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.

Abstract

PHAS-I is a heat-and acid-stable protein that is phosphorylated on Sert" residues in response to insulin and growth factors."0 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 K,,, and V,, 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 11 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-~-Asn-Ser-Pro-Val-Ala-Lys-Tbr. was released in the seventh cycle of Edman degradation, identifying the Ser (SerM) as the phosphoryla~d residue.SerM was also phosphorylated in response to insulin i n rat adipocytes.W e conclude that PHAS-I is a substrate for MAP kinase both in uiuo and in vitro.As PHAS-I i s one of the most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these cells.Insulin and growth factors that signal via protein tyrosine kinase receptors share a common signaling pathway that involves the sequential activation of the S e r m r kinases, raf-1, MEK, and MAP' kinase (1,2).Raf-1 and MEK appear to have few if any substrates other than MEK and MAP kinase, respectively.In contrast, MAP kinase phosphorylates several proteins including the epidermal growth factor receptor (31, cytoplasmic