Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2017 Mar 17;37(7):e00627-16. doi: 10.1128/MCB.00627-16

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2017 Roelants et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

PMC Copyright notice

FIG 2 — Fpk1 phosphorylates Akl1 at S960 and S1072. (A) Schematic representation of the Akl1, Ark1, and Prk1 protein kinases. The catalytic domain (red rectangle) is near the amino terminus. Akl1 has the longest C-terminal segment, and only Akl1 contains consensus Fpk1 phospho-acceptor site motifs, both near its C terminus. (B) GST-Fpk1 (pAX15, WT) or catalytically inactive (kinase-dead [KD]) mutant GST-Fpk1(D621A) (pJY10) were purified from E. coli, incubated with [γ-³²P]ATP and either GST-Akl1(767-1108) (pFR290), GST-Akl1(767-1108; S960A) (pFR293), GST-Akl1(767-1108; S1072A) (pFR294), or GST-Akl1(767-1108; S960A S1072A) (pFR297), also purified from E. coli. The resulting products were resolved by SDS-PAGE and analyzed by autoradiography and staining with Coomassie dye. (C) A wild-type (WT) strain (BY4741) and an isogenic fpk1Δ fpk2Δ mutant (YFR205) expressing from the GAL1 promoter either GFP-Akl1 (pDD0938) or GFP-Akl1(S960A S1072A) (pFR303) were grown to mid-exponential phase and lysed. The resulting extracts were resolved on a Phos-tag gel and analyzed by immunoblotting with anti-GFP antibodies. (D) Otherwise wild-type cells expressing full-length Akl1-mCherry from its endogenous promoter at its normal chromosomal locus (YFR437) were grown, and extracts were prepared, treated with calf intestinal phosphatase, and then resolved and analyzed as for panel C. (E) Same as in panel C except that the strains were expressing GFP-Akl1 in which residues 30 to 751 were deleted (pFR304) or the same construct with the S960A (pFR329), S1072A (pFR328), or S960A S1072A mutations (pFR334). (F) A wild-type strain (BY4741) expressing GFP-Akl1(Δ30-751) (pFR304) was grown and extracts were prepared, treated with calf intestinal phosphatase, and then resolved and analyzed as for panel E. (G) Strains BY4741 (WT) and avo3^ΔCT TOR1-1 were transformed with GFP-Akl1(Δ30-751) (pFR304), grown to mid-exponential phase, and left untreated (−) or treated (+) with rapamycin (0.2 μM) for 10 min, which in this strain specifically inhibits TORC2, before being lysed and analyzed as for panel C. (H) avo3^ΔCT TOR1-1 cells transformed with GFP-Akl1(Δ30-751) (pFR304), GFP-Akl1(Δ30-751; S960A) (pFR329), GFP-Akl1(Δ30-751; S1072A) (pFR328), or GFP-Akl1(Δ30-751; S960A S1072A) (pFR334) were grown and analyzed as for panel G.