated from HEK293 or CHO cells following MAPK pathway activation as described in EXPERIMENTAL PROCEDURES. The same quantity of kinase was used in each assay. A) MKK4 and MKK7 activity assayed with MBP as a substrate after pre-treatment with 1 mM of DMSO (lane 1), 400 µM berberine (lane 2), 1 mM HWY289, another protoberberine derivative from the library (lane 4), or 1 mM HWY336 (lane 5). DMSO was used as a negative control (lane 1) and 3 mM U0126 was used as a positive control (lane 3). B, berberine; U, U0126; 289, HWY289; 336, HWY336. B) MEK1, MKK3/MKK6, JNK1/2, p38, and ERK1/2 were immunoprecipitated from activated HEK293 and CHO cells as described in Materials and Methods. The activity of each immunoprecipitated kinase was assayed using MBP as a substrate after pre-treatment with 1 mM DMSO (lane 1), 400 µM berberine (lane 2), 1 mM HWY289 (lane 4), or 1 mM HWY336 (lane 5). Established inhibitors against each kinase (U0126 25 µM for MEK1, 3 mM for MKK3/MKK6, ERK1/2, and SP600125, 25 µM for JNK1/2 and SB203580, 25 µM for p38) were used as positive controls (lane 3). An equal amount of each kinase was used in all assays. B, Berberine; SP, SP600125; SB, SB203580; U, U0126; 289, HWY289; 336, HWY336. Figure S2, The mechanism of HWY336-mediated inhibition of MKK4 and MKK7. Stress-activated MKK4 and MKK7 were immunoprecipitated from HEK293 cells, and the same amount of each kinase was used in the assays as confirmed by Coomassie staining or western blotting. A) Reversibility of HWY336 binding to MKK4 and MKK7. MKK4 or MKK7 was pre-incubated with 600 µM HWY336 for 10 min and washed with kinase buffer containing increasing concentrations of NaCl prior to kinase assays. B, C) The ability of ATP (B) or MBP substrate (C) to compete with HWY336 for binding to MKK4 and MKK7. Immunoprecipitated MKK4 and MKK7 were pre-incubated with different concentrations of either ATP (B) or MBP (C) and their kinase activities were assayed in the presence of 600 µM HWY336 or DMSO (lane 1). Figure S3, HWY336 interacts with MKK4 reversibly, and inhibits MKK4 by competing with JNK, a physiological protein substrate, and not with ATP. Stress-activated MKK4 were immunoprecipitated from HEK293 cells as described in Materials and Methods. A) HWY336 interacts with MKK4 reversibly. Immunoprecipitated MKK4 was treated with 50 µM HWY336 for 10 min and washed with kinase buffer containing different concentrations of NaCl as in Figure S2A, B, C of Supplemental Figures. Competition of (B) ATP or (C) JNK substrate with HWY336 for binding to MKK4. MKK4 was immunopreciptiated and pre-incubated with different concentrations of either (B) ATP (C) or JNK, and their kinase activities were assayed in the presence of 50 µM HWY336 or DMSO (lane 1). Figure S4, Computer modeling predicts the conserved three-dimensional structures of MKK4 over MKK7. A) Three-dimensional structure of MKK7 (magenta) is superimposed onto the MKK4 (cyan) model structure. B) Three-dimensional modeling of HWY336 docking with MKK7. A proposed docked pose of ATP inside the MKK7 structure. The residues that vary (Arg283, Lys288, Thr291 and Arg292) within the activation loop are indicated. HWY336 forms a hydrogen bond with Arg292 and the conserved Thr291, which is important in the phosphorylation process of MKK7 (generated with the Pymol program; www.pymol.org). C) Activation loop sequences and critical residues are highly conserved among MKK4/MKK7 and Wis1, which are all inhibited by HWY336. Figure S5, Prediction of a HWY336-binding site within the activation loop of MKK4. All possible solvent pockets were simulated and a HWY336-binding site was predicted within the activation loop of MKK4 using SITE ID module of SYBYL 8.1.
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