Differential dephosphorylation of the insulin receptor and its 160-kDa substrate (pp160) in rat adipocytes.
A permeabilized rat adipocyte model was developed which permitted an examination of: 1) insulin receptor autophosphorylation, 2) phosphorylation of a putative insulin receptor substrate of 160 kDa, pp160, and 3) the dephosphorylation reactions associated with each of these phosphoproteins. Rat adipocytes, preincubated with [32P]orthophosphate for 2 h, were exposed to insulin (10(-7) M) at the time of digitonin permeabilization. Phosphorylation of pp160 and autophosphorylation of the insulin receptor increased as a function of Mn2+ concentration in the media with near maximum responses at 10 mM. Maximum response was at least as large as the intact cell response to 10(-7) M insulin. In contrast, magnesium did not increase phosphorylation of pp160 although an increase in receptor autophosphorylation was observed. Autophosphorylation was preserved at digitonin concentrations of 20-100 micrograms/ml, but pp160 phosphorylation was negligible beyond 40 micrograms/ml. Our previous work demonstrated that the insulin receptor was associated with a phosphotyrosine phosphatase activity in permeabilized adipocytes (Mooney, R., and Anderson, D. (1989) J. Biol. Chem. 264, 6850-6857). The current permeabilized adipocyte model made possible an examination of the effects of phosphotyrosine phosphatase inhibitors, including several divalent metal cations (Zn2+, Co2+, and Ni2+), vanadate, and molybdate on both net phosphorylation of pp160 and autophosphorylation of the insulin receptor. Zn2+ at 100 microM, Ni2+ at 1 mM, and Co2+ at 1 or 5 mM increased insulin-dependent phosphorylation of pp160 at least 5-fold and autophosphorylation 2-fold. At higher concentrations of Zn2+ (1 mM) and Ni2+ (5 mM), however, no increase in phosphorylation of pp160 was observed and autophosphorylation was inhibited. Vanadate (1 mM) and molybdate (100 microM) increased insulin-dependent phosphorylation of pp160 by 3-fold when tested separately and 7-fold in combination. Insulin receptor autophosphorylation was increased 50% by each and 3-fold when the agents were combined. Dephosphorylation of pp160 and the insulin receptor was analyzed directly by permeabilizing prelabeled insulin-treated adipocytes in the presence of EDTA (10 mM). Dephosphorylation of pp160 was especially rapid with a t1/2 of approximately 10 s. The t1/2 for the insulin receptor was 37 s. Zn2+ at 1 mM (a concentration that inhibited the insulin receptor kinase) was a strong inhibitor of dephosphorylation, prolonging the rate of pp160 dephosphorylation more than 12-fold and insulin receptor dephosphorylation 3-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
Mooney
R A
RA
Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
Bordwell
K L
KL
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002413:Cations, Divalent; D002463:Cell Membrane Permeability; D003035:Cobalt / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000378:metabolism*; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D000596:Amino Acids; D002413:Cations, Divalent; D007328:Insulin; D046911:Macromolecular Substances; D010750:Phosphoproteins; C044659:molybdate; D003035:Cobalt; D014638:Vanadates; D008345:Manganese; D009532:Nickel; D008982:Molybdenum; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D017027:Protein Tyrosine Phosphatases; D008274:Magnesium; D015032:Zinc