One of the well-characterized roles of the SCFGrr1
E3 ubiquitin ligase is to promote nutrient uptake by regulating nutrient permease gene expression. Here we describe a previously unrecognized target, Npr2, identified based upon a physical interaction with Grr1 observed by mass spectrometry-based MudPIT analysis. Although Npr2 is a relatively stable protein, its turnover is dependent upon Grr1. Previous genome-wide approaches identified Npr2 as a ubiquitinated protein but failed to reveal its regulation by Grr1, presumably because of its relatively low abundance (4
). As is typical of SCFGrr1
substrates, Npr2 is a phosphoprotein. Both its phosphorylation and instability are dependent upon CK1, a common feature of Grr1-dependent targets involved in nutrient regulation (1
). Accumulation of hyperphosphorylated Npr2 is associated with inactivation of Grr1.
Npr2 is required for efficient growth on some defined nitrogen sources and has been proposed to be a regulator of nitrogen permeases (24
). Although the precise role of Npr2 is unclear, it was originally identified based upon a defect of the mutant in growth on urea and proline as a nitrogen source, a property shared with Npr1, a protein kinase involved in the regulation of nitrogen permeases (5
). We have shown the phenotype of the npr2
mutant to include slow growth and compromised mass accumulation when it is grown on several nonoptimal nitrogen sources. These observations are consistent with a recent report from Neklesa and Davis (23
) showing that a complex of Npr2 and Npr3 plays an inhibitory role upstream of TORC1, an important regulator of protein synthesis and amino acid uptake. Genetic analysis performed in the context of that study suggests that Npr2 acts to restrict TORC1 function in the absence of glutamine, an optimal nitrogen source, thereby promoting the expression of nitrogen metabolite-repressible genes and decreasing the rate of ribosome biosynthesis. The metabolism of glutamine is unaffected by inactivation of Npr2. We hypothesized that the defect in growth on ammonia might be a consequence of misregulation of ammonium uptake rather than in its metabolism. In fact, inhibition of TOR by rapamycin has been reported to induce expression of the major ammonium permease encoded by MEP2
). However, we find that regulation of MEP2
expression is intact in the npr2
Δ mutant (data not shown), suggesting that any defect must occur either at the level of the permease itself or in some other aspect of ammonium assimilation.
Npr2 is a relatively stable protein with a half-life of greater than an entire cell cycle. However, Npr2 turnover is dependent upon Grr1 and, like that of several other targets of SCFGrr1
, Npr2 turnover depends upon CK1. The long half-life of Npr2 is unexpected for targets of ubiquitin-mediated degradation but might reflect our failure to identify conditions that favor degradation. For example, Mth1 appears to be rather stable in cells growing on nonglucose carbon sources but is rapidly degraded via phosphorylation-dependent ubiquitination by SCFGrr1
when glucose is added to the growth medium, which, in turn, induces glucose permease gene expression (10
). A similar phenomenon involving Grr1 may accelerate Npr2 turnover. Although Npr2 appears to be dispensable in cells growing in rich medium and therefore might be expected to be degraded under those conditions, neither the phosphorylation state nor accumulation of Npr2 appears to be strongly affected by the nitrogen source. We do know that hyperaccumulation of Npr2 is detrimental to cells and that it becomes lethal when Grr1 is inactivated. However, we do not know whether similar hyperaccumulation occurs under specific conditions in wild-type cells. Instead, Grr1 may be involved in a homeostatic mechanism regulating the overall abundance of Npr2 protein.
The requirement of casein kinase for the phosphorylation and instability of Npr2 is shared with several other Grr1 targets involved in the regulation of nutrient permease gene expression. In the case of Mth1, a corepressor of HXT
genes that is inactivated by glucose, phosphorylation by Yck1/2 is a prerequisite for recognition by SCFGrr1
). The target in the pathway leading to the activation of amino acid permeases is currently unknown. Both glucose and amino acid permeases are induced when nutrient uptake is desirable. Although our data point to Npr2 as a target of Yck1/2, neither the importance of turnover nor the stimulus leading to that turnover is understood.
Loss of Npr2 function has been reported to lead to resistance to the genotoxic agents doxorubicin, a topoisomerase II inhibitor, and cisplatin, which induces DNA damage by forming platinum-DNA adducts, two clinically important chemotherapeutics (11
). That phenotype is shared with mutants deficient in the Sky1 serine-rich protein-specific kinase. Resistance to a broad range of other compounds is not observed, and there is no evidence that this is related to a defect in uptake. Although the mechanism by which either of these genes confers resistance to the drugs is unknown, NPR2
are members of the same epistasis group, indicating that they function in the same pathway. This is consistent with the observation that both npr2
Δ and sky1
Δ mutants have a mild mutator phenotype (25
). Sky1 activity has been associated with mismatch repair defects and other aberrations of DNA metabolism. Although the NPR2 SKY2
epistasis could indicate a pathway in which Npr2 is phosphorylated by the Sky2 kinase, it seems unlikely because Npr2 lacks consensus sites for Sky2 (32
) and because we have shown that the majority of Npr2 phosphorylation is lost in the absence of CK1 activity, eliminating the need to invoke the involvement of another kinase. Whether CK1 or Grr1 contributes to the role of Npr2 in modulating sensitivity to genotoxic agents is not known.
The closest human homolog of Npr2 is the tumor suppressor encoded by NPRL2
). Inactivation of that protein, like that of its yeast counterpart, leads to cisplatin resistance, rendering cells refractory to this common cancer chemotherapeutic agent (29
). Also, like its yeast ortholog, Nprl2 has been shown to form a complex with Nprl3, although its role as a TOR pathway regulator has not been established (23
). Despite the fact that the physiological roles of Npr2 and Nprl2 are unknown, the shared phenotype of cisplatin resistance suggests that those roles at least partially overlap. Similarly, since the regulation of the Nprl2 protein has yet to be determined, it is not known whether it is a target for phosphorylation or ubiquitin-mediated proteolysis. However, based upon the similarity between these two proteins and the phenotypes of mutants in them, we suggest that Nprl2 may be a target for SCF-mediated ubiquitylation and subsequent turnover. If so, aberrant regulation of Nprl2 abundance could be important in the development and treatment of human cancer.