PHOSPHORYLATION OF PHAS-I BY MITOGEN-ACTIVATED PROTEIN (MAP) KINASE -IDENTIFICATION OF A SITE PHOSPHORYLATED BY MAP KINASE IN-VITRO AND INRESPONSE TO INSULIN IN RAT ADIPOCYTES

PHAS-I is a heat- and acid-stable protein that is phosphorylated on Se r/Thr residues in response to insulin and growth factors. To investiga te the phosphorylation of PHAS-I, the protein was expressed in bacteri a and purified for use as substrate in protein kinase reactions in vit ro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylat ed by mitogen-activated protein (MAP) kinase. At saturating MgATP, the K-m and V-max observed with PHAS-I were almost identical to those obt ained with myelin basic protein, one of the best MAP kinase substrates . PHAS-I was also phosphorylated at a significant rate by casein kinas e II and protein kinase C. To investigate sites of phosphorylation, PH AS-I was digested with collagenase and phosphopeptides were resolved b y reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase was recovered in the peptide, L eu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. P-32 was released in t he seventh cycle of Edman degradation, identifying the Ser (Ser(64)) a s the phosphorylated residue. Ser(64) was also phosphorylated in respo nse to insulin in rat adipocytes. We conclude that PHAS-I is a substra te for MAP kinase both in vivo and in vitro. As PHAS-I is one of the m ost prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these cells.