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 Kahn
The Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
FaO hepatoma cells
+/- insulin (1 mM), 1 min
pp185
INR(b)
(1) SDS(100oC) extraction/TCA ppt
or (2) Liquid N2 quick-freezing
Triton X-100 extraction
IP by anti-PY antibody
SDS-PAGE
Western blot by anti-PY antibody
Rats
Liver
+/- insulin (from portal vein)
1 min or various time points
pp185
Tissue removed
INR(b)
SDS/TCA extraction
IP by anti-PY antibody
SDS-PAGE
Western blot by anti-PY antibody
Fat pads
pp185
INR(b)
TCA ppt/washed by organic solvent/
lypholized/0.1 M NaOH solubilized/
filtered with 0.45 mM membrane
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)
+
insulin
-
(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
CPS: carbamyl phosphate synthase
as major contaminant in pp185
fraction
IP: aPY
WB: aPY
- +
aPY
aPep80
- +
aPep80
aPep80
-
+
aPep80
aPY
Production of
anti-peptide 80 Ab
- + (insulin)
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
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 motif
3 YXXM motif
1 EYYE motif
Synthetic YMXM-containing peptide can be phosphorylated
by purified INSR, Km~50 mM
CHO/IR
cells
CHO/IRS-1
CHO/Neo
metabolic 32P-labelling
+/- insulin (100 nM, 1 min)
cell extractes
IP by PY-Ab
SDS-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 Ab
SDS-PAGE/autoradiography
cut out 32P-labelled pp185 and IRS-1
Phosphoamino acid analysis
Y960:
the aa required for insulin
signaling
CHO/IR cells
+/- insulin (100 nM, 10 min)
cell extractes
IP by IR Ab or PY Ab or IRS-1 Ab
PI3 kinase assay in the IPP
Role of IRS-1 in insulin-mediated signal transduction