Abstract
It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.
Highlights
Cytes to trypsinis known to result ian time- and dose- The serineproteasetrypsinisaninsulin-mimeticagent dependentactivation of intracellular glycogen syn- which stimulates glucose uptake [10] and activates pyruvate okothfiuantrsayessr.yeosEstiaxencmaetmivikininitdnyabitacyisoaettnreyosapfntshgdtihalnyetctoothicgmeceusentrisscmyonwuutlrihastahetissoeosnfuafsocftftiiimcvtiyeuarntloiatostiniornaneipnidd-taolesrhoyyedbnrezoeygnmedneeasmseoon(f1s0ltir)paaitdenddangtdoleyvcgoolykgceeonagusetynonpstyhhnoatsshpeeh(soliorsy.)T,latrtwyioponsironefhgpauaslar-ity and magnitude to be consistent waitrhole of phos- tially and highly purified insulin receptors [11]and to stimphorylationintheactivation of glycogensynthase. ulate insulin receptor-associated tyrosine kinase activity to
The experiments demonstrating trypsin’sstimulat,ion of receptor phosphorylation were in vitro studies usingsolubilized insulin receptors [11].Forphosphorylation toberelevant to the sin treatment, respectively.We conclude that trypsin- insulin-like anaboliceffects of trypsin, stimulation of insulin induced cleavage of the insulin receptor a-subunit is receptor autophosphorylation and tyrosine kinase activation relevant to the abiliotyf trypsin to activate the insulinmust occur in intact cells
We fur- fore undertaken to determine whether trypsin causesin situ ther conclude that autophosphorylation of the insulin phosphorylation of the insulin receptor and activationof the receptor and activationof its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic insulin receptor tyrosine kinase
Summary
$ Supported by Research Grant 5T32 GM07055 from the National Institutes of Health. § T Owhom correspondence andreprint requests should be addressed. $ Supported by Research Grant 5T32 GM07055 from the National Institutes of Health. E. Vandenhoff (Diabetes Center, University of Virginia, Charlottesville, VA). UDP-[U-14C]glucosewas synthesized in our laboratory according to published methods [14]. Human serum containing autoantibodies against the insulin receptor was a gift from Dr G. H. Boden (Health Sciences Center, Temple University, Philadelphia, PA). Rabbit serum containing autoantibodies against amino acids 1313-1325 of the insulin receptor 8subunit was a gift from Lynn Kozma (University of Virginia, Charlottesville, VA). Wheat germ agglutinin coupled to agarose was from \’ector Laboratories. Trypsin Activates Insulin Receptor Tyrosine Kinase in Situ was from Behring Diagnostics.
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