Results: 4

1.
FIG. 3.

FIG. 3. From: Integration of Stress Responses: Modulation of Calcineurin Signaling in Saccharomyces cerevisiae by Protein Kinase A.

PKA regulates Crz1p activity in vivo. (A) Mutation of serines 409, 410, 423, 427, and 429 to alanine results in increased Crz1p activity. KWY251 cells (crz1Δ 4xCDRE::LacZ) expressing wild-type GFP-Crz1p (pKK249), GFP-Crz1pS409,410,423A (pKK251), or GFP-Crz1pS409,410,423,427,429A (pKK267) were treated with 100 mM CaCl2 where indicated. β-Galactosidase activity was measured and is reported as the maximum rate OD415 change/minute/microgram of protein. (B) PKA activation reduces Ca2+-dependent Crz1p activation. KKY436 cells (cdc35Δ pde2Δ yak1Δ 4xCDRE::LacZ) were incubated with 100 mM CaCl2 in the absence or presence of 3 mM cAMP as indicated, and the β-galactosidase activity was determined. (C) Constitutive PKA activity results in decreased Crz1p activity. Strains ASY832 (4xCDRE::LacZ) and KKY385 (bcy1Δ 4xCDRE::LacZ) were treated with 100 mM CaCl2 as indicated, and the β-galactosidase activity was determined.

Kimberly A. Kafadar, et al. Eukaryot Cell. 2004 October;3(5):1147-1153.
2.
FIG. 2.

FIG. 2. From: Integration of Stress Responses: Modulation of Calcineurin Signaling in Saccharomyces cerevisiae by Protein Kinase A.

Tpk1p phosphorylates residues within the Crz1p NLS. (A) Phosphorylation of Crz1p fragments by Tpk1p. Recombinant GST-Tpk1p was combined with 2 μg each of full-length GST-Crz1p (FL), GST-Crz1p1-180 (I), GST-Crz1p181-340 (II), GST-Crz1p341-440 (III), or GST-Crz1p441-678 (IV) and then incubated with ATP and [γ-32P]ATP for 40 min at 30°C. Samples were analyzed by SDS-PAGE and autoradiography. Fragment III (residues 341 to 440) contains the NLS. (B) Sequence of the Crz1p NLS, extended from residues 422 to 429 (28). Putative PKA phosphorylation sites are underlined (19). Residues mutated to alanine are marked with an asterisk. (C) Mutation of residues 409, 410, 423, 427, and 429 disrupts Tpk1p phosphorylation of Crz1p in vitro. Wild-type and mutant versions of recombinant GST-Crz1p341-440 were incubated with GST-Tpk1p, ATP, and [γ-32P]ATP. Samples were analyzed by SDS-PAGE and imaged on a Typhoon scanner for quantitation.

Kimberly A. Kafadar, et al. Eukaryot Cell. 2004 October;3(5):1147-1153.
3.
FIG. 4.

FIG. 4. From: Integration of Stress Responses: Modulation of Calcineurin Signaling in Saccharomyces cerevisiae by Protein Kinase A.

PKA regulates Crz1p nuclear import (A) Localization of 3× GFP-Crz1p341-440 in BJ5459 cells is affected by the mutation of serines 409, 410, and 423 to alanine. The wild-type fragment (pKK260) is cytosolic, whereas the S409,410,423A mutant (pKK261) is strongly nuclear. (B) Full-length GFP-Crz1pS409,410,423A constitutively binds the importin Nmd5p. (Top) Extracts were prepared from cells (BJ5459 or KKY420) treated with or without FK506 from which GST-Nmd5p was isolated by using glutathione-Sepharose. Association of wild-type GFP-Crz1p (pKK249) and GFP-Crz1pS409,410,423A (pKK251) was analyzed by GST-pulldown, SDS-PAGE, and Western blot analysis with an α-Crz1p antibody, which also recognizes GST. Lane 1, BJ5459 with pKK249; lane 2, KKY420 with pUG36; lanes 3 and 4, KKY420 with pKK249 (wild-type GFP-Crz1p) ± FK506; lanes 5 and 6, KKY420 with pKK251 (GFP-Crz1pS409,410,423A) with or without FK506. (Bottom panel) Western blot analysis of extracts showing equal expression of GFP-Crz1p fusions. A total of 40 μg of each extract was analyzed by SDS-PAGE and Western blot analysis with an α-GFP antibody.

Kimberly A. Kafadar, et al. Eukaryot Cell. 2004 October;3(5):1147-1153.
4.
FIG. 1.

FIG. 1. From: Integration of Stress Responses: Modulation of Calcineurin Signaling in Saccharomyces cerevisiae by Protein Kinase A.

PKA and Crz1p interact in vivo and in vitro. (A) Crz1p is phosphorylated by Tpk1p in vitro and can subsequently be dephosphorylated by calcineurin. Recombinant GST-Crz1p and GST-Tpk1p were incubated with ATP and [γ-32P]ATP for 40 min at 30°C, followed by incubation with buffer, calcineurin-calmodulin-Ca2+, or calcineurin-calmodulin-EGTA. Samples were analyzed by SDS-PAGE and autoradiography. (B) Crz1p phosphorylation is reduced in extracts lacking PKA. A total of 5 μg of GST-Crz1p was immobilized on glutathione-Sepharose and then incubated with extracts of msn2Δ msn4Δ cells or msn2Δ msn4Δ tpk1Δ tpk2Δ tpk3Δ cells (12), [γ-32P]ATP, and an ATP-regenerating system for 40 min at 30°C. Samples were analyzed by SDS-PAGE and imaged on a Typhoon scanner for quantitation. (C) Crz1p interacts with Tpk1p and Bcy1p in vivo. IgG-Sepharose was used to isolate Crz1p-ZZ from extracts of cells (BJ5459 and KKY271) expressing GST (pRD56), GST-Tpk1p, or GST-Bcy1p (42). Samples were analyzed by SDS-PAGE and Western blotting. Proteins were visualized with an α-Crz1p antibody that also recognizes GST. The asterisk marks a nonspecific band that cross-reacts with the α-Crz1p antibody.

Kimberly A. Kafadar, et al. Eukaryot Cell. 2004 October;3(5):1147-1153.

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