Prk1p Could Not Phosphorylate Its Target Sequence with Threonine Replaced by Serine
Our previous studies have characterized the consensus phosphorylation site of Prk1p as LxxQxTG (Zeng and Cai, 1999
). It is conspicuous that Pan1p contains 15 copies of this motif but not a single copy of LxxQxSG, suggesting that Prk1p may only be able to phosphorylate threonine but not serine in this context. To test this possibility, we carried out in vitro kinase assays using a mutated substrate of Prk1p that had the threonine replaced by a serine in this motif. This substrate was derived from R15, a GST-fused Pan1p fragment (amino acids 564–846) that contained a single LxxQxTG motif and had been used as a standard substrate for Prk1p in the previous in vitro kinase assays (Zeng and Cai, 1999
). For convenience of description, the threonine in the LxxQxTG motif was given a position number P0, and the residues upstream or downstream of P0 were designated as P–n or P+n, respectively (). In the control experiments, the immunoprecipitated HA-Prk1p (, lane 3) was able to phosphorylate the GST-fusion protein R15-WT (, lane 3), whereas the anti-HA immunoprecipitates from cells containing either untagged Prk1p (, lane 1), or HA-tagged kinase-dead mutant of Prk1p (HA-Prk1D158Y
p; , lane 2), failed to phosphorylate the same protein (, lanes 1 and 2). Prk1p, on the other hand, was unable to phosphorylate R15-T/S, an R15 variant with a T-to-S mutation at P0 (, lane 4). This result confirmed our speculation that the Prk1p phosphorylation motif, as had been identified, was threonine-specific.
Figure 1. Inability of Prk1p to phosphorylate serine in its recognition motif. (A) Western blotting of Prk1p and its kinase-dead mutant Prk1D158Yp (Zeng and Cai, 1999 ). The HA-tagged proteins were immunoprecipitated from equal amounts of cell extracts with the (more ...)
Hydrophobic Residues at P-5 Are Required for Prk1p Recognition
So far, we have determined the importance of Q, T, and G of the LxxQxTG motif in Prk1p phosphorylation. To examine the stringency of L for Prk1p recognition, the L residue of R15-WT at P-5 was mutated into A and subjected to in vitro kinase assay. As shown in , the resulting protein R15-L/A could not be phosphorylated by Prk1p (, lane 1). In addition, introduction of an L into other positions (P-6, P-4, and P-3) of R15-L/A also failed to confer Prk1p phosphorylation (, lanes 2, 4, and 5). These results indicate that there is a stringent requirement for leucine at P-5, but not other positions, for the motif to be recognized by Prk1p.
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 (more ...)
The Prk1p recognition motif LxxQxTG occurs numerous times in Pan1p and Sla1p, clustered within certain domains that are involved in protein–protein interactions (Zeng and Cai, 1999
; Zeng et al., 2001
). Interestingly, multiple additional copies of QxTG, in which the P-5 position is other residues than L, are also present within these domains. These multiple QxTG motifs in Pan1p and Sla1p may represent additional patterns of Prk1p recognition sequence. To investigate this possibility, the L residue of R15-WT at P-5 was mutated into each of the every P-5 variation found in Pan1p and Sla1p, namely, I, M, P, V, T, A, S, and N. The in vitro kinase assays showed that three of these residues, I, M, and V, were able to confer Prk1p phosphorylation (, lanes 3, 4, and 6). As these residues are all hydrophobic, two additional hydrophobic residues, F and W, were also tested for their ability to support Prk1p phosphorylation. As shown in , neither R15-L/F nor R15-L/W could serve as an efficient substrate for Prk1p (, lanes 10 and 11). Furthermore, introduction of other residues to P-5 did not result in identification of additional Prk1p recognition patterns (our unpublished data).
Although all the P-5 residues suitable for Prk1p phosphorylation were hydrophobic, they differed in phosphorylation efficiency (). Among them, L is most optimal. This is consistent with the fact that LxxQxTG is present most frequently in the known Prk1p substrates Pan1p and Sla1p (Zeng and Cai, 1999
; Zeng et al., 2001
). After L, in a descending order, are I, V, and M, with recognition efficiencies of 36, 15, and 13%, respectively, of that of L.
