Identification of novel recognition motifs and regulatory targets for the yeast actin-regulating kinase Prk1p

Abstract

Prk1p is a serine/threonine kinase involved in the regulation of the actin cytoskeleton organization in the yeast Saccharomyces cerevisiae. Previously, we have identified LxxQxTG as the phosphorylation site of Prk1p. In this report, the recognition sequence for Prk1p is investigated more thoroughly. It is found that the presence of a hydrophobic residue at the position of P-5 is necessary for Prk1p phosphorylation and L, I, V, and M are all able to confer the phosphorylation at various efficiencies. The residue flexibility at P-2 has also been identified to include Q, N, T, and S. A homology-based three-dimensional model of the kinase domain of Prk1p provided some structural interpretations for these substrate specificities. The characterization of the [L/I/V/M]xx[Q/N/T/S]xTG motif led to the identification of a spectrum of potential targets for Prk1p from yeast genome. One of them, Scd5p, which contains three LxxTxTG motifs and is previously known to be important for endocytosis and actin organization, has been chosen to demonstrate its relationship with Prk1p. Phosphorylation of Scd5p by Prk1p at the three LxxTxTG motifs could be detected in vitro and in vivo, and deletion of PRK1 suppressed the defects in actin cytoskeleton and endocytosis in one of the scd5 mutants. These results allowed us to conclude that Scd5p is likely another regulatory target of Prk1p.

Figure 1.

Inability of Prk1p to phosphorylate serine in its recognition motif. (A) Western blotting of Prk1p and its kinase-dead mutant Prk1^D158Yp (Zeng and Cai, 1999). The HA-tagged proteins were immunoprecipitated from equal amounts of cell extracts with the rabbit anti-HA antibody and analyzed on immunoblots. (B) Prk1p was unable to phosphorylate LxxQxSG. The immunoprecipitated proteins and substrates used in each reaction are as indicated. Phosphorylation results were shown as autoradiography, and the input substrates were visualized by Coomassie Blue staining. The amino acid sequence of the 15th LxxQxTG motif of Pan1p (R15-WT) is shown on the top with each residue labeled according to their position relative to the phosphorylation target T.

Figure 2.

Analysis of sequence requirement at P-5 for Prk1p recognition. (A) The requirement for leucine at P-5 for Prk1p recognition. GST-fusion proteins of R15-WT and its mutants were prepared and subjected to in vitro kinase assay. R15-L/A (lane 1) carried an L to A mutation at P-5, whereas mutants in lanes 2, 4, and 5 had an additional mutation that introduced L into positions of P-6, P-4, and P-3, respectively. (B) Phosphorylation of the [L/I/V/M]xxQxTG motifs by Prk1p. GST-fusion proteins of R15-WT and its mutants bearing various substitutions at P-5 as indicated were prepared and subjected to in vitro kinase assay. The immunoprecipitated HA-Prk1p was used as the kinase in all reactions of A and B. Phosphorylation results were shown as autoradiography and the input substrates were visualized by the Coomassie Blue staining. (C) Comparison of phosphorylation efficiency of the [L/I/V/M/F/P/W/A]xxQxTG motifs. The bands of the substrates were excised from the gel for scintillation counting after autoradiography and the results were normalized against the amounts of the substrates. Phosphorylation efficiencies of each motif were calculated as the percentage of reading relative to that of the LxxQxTG motif.

Figure 3.

Identification of N, T, and S as additional P-2 residues for Prk1p recognition. (A) Schematic diagrams of Yap1801p and Ent1p showing the distribution of Prk1p phosphorylation sites. The QxTG-containing (•) and [N/T/S]xTG-containing (▴) motifs are found in the Pan1p EH domain-binding regions (gray boxes) of each protein, in the vicinity of the NPF motifs (solid boxes). The bars beneath each of the diagrams indicate the region from each protein expressed as GST-fusion proteins. (B and C) Phosphorylation of Yap1801p, Ent1p, and their mutants by Prk1p. The substrates used in each reaction are as indicated. Substrates in C are labeled with the identities at P0 of the motifs arranged in a sequential order. For example, Yap1801^AAT (C, lane 4) is a mutant carrying the T-to-A mutations in the first two of the three motifs. Phosphorylation results were shown as autoradiography and the input substrates were visualized by the Coomassie Blue staining.

Figure 4.

Comparison of phosphorylation efficiency of the [L/I/V/M]xx[Q/N/T/S]xTG motifs. (A) Phosphorylation of the [L/I/V/M]xxNxTG motifs by Prk1p. (B) Phosphorylation of the [L/I/V/M]xxTxTG motifs by Prk1p. (C) Phosphorylation of the [L/I/V/M]xxSxTG motifs by Prk1p. The substrates used in lanes 1–4 are labeled to show their residue identities at P-5 and P-2. For example, R15-LN (A, lane 1) represents the motif of LxxNxTG. The substrates used in the lanes 5 are the negative controls and labeled with residue identities at P-2 and P0, e.g., R15-NA (A, lane 5) represents the motif of LxxNxAG. (D) Comparison of Prk1p phosphorylation efficiencies on the [L/I/V/M]xx[Q/N/T/S]xTG motifs. The phosphorylation results were measured by scintillation counting and normalized against the quantities of the substrates.

