Identification of Phosphorylation Sites in the Translational Regulator, PHAS-I, That Are Controlled by Insulin and Rapamycin in Rat Adipocytes

doi:10.1074/jbc.272.15.10240

Identification of Phosphorylation Sites in the Translational Regulator, PHAS-I, That Are Controlled by Insulin and Rapamycin in Rat Adipocytes*

From the Departments of ^‡ Molecular Physiology and Biological Physics,
^§ Pharmacology, and
^¶ Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908

^∥ To whom correspondence should be addressed:
Dept. of Pharmacology, Health Sciences Center Box 448, University of Virginia School of Medicine, 1300 Jefferson Park Ave. Charlottesville, VA 22908.
Tel.: 804-924-1584; Fax: 804-982-3878; E-mail: jcl3p{at}avery.med.virginia.edu

Abstract

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase in vitro decreased PHAS-I binding to eukaryotic initiation factor (eIF)-4E. The decrease in binding lagged behind the phosphorylation of PHAS-I in Ser⁶⁴, the preferred site of MAP kinase. Binding of the Ala⁶⁴ mutant of PHAS-I to eIF-4E was abolished by MAP kinase, indicating that phosphorylation of sites other than Ser⁶⁴ control binding. To identify such sites, PHAS-I was phosphorylated with MAP kinase and [γ-³²P]ATP and then cleaved proteolytically before the resulting phosphopeptides were isolated by reverse phase chromatography and directly identified by amino acid sequencing. Phosphorylated residues were located by determining the cycles in which ³²P was released when phosphopeptides were subjected to sequential Edman degradation. With an extended incubation in vitro, MAP kinase phosphorylated Thr³⁶, Thr⁴⁵, Ser⁶⁴, Thr⁶⁹, and Ser⁸². In rat adipocytes, the phosphorylation of all five sites was increased by insulin and decreased by rapamycin although there were differences in the magnitude of the effects. A form of PHAS-I phosphorylated exclusively in Thr³⁶ remained bound to eIF-4E, indicating that phosphorylation of Thr³⁶ is insufficient for dissociation of the PHAS-I·eIF-4E complex. In summary, our results indicate that multiple phosphorylation sites are involved in the control of PHAS-I. All five sites identified fit a (Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in cells is mediated by a proline-directed protein kinase.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants DK52753 and DK28312 and the Markey Center for Cellular Signaling. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MAP kinase

mitogen-activated protein kinase

ERK

extracellular receptor regulated protein kinase

HPLC

high performance liquid chromatography

mTOR

mammalian target of rapamycin

MEK

MAP/ERK kinase

PAGE

polyacrylamide gel electrophoresis.
↵² P. Fadden, T. A. J. Haystead, and J. C. Lawrence, Jr., unpublished data.
↵³ The Ser⁶⁴ phosphopeptide contains a cysteine residue, which may be oxidized to cysteic acid. Partial oxidation of this residue was previously shown to account for the resolution of two peaks of a comparable Ser⁶⁴ phosphopeptide (24).
- Received December 2, 1996.
- Revision received January 21, 1997.