In vitro phosphorylation of the adipocyte lipid-binding protein (p15) by the insulin receptor: Effects of fatty acid on receptor kinase and substrate phosphorylation

Phosphorylation of the adipocyte lipid-binding protein (ALBP) isolated from 3T3-L1 cells has been studied in vitro utilizing the wheat germ agglutinin-purified 3T3-L1 adipocyte insulin receptor and the soluble kinase domain of the human insulin receptor. Following insulin-stimulated, ATP-dependent autophosphorylation of the wheat germ agglutinin-purified receptor β-subunit, ALBP was phosphorylated exclusively on tyrosine 19 in the sequence Glu-Asn-Phe-Asp-Asp-Tyr¹⁹, analogous to the substrate phosphorylation consensus sequence observed for several tyrosyl kinases. The concentration of insulin necessary for half-maximal receptor autophosphorylation (K_0.5^IR) was identical to that necessary for half-maximal ALBP phosphorylation (K_0.5^ALBP), 10 nM. Kinetic analysis indicated that stimulation of ALBP phosphorylation by insulin was attributable to a 5-fold increase in the V_max (to 0.33 fmol/min/fmol insulin-binding sites) while the K_m for ALBP was largely unaffected. By utilizing the soluble kinase domain of the human receptor β-subunit, the presence of oleate bound to ALBP increased the k_cat/ K_m greater than 3-fold. Oleate dramatically inhibited autophosphorylation of the 38-kDa fragment of the soluble receptor kinase in a concentration dependent fashion (I_0.5 ∼ 4 μM). The 48-kDa kinase exhibited much less sensitivity to the effects of oleate (I0.5 ∼ 190 μM). The inhibition of autophosphorylation of the 48-kDa soluble kinase by oleate was reversed by adding saturating levels of ALBP. These results demonstrate that in vitro the murine adipocyte lipid-binding protein is phosphorylated on tyrosine 19 in an insulin-stimulated fashion by the insulin receptor and that the presence of a bound fatty acid on ALBP increases the affinity of insulin receptor for ALBP. Inhibition of insulin receptor kinase activity by unbound fatty acids suggests that the end products of the lipogenic pathway may feedback inhibit the tyrosyl kinase and that fatty acid-binding proteins have the potential to modulate such interaction.