Roles of phosphorylation sites in regulating activity of the transcription factor pho4

Science

Volumn 284, Issue 5416, 1999, Pages 977-980

  Komeili, Arash ^a   O'Shea, Erin K ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

FUNGAL PROTEIN; TRANSCRIPTION FACTOR;

ARTICLE; NONHUMAN; NUCLEAR LOCALIZATION SIGNAL; POLYACRYLAMIDE GEL ELECTROPHORESIS; PRIORITY JOURNAL; PROTEIN LOCALIZATION; PROTEIN PHOSPHORYLATION; REGULATORY MECHANISM; SIGNAL TRANSDUCTION; YEAST;

ACID PHOSPHATASE; AMINO ACID SUBSTITUTION; CELL NUCLEUS; CYCLIN-DEPENDENT KINASES; CYCLINS; DNA-BINDING PROTEINS; FUNGAL PROTEINS; HOMEODOMAIN PROTEINS; KARYOPHERINS; MEMBRANE TRANSPORT PROTEINS; NUCLEAR LOCALIZATION SIGNALS; PHOSPHORYLATION; RECEPTORS, CYTOPLASMIC AND NUCLEAR; RECOMBINANT FUSION PROTEINS; REPRESSOR PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; TRANS-ACTIVATORS; TRANSCRIPTION FACTORS; TRANSCRIPTION, GENETIC;

SACCHAROMYCETALES;

EID: 0033532281     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5416.977     Document Type: Article

References (35)

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9. A. Komeili and E. O'Shea, unpublished results.
10. Previous studies demonstrate that Pho4 mutants containing Ser→Ala substitutions of a subset of the phosphorylation sites can be efficiently phosphorylated in vitro (6).
11. SA1465D23-GFP, containing Ser→Ala substitutions at sites 1, 4, and 6, and Ser→Asp substitutions at sites 2 and 3, is not exported from the nucleus (8).
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13. Three tandem copies of GFP were used in order to (i) enhance the fluorescence signal and (ii) slow down the import of the two proteins to facilitate detection of a difference in their nuclear import.
14. 3 indicate there is a similar amount of each full-length protein and a minor population of degraded protein (8).
15. SE4-GFP expressed under the control of the PHO4 promoter on a CEN/ARS plasmid was still cytoplasmic after 2 hours of phosphate starvation. In contrast, wild-type Pho4-GFP was imported into the nucleus in less than 30 min after phosphate starvation (8). Additionally, a pse1-1 strain, defective for import of Pho4, cannot induce expression of acid phosphatase in response to phosphate starvation.
16. SA1-GFP mutant has no defect in localization or regulation of acid phosphatase expression (8).
17. SA4-GFP, which can be exported but whose import cannot be inhibited by phosphorylation, regulated acid phosphatase expression appropriately (8).
18. SA1234PA6 does not undergo a shift in its electrophoretic mobility after incubation with Pho80/ Pho85 and adenosine 5′-triphosphate (ATP), suggesting that it cannot be phosphorylated (8).
19. SA12346 is a weak activator of transcription (6).
20. SA1234 also produces fully induced levels of acid phosphatase when grown in phosphate-rich medium (Fig. 3B). Thus, the additional mode of regulation requires Pho80, as expected if the regulation involves phosphorylation.
21. PA6-GFP occurs with the same kinetics as wild-type Pho4 (8), suggesting that the ability of this mutant to interact with and be phosphorylated by Pho80 is similar to that of wild-type Pho4. (ii) Phosphorylation site 6 is in a domain involved in interaction with Pho2 (21), not with Pho80 (P. S. Jayaraman et al., above).
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25. SA1234PA6 interacted efficiently with Pho2 (Fig. 3C) (8) and activated transcription of acid phosphatase (Fig. 3B). Thus, Ala substitutions at Ser 1, 2, 3, and 4 do not affect the interaction with Pho2, and the Ser at site 6 is important for the interaction between Pho4 and Pho2.
26. All yeast strains are derived from K699 MATa [K. Nasmyth, G. Adolf, D. Lydall, A. Seddon, Cell 62, 631 (1990)]. PHO3 encodes an acid phosphatase expressed in phosphate-rich conditions [A. Toh-e, Y. Ueda, S.-I. Kakimoto, Y. Oshima, J. Bacteriol. 113, 727 (1973)].
27. All yeast strains are derived from K699 MATa [K. Nasmyth, G. Adolf, D. Lydall, A. Seddon, Cell 62, 631 (1990)]. PHO3 encodes an acid phosphatase expressed in phosphate-rich conditions [A. Toh-e, Y. Ueda, S.-I. Kakimoto, Y. Oshima, J. Bacteriol. 113, 727 (1973)].
28. 4 was added to a final concentration of 20 mM. Photos were taken 5 to 10 min after the addition of phosphate.
29. 6 (11).
30. 3 (12) under the control of the GAL1-10 promoter was grown in synthetic raffinose medium at 30 °C to log phase. No fluorescence is visible under noninducing conditions when cells are grown in raffinose. Expression of each fusion protein was induced by addition of galactose to a final concentration of 2%, and localization was monitored as a function of time by fluorescence microscopy (11). The photo was taken 1.5 hours after the addition of galactose.
31. 600 values.
32. 2 elutions were run on 7.8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Pho2 was detected by protein immunoblotting with polyclonal anti-Pho2.
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35. 6-Gsp1Q71L; A. Kaffman for helpful discussions; and C. Gross, J. Weissman, R. Tjian, and members of the O'Shea lab for helpful comments on the manuscript Supported by the David and Lucile Packard Foundation (E.K.O.). A.K. was supported by a Howard Hughes Medical Institute Predoctoral Fellowship.