Figure 5.

Interactions between Prk1p and its recognition motifs depicted by homology-based modeling. (A) Two views of the Prk1p kinase domain. The left view has ribbons and loops colored by B-factor. Blue indicates regions with high degree of conservation and red low conservation. The major secondary structures such as β strand and α helix are also labeled. The right view highlights the important features of the kinase domain. The light green region represents the N-terminal lobe and the light blue region the C-terminal lobe. The glycine loop responsible for binding the nontransferable phosphates of ATP is located in the N-terminal lobe and colored in dark blue. The C-terminal lobe includes an activation segment in orange, four helices (D, E, F, and H) in dark green and the conserved catalytic residue ASP158 in dark blue. A P-5 binding pocket formed by ILE117, MET120, and ILE238 from the helices D and H is also shown. (B). A general view of the kinase domain of Prk1p with a substrate peptide (PLTAQKTG) bound. The kinase domain is visualized with a MOLCAD surface colored by lipophilic potential. The positions of the activation segment, the ATP-binding pocket, the catalytic residue ASP158, and the P-5 binding pocket are as indicated. (C) Schematic representation of the molecular interactions between Prk1p and its substrate peptide. The substrate peptide colored in black is shown together with the kinase residues essential for the interaction. Residues involved in docking the QxTG motif (ASP158, LYS160, and GLN207) are colored in blue and residues making up the P-5 binding pocket (ILE117, MET120, and ILE238) are colored in yellow. The dashed lines represent the hydrogen bonds between the carboxyl side chain of ASP158 and the hydroxyl group of P0, the amine side chain of LYS160 and the backbone carbonyl group of P-1, and the amide group of GLN207 and the amine or hydroxyl group of P-2, respectively.

Figure 6.

Identification of Scd5p as a new phosphorylation target of Prk1p. (A) Schematic diagram of Scd5p showing the locations of three LxxTxTG motifs. In addition to these motifs, Scd5p also contains two putative PP1-binding motifs (KVDF and KKVRF), three copies of a 20-aa sequence (solid boxes) and nine copies of a 12-aa sequence (hatched boxes). The bar below the diagram represents the region used for GST-fusion protein expression. (B) Phosphorylation of Scd5p and its mutant forms by Prk1p in vitro. The substrates used in each reaction are as indicated. (C) Phosphorylation of Scd5p by Prk1p in vivo. Scd5p from YMC448 (scd5::SCD5-HA) containing vector control pRS316 (lane 1), pGAL-PRK1^D158Y (lane 2), or pGAL-PRK1 (lane 3), and Scd5^AAAp from YMC449 (scd5::SCD5^AAA-HA) containing either pGAL-PRK1 (lane 4) or pRS316 (lane 5) were precipitated by TCA and analyzed by SDS-PAGE and immunoblotting.

Figure 7.

Suppression of the scd5-1 mutation by prk1Δ. (A) Temperature sensitivity test of the scd5-1 and prk1Δ scd5-1 mutants. The strains YMC446 (scd5-1) and YMC447 (prk1Δ scd5-1) were streaked on YEPD plates and incubated at respective temperatures (25°C and 37°C) for 3 d. (B) LY uptake in wild-type (WT), scd5-1, and prk1Δ scd5-1 cells grown at 25°C or incubated at 37°C for 2 h. After 2 h of incubation with LY, cells were examined under a microscope with fluorescein isothiocyanate and Nomarski optics. (C) Internalization of Ste3-EGFP in WT, scd5-1, and prk1Δ scd5-1 cells grown at 25°C. Cells containing pGAL-STE3-EGFP were induced to express Ste3-EGFP for 1 h and samples were taken for observation at 15-min intervals after shutdown of Ste3p expression. (D) Actin staining of WT, scd5-1, and prk1Δ scd5-1 cells grown at 25°C or incubated at 37°C for 4 h. Cells were fixed and incubated with rhodamine-phalloidin for 30 min before the visualization. Bars, 5 μm.

Figure 8.

Regulation of protein–protein interactions by Prk1p phosphorylation. (A) Three possible protein modules formed by the potential phosphorylation targets of Prk1p. Solid and dashed lines indicate physical and genetic interactions, respectively. The physical interactions probably regulated by Prk1p phosphorylation are highlighted with thick lines. (B) The location of Prk1p recognition motifs on some of its potential phosphorylation targets. The [L/I/V/M]xx[Q/N/T/S]xTG motifs are shown as solid dots and NPF motifs as solid boxes. The regions involved in protein–protein interactions are indicated as gray boxes.