p85α gene generates three isoforms of regulatory subunit for phosphatidylinositol 3-kinase (PI 3-kinase), p50α, p55α, and p85α, with different PI 3-kinase activity elevating responses to insulin

Abstract

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine- phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85α, but the SH3 and ber homology domains of p85α were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85α gene product. We suggest that this protein be called p50α. Northern blotting using a specific DNA probe corresponding to p50α revealed 6.0- and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50α protein and its associated Pl 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50α. Taken together, these observations indicate that the p85α gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85α, p55α, and p50α), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50α forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85α and p55α associate only with p110 that can be wortmannin labeled. Furthermore, p50α exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosine-phosphorylated IRS-1 than the other isoforms. Considering the high level of p50α expression in the liver and its marked responsiveness to insulin, p50α appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three α isoforms has a different function and may have specific roles in various tissues.