Identification of N, T, and S as Additional P-2 Residues
Using R15 variants as substrates, we identified [I/V/M]xxQxTG motifs as the new phosphorylation site of Prk1p. To verify this finding, we chose Yap1801p and Ent1p, two yeast proteins that contain some of these newly identified motifs, to test their ability to act as substrates of Prk1p in vitro. Yap1801p was originally identified as one of the yeast homologs of the mammalian clathrin assembly protein AP180 from a yeast two-hybrid screen by using the Eps15-homology (EH) domains of Pan1p as a bait (Wendland and Emr, 1998
). The same screen also identified a pair of yeast epsins, Ent1p and Ent2p (Wendland et al., 1999
). Yap1801p contains one (VxxQxT413
G), whereas Ent1p has two (IxxQxT395
G and LxxQxT415
G) Prk1p recognition motifs in their Pan1p EH domain-binding regions (). Recently, Ent1p has been shown to exhibit Prk1p-dependent phosphorylation on its [L/I]xxQxTG motifs in vivo (Watson et al., 2001
). Nevertheless, it is of interest to test whether it can be directly phosphorylated by Prk1p in vitro.
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 () (more ...)
As expected, a GST-fusion protein containing the VxxQxTG motif of Yap1801p (Yap1801WT) was efficiently phosphorylated by Prk1p (, lane 1). Surprisingly, however, when the T residue within VxxQxTG was mutated to A (T413A), the resulting protein Yap1801T/A could still be phosphorylated by Prk1p (, lane 2). Similar observation was also made with Ent1p. Although GST-fusion proteins containing both, or either one, of the [L/I]xxQxTG motifs of Ent1p could be efficiently phosphorylated by Prk1p (, lanes 3–5), disruption of both motifs by site-directed mutagenesis (T395A and T415A) failed to abolish the Prk1p-dependent phosphorylation (, lane 6). These results indicate that there must be additional Prk1p phosphorylation sites on Yap1801p and Ent1p.
On a closer examination of the sequences of Yap1801p and Ent1p, several motifs approximately resembling the phosphorylation targets of Prk1p were spotted. They were LxxTxT427
G and VxxSxT453
G on Yap1801p, and LxxNxT346
G, and VxxTxT427
G on Ent1p (). These motifs differ in that they have residues N, T, and S rather than Q at the P-2 position. Two observations suggested that these motifs may also be phosphorylation sites of Prk1p. First, these motifs are clustered within a short region in both Yap1801p and Ent1p known to be involved in protein–protein interactions, i.e., the Pan1p EH domain-binding region (Wendland and Emr, 1998
; Wendland et al., 1999
), similarly to the distribution of the LxxQxTG motifs in Pan1p and Sla1p. Second, several such motifs were also found in Pan1p (LxxTxT241
G, and LxxTxT944
G) and Sla1p (IxxNxT730
G and IxxSxT923
G). Indeed, as shown in , both VxxQxT413
G and LxxTxT427
G motifs from Yap1801p could be phosphorylated by Prk1p (, lanes 2 and 3). There was no detectable phosphorylation on the VxxSxT453
G motif (, lane 4), although scintillation counting revealed a reading two-fold higher than that of the background (our unpublished data). After all these sites were mutated, the phosphorylation on Yap1801p by Prk1p was completely eliminated (, lane 1). Similarly, any one of these motifs from Ent1p was able to support phosphorylation by Prk1p, albeit to various degrees (, lanes 6–10), whereas the mutant with all the motifs disrupted showed no Prk1p phosphorylation (, lane 5). It is concluded, therefore, that N, T, and S are the additional P-2 residues in the Prk1p recognition motif.
Comparison of Phosphorylation Efficiency of All [L/I/V/M]xx[Q/N/T/S]xTG Motifs
Combining all the sequence requirements at P-2 and P-5, the Prk1p phosphorylation motif is now consolidated to be [L/I/V/M]xx[Q/N/T/S]xTG. Again, using R15 as a template, we generated a series of motifs to compare their efficiency of serving as the target sequence of Prk1p, as we did with the set of mutations on P-5 of LxxQxTG shown in . showed the in vitro kinase assay result of the test on the [L/I/M/V]xxNxTG motifs. It is clear that different combinations gave rise to different phosphorylation efficiencies (, lanes 1–4). Similar tests were also performed on [L/I/M/V]xxTxTG, and [L/I/M/V]xxSxTG, as shown in , respectively. Finally, these results were compared in . Consistent with our result obtained with native substrate, namely, Yap1801AAT (, lane 4), the VxxSxTG motif was the weakest Prk1p phosphorylation site (, R15-VS).
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 (more ...)
