A Hepatocyte Growth Factor Receptor (Met) Insulin Receptor hybrid governs hepatic glucose metabolism Arlee Fafalios, Jihong Ma, Xinping Tan, John Stoops, Jianhua Luo, Marie C. DeFrances and Reza Zarnegar SUPPLEMENTARY FIGURES, LEGENDS AND METHODS Supplementary Figure 1. Amino acid sequence similarities between Met and Insulin Receptor (INSR) kinase domains. Shown is the amino acid alignment of human INSR, Met and EGFR. Note the presence of the known regulatory triple Tyr (Y) residues in Met and INSR (box with solid line a motif which binds IRS2 when fully activated in the case of INSR 1 ) and the QPEY motif (box with dashed line) in Met, which is similar to the IRS binding site in INSR (NPEY not shown). EGFR lacks these similarities. 1
Supplementary Figure 2. Met phosphorylates IRS1 and -2. Hepatocytes were stimulated with EGF (20 ng ml -1 for 5 min.), insulin (200 ng ml -1 for 5 min.) or HGF (20 ng ml -1 for 1, 5, or 15 min.). Lysates were prepared and subjected to IP with py20 and IB with anti-irs1 or anti-irs2 as indicated. Fibroblast lysate (NIH3T3) provided by the supplier of IRS1/2 antibodies was included as a positive control. A pool of HGF treated samples was subjected to IP with normal mouse IgG as a negative control. 2
Supplementary Figure 3. Met activates INSR in a cell-free system. (a) Recombinant Met (GST-tagged, Mr 78 kda) and INSR (His-tagged, Mr approximately 37 kda) kinases were incubated in kinase buffer at the indicated molar ratios in an in vitro kinase assay and subjected to IB studies using py20 antibody. INSR concentration was 3 pmole and kept constant while Met concentration was increased as shown. (b) A hot kinase assay was performed by incubating recombinant Met, INSR (both GST-tagged, Mr, 78 and 70 kda, respectively) and EGFR kinases for 15 min. in kinase buffer containing 32 P-γATP. The reaction products were subjected to SDS-PAGE, transferred to PVDF membrane and processed for autoradiography (48 h. exposure). The blot was subsequently probed with anti-insrβ and ECL detection to document the presence of INSR in the reaction. Numbers to right of blots are size markers in kda. The bars depict relative normalized de novo pinsr/total INSR (i.e. corresponding to pinsr/insr signals from lanes 3, 4, 7 and 9). 3
Supplementary Figure 4. Met activates INSR in human hepatocytes. Normalized data corresponding to IB results shown in Figure 2c. Human primary hepatocyte cultures were treated with HGF and/or insulin for various times and assessed for Met and INSR activation by IP and IB as indicated. Error bar represents mean ± s.e.m. 4
Supplementary Figure 5. Recruitment of IRS2 to Met requires functional Met. Hepa1-6 cells expressing Tet regulatable DN-Met were cultured overnight with or without doxycycline, then stimulated with HGF (40 ng/ml) and subjected to IP with anti- Met antibodies and IB with anti-irs2 and anti-met as indicated. Input lysates were assessed for DN-Met expression and IRS2 by IB. 5
Supplementary Figure 6. Met and INSR and Met and IRS form a complex in the livers of mice. (a) Wild type CD1 mice (n = 2) were fasted overnight and then injected with insulin. At various times, livers were harvested and subjected to IP and IB using the indicated antibodies. The data shown are from insulin given at 1 mu g -1 i.p. (b) Livers from AlbDN-Met and wild type mice were subjected to IP with anti-met antibody and IB with anti-insr antibody as indicated. 6
Supplementary Figure 7. sirna-mediated knock down of Met in primary cultures of mouse hepatocytes. Primary mouse hepatocytes were transfected with Met or control sirna for 24 72 h. Lysates were subjected to IB analysis and knockdown of Met was confirmed using an anti-met antibody. 7
Supplementary Figure 8. Relative abundance of Met and INSR proteins in mouse tissues. Ob/ob mice were injected with HGF or saline control, and 15 min. later, tissues were harvested and analyzed for Met and INSR expression by IB. Liver from lean mice injected with HGF is included as positive control. Error bars represents mean ± s.e.m. 8
