Phosphorylation of PHAS-I by Mitogen-Activated Protein (MAP) Kinase. Identification of a Site Phosphorylated by MAP Kinase in Vitro and in Response to Insulin in Rat Adipocytes

J Biol Chem. 1994 Sep 16;269(37):23185-91.


PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors. To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein (MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these cells.

Publication types

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

MeSH terms

  • Adipocytes / drug effects
  • Adipocytes / metabolism*
  • Amino Acid Sequence
  • Animals
  • Carrier Proteins*
  • Insulin / pharmacology*
  • Intracellular Signaling Peptides and Proteins
  • Male
  • Mitogen-Activated Protein Kinase 1
  • Molecular Sequence Data
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Tyrosine Kinases / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Recombinant Proteins / metabolism


  • Carrier Proteins
  • Eif4ebp1 protein, rat
  • Insulin
  • Intracellular Signaling Peptides and Proteins
  • Phosphoproteins
  • Recombinant Proteins
  • Protein-Tyrosine Kinases
  • Protein-Serine-Threonine Kinases
  • Mitogen-Activated Protein Kinase 1