Multisite phosphorylation of the Saccharomyces cerevisiae filamentous growth regulator Tec1 is required for its recognition by the E3 ubiquitin ligase adaptor Cdc4 and its subsequent destruction in vivo.
In Saccharomyces cerevisiae, the pheromone-induced ubiquitylation and degradation of the filamentation pathway-specific activator, Tec1, suppresses cross talk between the mating and filamentous growth mitogen-activated protein kinase (MAPK) pathways. The mating pathway MAPK, Fus3, phosphorylates Tec1, resulting in its recognition by the SCF (for Skp1, Cullin, F-box containing) E3 ubiquitin ligase complex, leading to its proteolysis. Previously, it was found that Tec1 destruction requires phosphorylation on threonine 273 (T273). T273 is embedded in the sequence LLpTP, which is identical to the canonical binding site for Cdc4, a conserved F-box substrate adaptor for the SCF complex. However, recent work on both Cdc4 and the human Cdc4 ortholog Fbw7 has shown that a second substrate phosphorylation can be required for optimal Cdc4 binding in vitro. We report here that high-affinity binding of recombinant Cdc4 to Tec1 phosphopeptides requires phosphorylation of not only T273 but also a second site, T276. Significantly, both phospho-sites on Tec1 and a conserved basic pocket on Cdc4 are critical for Tec1 proteolysis in response to pheromone treatment of cells, establishing a role for two-phosphate recognition by yeast Cdc4 in substrate targeting in vivo.
Bao
Marie Z
MZ
Department of Biochemistry and Biophysics, University of California, 600 16th St., MC2200, San Francisco, CA 94158-2200, USA.
Shock
Teresa R
TR
Madhani
Hiten D
HD
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
C080443:CDC4 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D044783:F-Box Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C069926:TEC1 protein, S cerevisiae; D014157:Transcription Factors; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases