Biomedical subjects

Jeremy Thorner

Publications and source records attributed to Jeremy Thorner.

21 records · Page 2Linked to original sources

PubMed2003

Regulation of Ste7 ubiquitination by Ste11 phosphorylation and the Skp1-Cullin-F-box complex.

Ste7 is a mitogen-activated protein kinase kinase that mediates pheromone signaling in Saccharomyces cerevisiae. We showed previously that Ste7 is ubiquitinated upon prolonged stimulation by pheromone and that accumulation of ubiquitinated Ste7 results in enhanced transcription and cell division arrest responses (Wang, Y., and Dohlman, H. G. (2002) J. Biol. Chem. 277, 15766-15772). We now report that ubiquitination of Ste7 requires Ste11 kinase and Skp1/Cullin/F-box (SCF) ubiquitin-conjugating activities. Ste7 is not ubiquitinated in Ste11-deficient cells or when the Ste11 phosphorylation sites have been mutated. Ste7 ubiquitination and degradation (but not phosphorylation) is specifically blocked in mutants defective for the E2 ubiquitin-conjugating enzyme Cdc34 or the cullin homologue Cdc53. Both are components of the SCF complex that ubiquitinates proteins during the G1-S transition of the cell cycle. Our findings suggest that SCF promotes the ubiquitination and degradation of Ste7, thereby favoring the resumption of cell division cycling after pheromone-induced growth arrest.

Cell Cycle Proteins↗

PubMed2002

Molecular interactions of yeast frequenin (Frq1) with the phosphatidylinositol 4-kinase isoform, Pik1.

Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis of an Frq1-binding fragment, Pik1-(10-192), showed that residues within 80-192 are necessary and sufficient for Frq1 association in vitro. A synthetic peptide (residues 151-199) competed for binding of [(35)S]Pik1-(10-192) to bead-immobilized Frq1, whereas shorter peptides (164-199 and 174-199) did not. Correspondingly, a deletion mutant, Pik1(delta152-191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature. Site-directed mutagenesis of Pik1-(10-192) suggested that recognition determinants lie over an extended region. Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110-192), or the 151-199 peptide to Frq1 shows high affinity (K(d) approximately 100 nm) and is largely entropic, consistent with hydrophobic interaction. Stoichiometry of Pik1-(110-192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography. Cys-15 in Frq1 is not S-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Delta cells. This study defines the segment of Pik1 required for high affinity binding of Frq1.

1-Phosphatidylinositol 4-Kinase↗

PubMed2002

Pkh1 and Pkh2 differentially phosphorylate and activate Ypk1 and Ykr2 and define protein kinase modules required for maintenance of cell wall integrity.

Saccharomyces cerevisiae Pkh1 and Pkh2 are functionally redundant homologs of mammalian protein kinase, phosphoinositide-dependent protein kinase-1. They activate two closely related, functionally redundant enzymes, Ypk1 and Ykr2 (homologs of mammalian protein kinase, serum- and glucocorticoid-inducible protein kinase). We found that Ypk1 has a more prominent role than Ykr2 in mediating their shared essential function. Considerable evidence demonstrated that Pkh1 preferentially activates Ypk1, whereas Pkh2 preferentially activates Ykr2. Loss of Pkh1 (but not Pkh2) reduced Ypk1 activity; conversely, Pkh1 overexpression increased Ypk1 activity more than Pkh2 overexpression. Loss of Pkh2 reduced Ykr2 activity; correspondingly, Pkh2 overexpression increased Ykr2 activity more than Pkh1 overexpression. When overexpressed, a catalytically active C-terminal fragment (kinase domain) of Ypk1 was growth inhibitory; loss of Pkh1 (but not Pkh2) alleviated toxicity. Loss of Pkh2 (but not Pkh1) exacerbated the slow growth phenotype of a ypk1Delta strain. This Pkh1-Ypk1 and Pkh2-Ykr2 dichotomy is not absolute because all double mutants (pkh1Delta ypk1Delta, pkh2Delta ypk1Delta, pkh1Delta ykr2Delta, and pkh2Delta ykr2Delta) were viable. Compartmentation contributes to selectivity because Pkh1 and Ypk1 were located exclusively in the cytosol, whereas Pkh2 and Ykr2 entered the nucleus. At restrictive temperature, ypk1-1(ts) ykr2Delta cells lysed rapidly, but not in medium containing osmotic support. Dosage and extragenic suppressors were selected. Overexpression of Exg1 (major exoglucanase), or loss of Kex2 (endoprotease involved in Exg1 processing), rescued growth at high temperature. Viability was also maintained by PKC1 overexpression or an activated allele of the downstream protein kinase (BCK1-20). Conversely, absence of Mpk1 (distal mitogen-activated protein kinase of the PKC1 pathway) was lethal in ypk1-1(ts) ykr2Delta cells. Thus, Pkh1-Ypk1 and Pkh2-Ykr2 function in a novel pathway for cell wall integrity that acts in parallel with the Pkc1-dependent pathway.

3-Phosphoinositide-Dependent Protein Kinases↗