Protein kinase C-ζ phosphorylates insulin-responsive aminopeptidase in vitro at Ser-80 and Ser-91

Insulin-responsive aminopeptidase (IRAP) colocalizes with glucose transporter type 4 (GLUT4) in adipocytes and is recruited to the plasma membrane in response to insulin. Microinjection of peptides corresponding to the IRAP cytoplasmic domain sequences causes GLUT4 recruitment in adipocytes. Inhibitors of protein kinase C-ζ (PKC-ζ) abolish the insulin-induced GLUT4 recruitment in rat adipocytes. These findings suggest an interesting possibility that PKC-ζ may phosphorylate IRAP, playing a key role in GLUT4/IRAP recruitment. To test this possibility, here we studied the 32P incorporation into IRAP catalyzed by PKC-ζ in insulin-stimulated cells. There was a small but significant 32P incorporation into IRAP in rat adipocytes, which was partly abolished upon addition of a PKC-ζ pseudosubstrate, suggesting that PKC-ζ may be responsible in part for the IRAP phosphorylation in adipocytes. PKC-ζ also catalyzed the incorporation of 32P not only into IRAP in GLUT4 vesicles isolated from rat adipocytes but also into the IRAP cytoplasmic domain inserts in glutathione S-transferase-fusion proteins, demonstrating direct IRAP phosphorylation by PKC-ζ. Reversed-phase HPLC, matrix-assisted laser desorption ionization mass spectrometry, and radiosequencing of the tryptic digests of the 32P-labeled IRAP fusion proteins identified Ser-80 and Ser-91 as major phosphorylation sites. In GLUT4 vesicles, the 32P incorporation into IRAP was exclusively localized at a 6.9-kDa tryptic fragment identified as IRAP76–138 and the 32P labeling at Ser-80 accounted for 80–90% of the total IRAP labeling, suggesting that Ser-80 is the major phosphorylation site in intact IRAP. These findings are consistent with the possibility that the IRAP cytoplasmic domain phosphorylation by PKC-ζ plays a key role in insulin-induced IRAP or GLUT4 recruitment in adipocytes.