A Homology-based Three-dimensional (3D) Model of Prk1p
Prk1p belongs to a distinct family of kinases and shares significant sequence homology to other kinases in the kinase domain (Cope et al., 1999
). The 3D structures of Prk1p or any member of this family have not been resolved. To gain some insights on the structural basis of Prk1p's function, a homology-based three-dimensional model of the Prk1p kinase domain (residues 23–298) was generated as described in MATERIALS AND METHODS. Based on this model, the Prk1p kinase domain comprises two main portions: an N-terminal lobe and a C-terminal lobe (). The N-terminal lobe (residues 23–110) consists of five antiparallel β strands (β1–β5) and one α helix (helix C) and is responsible for binding ATP through a glycine loop located between β1 and β2. The C-terminal lobe (residues 111–298) consists of a four-helix bundle (helices D, E, F, and H) that is flanked by two smaller helices and two short antiparallel β sheets. The catalytic loop with the conserved catalytic ASP158 residue and the activation segment (residues 176–212) are both located in this lobe. Using the sequence of PLTAQKTG (the LxxQxTG motif present in the in vitro substrate R15) as the substrate peptide, a structural model of the kinase–substrate complex was further constructed (). In the docked state, the substrate peptide binds in an extended conformation across the kinase catalytic site, contacting only the C-terminal lobe of the kinase domain ().
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 (more ...)
Studies on the substrate–kinase complex model revealed four sets of essential interactions between Prk1p and the [L/I/V/M]xx[Q/N/T/S]xTG motif (). Among these interactions, three of them are the hydrogen bonds between the carboxyl side chain of ASP158 and the hydroxyl group of P0 residue T, the amine side chain of LYS160 and the backbone carboxyl group of P-1 residue, and the amine group of GLN207 and the amine group (Q and N) or the hydroxyl group (T and S) of the P-2 residue (, dashed lines). The other interaction is the van der Waals force between a hydrophobic binding pocket consisting of three kinase residues ILE117, MET120, and ILE238 and the P-5 hydrophobic residue ().
The interaction between the hydrophobic pocket of the kinase and the P-5 residue of the substrate explains why only hydrophobic residues should be present at P-5. Among all the hydrophobic residues, i.e., A, I, L, M, F, P, W, and V, only L, I, V, and M are the suitable P-5 residues, because A would be too small and F too bulky to be well docked inside the pocket, whereas both P and W are not flexible enough to be compacted into the pocket due to their rigid structures.
Potential Phosphorylation Targets of Prk1p in the Yeast Genome
The identification of [L/I/M/V]xx[Q/N/T/S]xTG motifs as the Prk1p recognition sequence has increased the number of the potential Prk1p phosphorylation sites in Pan1p and Sla1p to 19 and 10, respectively. A search of the S. cerevisiae
genome database (http://seq.yeastgenome.org/cgi-bin/SGD/PATMATCH/nph-patmatch
) with a peptide sequence of [LIMV]xx[QNTS]xTG gave rise to ~500 proteins that contain one or more copies of the [L/I/M/V]xx[Q/N/T/S]xTG motifs. Some of these proteins stand out as more likely to be potential regulatory targets of Prk1p than others because their functions in actin cytoskeleton organization, cell polarity, or endocytosis have been well documented. Twenty-three of them, including the known Prk1p targets Pan1p and Sla1p, are listed in . They are tentatively divided into three groups: proteins that are functionally related to Pan1p (, group I), the ones that are involved in the regulation of actin dynamics (, group II), and the ones that have important roles in bud site selection and cell polarity (, group III).
List of confirmed and potential phosphorylation targets of Prk1p in yeast genome
Scd5p Is a Novel Phosphorylation Target of Prk1p
Among the above-mentioned proteins, Scd5p, a protein containing three copies of LxxTxTG, seems particularly promising as a new Prk1p phosphorylation target (). Scd5p was first identified from a genetic screen searching for multicopy suppressors of the clathrin heavy chain-deficient yeast strains (Nelson and Lemmon, 1993
; Nelson et al., 1996
). Subsequent studies revealed that Scd5p plays an important role in cortical actin organization and endocytosis (Henry etal., 2002
). More recently, two potential protein phosphatase-1 (PP1) binding motifs were identified near the N terminus of Scd5p (KVDF and KKVRF; ), one of which, the second one, was shown to mediate physical interaction between Scd5p and Glc7p, the yeast PP1 known to have a function in actin organization and endocytosis (Chang et al., 2002
). In addition to the PP1-binding motifs, Scd5p also contains a 20-aa sequence (, solid boxes) repeated three times in the central region and a 12-aa sequence (, hatched boxes) repeated nine times at the C-terminal region (Nelson et al., 1996
). The newly identified LxxTxTG motifs are embedded in each of the 20-aa repeats (). A resemblance between these central motifs and our previously defined Prk1p phosphorylation site has also been noted by Chang et al
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 (more ...)
To test whether the LxxTxTG motifs in Scd5p could be recognized by Prk1p in vitro, GST-fusion proteins of Scd5p were prepared for kinase assays. As shown in , the motif-containing substrate could indeed be strongly phosphorylated by Prk1p (, lane 1). The phosphorylation was completely abolished when all three motifs were mutated (, lane 5). Any single motif of the three was able to confer phosphorylation (, lanes 2–4), indicating that each of them was recognized by Prk1p.
