The uptake and processing of nutrients is highly regulated. Cells adapt to changes of the availability of nutrients to provide a complete set of transporters and metabolizing enzymes for optimal use of the available nutrients. The Saccharomyces cerevisiae nitrogen permease reactivator protein (NPR1) plays an important role in nitrogen regulation by controlling the sorting and stability of several amino acid permeases. Npr1 is a hyperphosphorylated protein that belongs to a fungusspecific family of Ser/Thr protein kinases. Its activity is regulated by the TOR (target of rapamycin) signaling pathway. Inhibition of TOR by the immunosuppressant drug rapamycin or growth on a poor nitrogen source causes dephosphorylation of Npr1 by the Sit4 phosphatase. Previously, the rapamycin sensitive phosphorylation sites had been determined. They are clustered in two regions of an N-terminal serine-rich domain of Npr1. Besides in vivo phosphorylation, Npr1 underwent intense autophosphorylation when assayed in vitro. Investigation of Npr1 autophosphorylation revealed three autophosphorylation sites previously mapped in untreated GST-Npr1. Autophosphorylation had no regulatory effect on Npr1 kinase activity. To learn more about Npr1 substrate requirements, a set of classical protein kinase substrates were tested. From a set of basic proteins, myelin basic protein (MBP) was found to be an optimal substrate for Npr1. To find physiologically relevant Npr1 substrates, a KESTREL-based approach disclosed ribosomal protein Rpl24a as an excellent substrate for Npr1. The Npr1 consensus sequence was investigated with a set of peptides designed around the phosphorylation site of Rpl24a. Basic residues at position P-3 and P+1 are crucial determinants of the consensus sequence. Since Rpl24a is unlikely to be a bona fide substrate, an interactor-based substrate screen with GST-Npr1 as bait was carried out. Physiologically relevant interactors were the ubiquitin ligase Rsp5 and the AMP-activated Ser/Thr protein kinase Snf1 and its two subunits Snf4 and Gal83. The Npr1-Rsp5 interaction was immunologically confirmed and shown by point mutations to be specific. However, Rsp5 turned out not to be a direct protein substrate for Npr1. On the other hand, Npr1 was not ubiquitinated when tested with anti-ubiquitin antibodies. Functional analysis of the N-terminal serine-rich domain of Npr1 indicated a substrate binding domain between residues 252-413 that anchors the substrate for phosphorylation by the catalytic domain. However, the significance of this finding needs to be clarified with respect to the function of Npr1 in vivo where the N-terminal domain proved to be dispensable for growth and permease sorting.