Use of tyrosine-containing polymers to characterize the substrate specificity of insulin and other hormone-stimulated tyrosine kinases.
Synthetic copolymers containing tyrosine residues were used to characterize the substrate specificity of the insulin receptor kinase and compare it to tyrosine kinases stimulated by epidermal growth factor, insulin-like growth factor-1 and phorbol ester. In partially purified receptor preparations from eight different tissues insulin best stimulated (highest V) phosphorylation of a random copolymer composed of glutamic and tyrosine residues at a 4:1 ratio (Glu/Tyr, 4:1). The insulin-stimulated phosphorylation of this polymer was highly significant also in receptor preparations from fresh human monocytes, where insulin binding and autophosphorylation were difficult to detect. Other tyrosine-containing polymers Ala/Glu/Lys/Tyr (6:2:5:1) and Glu/Ala/Tyr (6:3:1) were also phosphorylated by the insulin-stimulated kinase but to a lower extent. A tyrosine kinase stimulated by insulin-like growth factor-1, and one stimulated by phorbol ester also best phosphorylated the polymer Glu/Tyr (4:1). The three kinases differed only in their capability to phosphorylate Glu/Ala/Tyr (6:3:1) or Ala/Glu/Lys/Tyr (6:2:5:1). Glu/Tyr (4:1) was a poor substrate for the epidermal growth factor receptor kinase which best phosphorylated the polymer Glu/Ala/Tyr (6:3:1). Three additional polymers: Glu/Tyr (1:1), Glu/Ala/Tyr (1:1:1), and Lys/Tyr (1:1) failed to serve as substrates for all four tyrosine kinases tested. Taken together these findings suggest that. Hormone-sensitive tyrosine kinases have similar yet distinct substrate specificity and are likely to phosphorylate their native substrates on tyrosines adjacent to acidic (glutamic) residues. Tyrosine-containing polymer substrates are highly sensitive and convenient tools to study (hormone-sensitive) tyrosine kinases whose native substrates are unknown or present at low concentrations.
Zick
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Grunberger
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Rees-Jones
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Comi
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D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D006728:Hormones / Q000502:physiology*; D006801:Humans; D007328:Insulin / Q000502:physiology*; D008214:Lymphocytes / Q000201:enzymology; D051379:Mice; D010766:Phosphorylation; D011108:Polymers / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D051381:Rats; D011972:Receptor, Insulin; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D006728:Hormones; D007328:Insulin; D011108:Polymers; D014443:Tyrosine; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin