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

Arch Biochem Biophys. 2002 Jul 1;403(1):71-82. doi: 10.1016/S0003-9861(02)00261-8.

Abstract

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-zeta (PKC-zeta) abolish the insulin-induced GLUT4 recruitment in rat adipocytes. These findings suggest an interesting possibility that PKC-zeta may phosphorylate IRAP, playing a key role in GLUT4/IRAP recruitment. To test this possibility, here we studied the (32)P incorporation into IRAP catalyzed by PKC-zeta in insulin-stimulated cells. There was a small but significant (32)P incorporation into IRAP in rat adipocytes, which was partly abolished upon addition of a PKC-zeta pseudosubstrate, suggesting that PKC-zeta may be responsible in part for the IRAP phosphorylation in adipocytes. PKC-zeta also catalyzed the incorporation of (32)P 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-zeta. Reversed-phase HPLC, matrix-assisted laser desorption ionization mass spectrometry, and radiosequencing of the tryptic digests of the (32)P-labeled IRAP fusion proteins identified Ser-80 and Ser-91 as major phosphorylation sites. In GLUT4 vesicles, the (32)P incorporation into IRAP was exclusively localized at a 6.9-kDa tryptic fragment identified as IRAP(76-138) and the (32)P 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-zeta plays a key role in insulin-induced IRAP or GLUT4 recruitment in adipocytes.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adipocytes / enzymology*
  • Amino Acid Sequence
  • Aminopeptidases / metabolism*
  • Animals
  • Blotting, Western
  • Catalysis
  • Cell Membrane / enzymology
  • Chromatography, High Pressure Liquid
  • Cystinyl Aminopeptidase
  • Cytoplasm / enzymology
  • Enzyme Inhibitors / pharmacology
  • Glucose Transporter Type 4
  • Metalloendopeptidases / metabolism
  • Molecular Sequence Data
  • Monosaccharide Transport Proteins / metabolism
  • Muscle Proteins*
  • Phosphorylation
  • Precipitin Tests
  • Protein Binding
  • Protein Kinase C / metabolism*
  • Protein Structure, Tertiary
  • Rats
  • Serine / metabolism*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Subcellular Fractions

Substances

  • Enzyme Inhibitors
  • Glucose Transporter Type 4
  • Monosaccharide Transport Proteins
  • Muscle Proteins
  • Slc2a4 protein, rat
  • Serine
  • protein kinase C zeta
  • Protein Kinase C
  • Aminopeptidases
  • Cystinyl Aminopeptidase
  • leucyl-cystinyl aminopeptidase
  • Metalloendopeptidases
  • peptidyl-Lys metalloendopeptidase