Functional analysis of the "Saccharomyces cerevisiae" Npr1 protein kinase

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.