Phosphorylation of the RGS protein Sst2 by the MAP kinase Fus3 and use of Sst2 as a model to analyze determinants of substrate sequence specificity.
Previously, we used mass spectrometry to demonstrate pheromone-stimulated phosphorylation of Ser-539 in Sst2, a regulator of G protein signaling in yeast Saccharomyces cerevisiae [Garrison, T. R., et al. (1999) J. Biol. Chem. 274, 36387-36391]. Here, we show that Sst2 phosphorylation is mediated by the mitogen-activated protein (MAP) kinase Fus3. Phosphorylation occurs within a canonical MAP kinase phosphorylation site (Pro-X-Ser/Thr-Pro, where "X" at the -1 position can be any amino acid), a consensus sequence deduced earlier from analysis of synthetic peptide substrates. In a direct test of the model, we compared Sst2 phosphorylation following systematic substitution of the -1 residue His-538. Each of the substitution mutants was suitable as a MAP kinase substrate, as shown by phosphorylation-dependent mobility shifts in vivo and/or by direct phosphorylation in vitro followed by peptide mapping and mass spectrometry sequencing. This analysis documents phosphorylation of Sst2 by Fus3 and demonstrates that the prevailing model for MAP kinase recognition is valid for a native substrate protein in vivo as well as for small synthetic peptides tested in vitro.
Parnell
Stephen C
SC
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260, USA.
Marotti
Louis A
LA
Kiang
Lee
L
Torres
Matthew P
MP
Borchers
Christoph H
CH
Dohlman
Henrik G
HG
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D016384:Consensus Sequence; D025461:Feedback, Physiological / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010649:Phenylalanine / Q000235:genetics; D010766:Phosphorylation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D014364:Tryptophan / Q000235:genetics; D020710:RGS Proteins
D000409:Alanine; D020690:GTPase-Activating Proteins; D006639:Histidine; D020928:Mitogen-Activated Protein Kinases; D010649:Phenylalanine; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D014364:Tryptophan; C094301:SST2 protein, S cerevisiae; D020710:RGS Proteins; C075580:FUS3 protein, S cerevisiae