J. Biol. Chem. 266, pp. 8302-8311,1991 Purification and Partial Sequence Analysis of pp185,

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