To further confirm Scd5p as a phosphorylation target of Prk1p, we examined whether Scd5p could be phosphorylated by Prk1p in vivo in a host that overproduced the kinase. Scd5p (tagged with HA) was directly precipitated from cell lysates by TCA to maximally preserve its phosphorylated states, and dissolved in loading buffer for SDS-PAGE analysis. As shown in , in the absence of PRK1 overexpression, wild-type Scd5-HA migrated as a single band on the gel (, lane 1) with a same mobility as that of the nonphosphorylable Scd5AAA-HA mutant (, lane 5). In contrast, the band of Scd5p extracted from cells that had experienced PRK1 overexpression became smeared and retarded (, lane 3). This phenomenon was not observed for Scd5AAA-HA, which showed no changes in gel mobility upon Prk1p overproduction (, lane 4). Similarly, no mobility changes were observed for Scd5-HA precipitated from cells overexpressing the kinase-dead mutant of PRK1, PRK1D158Y (, lane 2). These results indicated that the gel mobility change of Scd5-HA extracted from Prk1p-overproducing cells was due to its phosphorylation by Prk1p on T416, T450, and T490 residues. It is concluded, therefore, that Scd5p could be phosphorylated by Prk1p in vivo.
Suppression of the scd5-1 Mutant by prk1Δ
The above-mentioned result suggested that the function of Scd5p may be subjected to regulation of Prk1p by phosphorylation. Furthermore, because Scd5p is also potentially under the regulation by the phosphatase Glc7p (Chang et al., 2002
), it is possible that Glc7p and Prk1p could be counter-acting each other on the LxxTxTG motifs. Scd5p interacts with Glc7p mainly through its second PP1-binding motif (PBM2), and disruption of this motif results in the dissociation of Glc7p from Scd5p accompanied by a phenotype of temperature-sensitive (ts) growth (Chang et al., 2002
). Hence, it is likely that the ts phenotype was due to over-phosphorylation of Scd5p by Prk1p and could be expected to be suppressed by deletion of PRK1
To test this hypothesis, we generated an scd5
, YMC446) that carries the same mutations as reported by Chang et al
) on the second PP1-binding motif of Scd5p. Consistent with the previous report, this PBM2-disrupted scd5
mutant was viable at 25°C and was temperature sensitive for growth at 37°C (). After introduction of the prk1
Δ mutation into this mutant, the temperature sensitivity could indeed be suppressed ().
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 (more ...)
Δ mutation not only suppressed the ts phenotype of the scd5-1
mutant but also corrected the deficiencies of the mutant in endocytosis and actin organization characterized previously (Chang et al., 2002
; Henry et al., 2002
). We performed two types of endocytosis assays. The first one was the fluid-phase endocytosis by using LY as a marker. As shown in , wild-type cells could efficiently internalize the dye at both 25°C and 37°C, and exhibited an unambiguous vacuolar staining (, left), whereas the scd5-1
cells failed to accumulate LY in vacuoles even at 25°C (, middle). In contrast, the prk1
cells showed normal LY uptake at both 25°C and 37°C (, right). We next examined the endocytosis of a plasma membrane protein Ste3p. Ste3p is the receptor of a
-factor and is known to undergo constitutive endocytosis, leading to degradation in the vacuole in the absence of its ligand (Davis et al., 1993
). As shown in , the majority of Ste3-EGFP in the wild-type cells growing at 25°C was found to be in the vacuole 45 min after the shutdown of its expression (). However, the scd5-1
mutant cells showed severe defects in this process (). Even at the last time point when no Ste3-EGFP was observed on the membranes of the wild-type cells (), the scd5-1
cells maintained significant Ste3-GEFP staining on the plasma membrane (). The prk1
mutant cells, on the other hand, exhibited a normal pattern of internalization of Ste3-EGFP (), essentially indistinguishable from that of wild-type cells. Similar results were also obtained with cells incubated at 37°C (our unpublished data). These results indicate that the endocytosis defect of scd5-1
could also be suppressed by prk1
The organization of the actin cytoskeleton was investigated by rhodamine-phalloidin staining. Wild-type cells exhibited normal actin structures throughout the cell cycle at either 25°C or 37°C (). At 25°C, the scd5-1 mutant cells exhibited a nearly normal actin organization, as far as the morphology and the distribution pattern of the cortical actin patches were concerned (). After temperature shift to 37°Cfor4h, ~30–40% of the scd5-1 cells obviously showed aberrant actin structures (). These include depolarized cortical actin patches and accumulation of abnormal actin aggregates. These aberrant actin structures, however, were no longer visible in the prk1Δ scd5-1 cells either at 25°C or 37°C ().