Work

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.

Year: 1994

Type: article

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

Source: Journal of Biological Chemistry

DOI: 10.1016/s0021-9258(17)31637-x

Authors: T.A. Haystead, C.M. Haystead, C. Hu, T.A. Lin, J.C. Lawrence

Institutions: Washington University in St. Louis, University of Virginia

Language: English

Topic: Protein Kinase Regulation and GTPase Signaling

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,430

Funders: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases