INSULIN-STIMULATED PHOSPHORYLATION OF RECOMBINANT PP120/HA4, AN ENDOGENOUS SUBSTRATE OF THE INSULIN-RECEPTOR TYROSINE KINASE

Insulin binding to the alpha-subunit of its receptor stimulates the receptor tyrosine kinase to phosphorylate the beta-subunit and several endogenous protein substrates, including pp120/HA4, a liver-specific plasma membrane glycoprotein of M(r) 120 000. Analysis of the deduced amino acid sequence of rat liver pp1201HA4 revealed two potential sites for tyrosine phosphorylation in the cytoplasmic domain (Tyr(488) and Tyr(513)), as well as a potential cAMP-dependent protein kinase phosphorylation site (Ser(503)). To determine which of these sites is phosphorylated in response to insulin, each of these amino acid residues was altered by site-directed mutagenesis. Mutant cDNAs were then expressed by stable transfection in NIH 3T3 cells. Two mutations (Phe(488) and Ala(503)) impaired insulin-induced phosphorylation of pp120/HA4, suggesting that pp120/HA4 undergoes multisite phosphorylation. It seems likely that Tyr(488) is phosphorylated by the insulin receptor kinase, and phosphorylation of Ser(513) may contribute to the regulation of tyrosine phosphorylation. Since pp120/HA4 is believed to be associated with a Ca2+/Mg2+-dependent ecto-ATPase activity, we determined the effects of insulin-induced phosphorylation on this enzymatic activity. In NIH 3T3 cells co-expressing the insulin receptor and pp120/HA4, insulin caused a 2-fold increase in ecto-ATPase activity. Moreover, elimination of the phosphorylation sites of pp120/HA4 impaired the ability of insulin to stimulate the ecto-ATPase activity. These data suggest that tyrosine phosphorylation of pp120/HA4 may regulate Ca2+/Mg2+-dependent ecto-ATPase activity.