j. biol. chem. 266, pp. 8302-8311,1991 purification and partial sequence analysis of pp185, the...
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J. Biol. Chem. 266, pp. 8302-8311,1991
Purification and Partial Sequence Analysis of pp185, the Major Cellular Substrate of the Insulin Receptor Tyrosine Kinase*Paul L. Rothenberg, William S. Lane, Avraham Karasik, Jonathan Backer, Morris White, & C. Ronald KahnThe Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
pp185
INR()
FaO hepatoma cells +/- insulin (1 M), 1 min
(1) SDS(100oC) extraction/TCA pptor (2) Liquid N2 quick-freezing Triton X-100 extraction IP by anti-PY antibody
SDS-PAGE
Western blot by anti-PY antibody
Rats +/- insulin (from portal vein) 1 min or various time points
Tissue removed
SDS/TCA extraction IP by anti-PY antibody
SDS-PAGE
Western blot by anti-PY antibody
TCA ppt/washed by organic solvent/lypholized/0.1 M NaOH solubilized/filtered with 0.45 M membrane
Liver
Fat pads
pp185
INR()
pp185
INR()
Rats insulin infusion (from portal vein) (0.5 min at 1 ml/min)
Liver removed
SDS/TCA extraction IP by anti-PY antibody
SDS-PAGE
Western blot by anti-PY antibody
Half maximum stimulation ~1-5 x 10-8 M insulin
Rats insulin infusion (from portal vein) (0.5 min at 1 ml/min)
Liver removed
SDS/TCA extraction IP by anti-PY antibody
Eluted by pNPP
Sup. + ppt.
SDS-PAGE
Western blot by anti-PY antibody & silver stained
50 Rats (starvation for 2-3 days) +/-insulin infusion (from portal vein) (0.5 min at 1 ml/min)
Liver removed
SDS/TCA extraction IP by anti-PY antibody
Eluted by pNPP
Sup. + ppt.
SDS-PAGE
Transferred to NC membrane & trypsin digested
RPC18 HPLC
Amino acid sequencer
+
-
insulin
CPS: carbamyl phosphate synthase as major contaminant in pp185 fraction
Production ofanti-peptide 80 Ab
Rats insulin infusion (from portal vein) (0.5 min at 1 ml/min)
Liver removed
SDS/TCA extraction IP by anti-PY or anti-pep 80 antibody
SDS-PAGE
Western blot by anti-PY or anti-pep 80 antibody
IP: aPY aPY aPep80 aPep80WB: aPY aPep80 aPep80 aPY
- + - + - + - + (insulin)
Nature. 1991 Jul 4;352(6330):73-7.Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein.
Sun XJ, Rothenberg P, Kahn CR, Backer JM, Araki E, Wilden PA, Cahill DA, Goldstein BJ, White MF.Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA
IRS-1 cloning strategy:(1)Two cDNA libraries(2)Screened by 32P-oligonucleotide probes made from peptide 80 and 138(3)Picked up positive clones and sequence determination (C18, C19 & P2-2)(4)Screened further by probes derived from C18 & P2-2 (14 clones were obtained)(5)Sequencing and overlapping DNA sequences of 14 clones
IRS-1 cDNA:(1) Hydrophilic protein, 131 kDa(2) 9 tryptic peptides in DNA sequence(3) No SH2/SH3 domains(4) Contain ATP-binding domain (GXGXXG)(5) No protein kinase activity(6) mRNA 9.5 kb
6 YMXM motif3 YXXM motif1 EYYE motif
Synthetic YMXM-containing peptide can be phosphorylated by purified INSR, Km~50 M
CHO/IR cellsCHO/IRS-1 CHO/Neo
metabolic 32P-labelling +/- insulin (100 nM, 1 min) cell extractes IP by PY-AbSDS-PAGE/autoradiography
IRS-1 expressed in CHO cells as 185 kDa PY-containing protein
CHO/IR or CHO/IRF960 or CHO/IRD960 cells metabolic 32P-labelling +/- insulin (100 nM, 1 min) cell extractes IP by PY-Ab or IRS-1 AbSDS-PAGE/autoradiography cut out 32P-labelled pp185 and IRS-1Phosphoamino acid analysis
Y960:the aa required for insulinsignaling
CHO/IR cells +/- insulin (100 nM, 10 min) cell extractes IP by IR Ab or PY Ab or IRS-1 AbPI3 kinase assay in the IPP
Role of IRS-1 in insulin-mediated signal transduction