Supplementary Figure 9. HGF is a potent activator of AKT and Gab1 in human hepatocytes. Primary cultures of human hepatocytes were treated with HGF, insulin or IGF1 (at the indicated doses) for 0 15 min., and lysates were assessed by IB with the indicated antibodies. 9
10
SUPPLEMENTARY METHODS Cell lines, culture conditions, and antibodies. HepG2, Hepa1-6, and primary hepatocyte cultures were maintained in DMEM supplemented with 10% FBS. Antibodies against pmet (Tyr1234/35), pinsr (Tyr1146), pinsr (Tyr1150/51), pirs (Tyr895), pfoxo1 (Ser256 which also cross-reacts with pfoxo4 Ser193), FoxO1, FoxO4, PARP, and Met (25H2) were obtained from Cell Signaling; Met (B2), G6Pase, PEPCK and INSR-beta were from Santa Cruz Biotechnology; IRS1 and -2, β-actin and GLUT-2 were from Millipore; pirs (Tyr612), and pinsr (Tyr972) were from Invitrogen; and py20 was from BD Biosciences. All antibodies were used at the manufacturer s recommended dilution. Protein isolation, SDS PAGE, immunoprecipitation and immunoblot analysis. Protein isolation, SDS-PAGE, immunoprecipitation (IP) and immunoblot (IB) analysis were carried out using standard procedures as previously described 2. Generally, 40 micrograms of protein lysate made in RIPA buffer was subjected to SDS-PAGE. For IP, 1 mg of total protein was incubated with the indicated antibodies. Membranes were probed with primary and corresponding secondary antibodies. Signals were illuminated by the Western Lightning Chemiluminescence Reagent PLUS (Perkin-Elmer) and captured on X-ray film. sirna-mediated knockdown. HepG2 cells were plated at 2 x 10 6 cells in a 100 mm cell culture dish. To knock down endogenous Met expression, 21 base pair doublestranded RNA oligonucleotides specific for Met (Sense: 5 - GGAGGUGUUUGGAAAGAUAtt; Antisense: 5 -UAUCUUUCCAAACACCUCCtg) were obtained from Ambion, Inc. (sirna ID#: 42825) and transfected into cells (100 ng per well) using Silencer siport Amine sirna Transfection kit (Ambion, Inc.). Negative control sirnas purchased from Ambion (#4637) were also transfected (100 ng per well). They consist of 19 bp nontargeting sequences with 3 dt overhangs and have no significant homology to any known gene sequences in mouse, rat, or human. The 11
transfected cells were incubated at 37 C for the indicated time and then harvested for IB analysis. Dominant negative Met expression in vitro. We utilized a Tet-regulated stable Hepa1-6 mouse hepatocytic cell line which expresses a dominant negative (DN-Met) form of Met as previously described 2. These cells were used in numerous experiments with or without doxycycline (i.e. Tet) and with varying treatments of insulin, HGF, and/or both at the indicated concentrations and time points in the figures. Histological studies. Histological analysis was performed on livers as previously described 3. Microarray analysis. Wild type male Balb/c mice (n = 4 per group) were injected systemically with either saline or recombinant human HGF (50 micrograms). Approximately, three hours later animals were sacrificed, RNA from their livers was prepared. Samples were subjected to microarray analyses using an Affymetrix Genechip platform performed by our departmental core facility at the University of Pittsburgh as recommended by the manufacturers and as previously described 4. Statistical analyses. Computations were performed using VassarStat website for statistical computation at http://faculty.vassar.edu/lowry/vassarstats.html and instat-2 software. A P value of 0.05 or less considered significant. 12
SUPPLEMENTARY REFERENCES 1. Hanke, S. & Mann, M. The phosphotyrosine interactome of the insulin receptor family and its substrates IRS-1 and IRS-2. Mol Cell Proteomics 8, 519-534 (2009). 2. Wang, X., et al. A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Mol Cell 9, 411-421 (2002). 3. Zou, C., et al. Lack of Fas antagonism by Met in human fatty liver disease. Nat Med 13, 1078-1085 (2007). 4. Luo, J.-H., et al. Transcriptomic and genomic analysis of human hepatocellular carcinomas and hepatoblastomas. Hepatology 44, 1012-1024 (2006). 13