Identification of the rapamycin-sensitive phosphorylation sites within the Ser/Thr-rich domain of the yeast Npr1 protein kinase.
The Saccharomyces cerevisae nitrogen permease reactivator Npr1 is a hyperphosphorylated protein that belongs to a fungus-specific family of Ser/Thr protein kinases dedicated to the regulation of plasma membrane transporters. Its activity is regulated by the TOR (target of rapamycin) signalling pathway. Inhibition of the TOR proteins by treating yeast cells with the immunosuppressant drug rapamycin promotes rapid dephosphorylation of Npr1. To identify the rapamycin-sensitive phosphorylation sites in yeast Npr1, glutathione-S-transferase (GST)-tagged Npr1 was isolated from untreated or rapamycin-treated cells, and analyzed by mass spectrometry. Here, we report for the first time 22 phosphorylation sites that are clustered in two regions of the N-terminal serine-rich domain. All phosphorylation sites, except two, were found to be rapamycin-sensitive. Some phosphorylation sites are contained in motifs that closely resemble those in mammalian S6K (serines followed by a tyrosine or a phenylalanine) and 4E-BP1 (serines followed by a proline). Other sites, such as serines followed by Ala, Asn, Gln, His, Ile, Leu, or Val, appear to define new motifs. Thus, TOR controls an unusually broad array of phosphorylation sites in Npr1. In addition to phosphorylation by upstream kinases, Npr1 undergoes autophosphorylation that was mapped to three distinct serines in the N-terminal domain of which Ser257 appears to be the main autophosphorylation site. Site-directed mutagenesis confirmed the mass spectral assignments of the autophosphorylation sites and shows that Ser257 is specifically involved in forming an in vitro substrate-binding site.
Gander
Stefan
S
Department of Biochemistry, Biozentrum of the University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland.
Bonenfant
Debora
D
Altermatt
Patrick
P
Martin
Dietmar E
DE
Hauri
Simon
S
Moes
Suzette
S
Hall
Michael N
MN
Jenoe
Paul
P
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002853:Chromatography, Liquid; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology*; D053719:Tandem Mass Spectrometry
D029701:Saccharomyces cerevisiae Proteins; C066958:NPR1 protein, S cerevisiae; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C500749:target of rapamycin protein, S cerevisiae; D009584:Nitrogen; D020123:Sirolimus