Isolation and characterization of the 160,000-Da phosphotyrosyl protein, a putative participant in insulin signaling.
The 160,000-Da protein (pp 160) which is rapidly phosphorylated on tyrosine in response to insulin and thus is a putative participant in signaling from the insulin receptor has been purified to homogeneity from 3T3-L1 adipocytes. Isolation of this protein was accomplished by chromatography on an immobilized monoclonal antibody against phosphotyrosine, followed by gel electrophoresis. Sufficient protein was obtained to allow the determination of the sequences of several peptides, which in turn enabled the development of anti-peptide antibodies that specifically recognize pp 160. Immunoblotting of 3T3-L1 adipocyte lysates, together with the purified pp 160 as a standard, indicate that an insulin-treated 3T3-L1 adipocyte possesses about 230,000 copies of tyrosine-phosphorylated pp160 and that this amount is approximately 25% of the total pp160 in the cell. The number of tyrosine-phosphorylated pp160s per cell is approximately the same as that of insulin receptor beta subunits. These results provide further evidence for a role of pp160 in insulin signaling. Moreover, the availability of purified protein and knowledge of peptide sequences will allow the elucidation of the structure and function of this protein.
Keller
S R
SR
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
Kitagawa
K
K
Aebersold
R
R
Lienhard
G E
GE
Garner
C W
CW
D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology*; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000502:physiology*; D019000:Phosphotyrosine; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000502:physiology*; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives
D007328:Insulin; D046911:Macromolecular Substances; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin