Identification of the rapamycin‐sensitive phosphorylation sites within the Ser/Thr‐rich domain of the yeast Npr1 protein kinase

Abstract

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.

Reciprocal Regulation of Target of Rapamycin Complex 1 and Potassium Accumulation

“…3F, the ratio of phosphorylated Npr1 is very similar in wild type strains treated with the TORC1 activator and in the strains lacking the Hal4 and Hal5 kinases. As stated, this phosphorylation has been correlated with a decrease in Npr1 activity and thus is consistent with the observations described above suggesting that Npr1 activity is reduced in hal4 hal5 mutants (10,32). Interestingly, this increased phosphorylation is also observed in low potassium conditions in the wild type strain (fifth lane, Fig.…”

“…Moreover, the accumulation of the Npr1 kinase is reduced in these mutants, and the phosphorylation levels of the remaining Npr1 protein are also increased. Phosphorylation of Npr1 has been shown to correlate with decreased activity of the kinase; therefore, the levels of active Npr1 are markedly reduced in the hal4 hal5 mutant (10,32). Moreover, we observed that a decrease in Npr1 activity leads to an over-accumulation of the active form of the Rsp5 adaptor, Art1.…”

“…Several reports have thoroughly demonstrated that the level of Npr1 phosphorylation is controlled by the TORC1 protein kinase complex, increasing its phosphorylation level under conditions of TORC1 activation and becoming dephosphorylated upon TORC1 inhibition (10,27,32). Moreover, these studies have shown that TORC1-dependent phosphorylation of Npr1 leads to decreased Nrp1 activity.…”

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…Npr1 is required for stabilization of Gap1 at the plasma membrane and induces the degradation of Tat2, possibly by regulating their ubiquitination (Schmidt et al 1998;Springael and Andre 1998;De Craene et al 2001;Helliwell et al 2001;Soetens et al 2001;Springael et al 2002). TORC1 activity, through control of the Tap42-Sit4 phosphatase complex, promotes the phosphorylation of Npr1 (Schmidt et al 1998;Jacinto et al 2001;Gander et al 2008). Presumably, this phosphorylation inhibits Npr1 which would promote Tat2 stabilization and Gap1 degradation.…”

The TORC1 effector kinase Npr1 fine tunes the inherent activity of the Mep2 ammonium transport protein

“…The Npr1 (nitrogen permease reactivator 1) kinase is a TORC1 effector responding to the quality of the nitrogen supply and controlling the stability of nitrogen source transporters 4,5 . During poor nitrogen supply or rapamycin treatment, namely under conditions that downregulate TORC1, Npr1 is weakly phosphorylated and assumed to be active, while the presence of a preferred nitrogen source leads to increased phosphorylation of Npr1 and to theoretical inactivation of the kinase 4,6,7 . Npr1-dependent phosphorylation of arrestin-like proteins prevents recruitment of the Rsp5 ubiquitin-ligase to its plasma-membrane targets, thus protecting them from ubiquitylation, endocytosis and lysosomal degradation 2,3 .…”