Asterless is a Polo-like kinase 4 substrate that both activates and inhibits kinase activity depending on its phosphorylation state

Abstract

Centriole assembly initiates when Polo-like kinase 4 (Plk4) interacts with a centriole "targeting-factor." In Drosophila, Asterless/Asl (Cep152 in humans) fulfills the targeting role. Interestingly, Asl also regulates Plk4 levels. The N-terminus of Asl (Asl-A; amino acids 1-374) binds Plk4 and promotes Plk4 self-destruction, although it is unclear how this is achieved. Moreover, Plk4 phosphorylates the Cep152 N-terminus, but the functional consequence is unknown. Here, we show that Plk4 phosphorylates Asl and mapped 13 phospho-residues in Asl-A. Nonphosphorylatable alanine (13A) and phosphomimetic (13PM) mutants did not alter Asl function, presumably because of the dominant role of the Asl C-terminus in Plk4 stabilization and centriolar targeting. To address how Asl-A phosphorylation specifically affects Plk4 regulation, we generated Asl-A fragment phospho-mutants and expressed them in cultured Drosophila cells. Asl-A-13A stimulated kinase activity by relieving Plk4 autoinhibition. In contrast, Asl-A-13PM inhibited Plk4 activity by a novel mechanism involving autophosphorylation of Plk4's kinase domain. Thus, Asl-A's phosphorylation state determines which of Asl-A's two opposing effects are exerted on Plk4. Initially, nonphosphorylated Asl binds Plk4 and stimulates its kinase activity, but after Asl is phosphorylated, a negative-feedback mechanism suppresses Plk4 activity. This dual regulatory effect by Asl-A may limit Plk4 to bursts of activity that modulate centriole duplication.

FIGURE 1:

Plk4 phosphorylates the N-terminal domain of Asl. (A, B) Linear maps of Drosophila Plk4 and Asterless (Asl) showing functional and structural domains. CC, coiled-coil; DRE, downstream regulatory element (contains the SCF^Slimb-binding motif); PB, Polo boxes; L1 and L2, linkers. The three regions of Asl are indicated with circled letters. Both Asl-A and Asl-C bind Plk4. (C) In vitro kinase assay of purified recombinant Plk4 (consisting of kinase domain + DRE), GST-tagged Asl regions, and γP³²-ATP. Plk4 phosphorylates itself and Asl-A but not Asl-B, Asl-C, or the GST control. Coomassie-stained SDS–PAGE gels and corresponding autoradiographs (or phosphorimage) are shown. (D) Linear map of Asl-A showing phosphorylated residues identified by MS/MS from samples of purified Asl-A incubated with Plk4 in vitro (top) or immunoprecipitated Asl from lysates of S2 cells coexpressing Plk4 (bottom). (E) Lysates of S2 cells coexpressing Asl-A-V5 and either GFP, kinase-dead (KD) Plk4-GFP, or active nondegradable (ND) Plk4-GFP were resolved by 2D electrophoresis and the Western blot probed with anti-V5 antibody. Resolution in the horizontal axis is by isoelectric point. Note that expressed Asl-A exists as multiple differently charged species and that coexpression of active Plk4 causes an acidic shift for the majority of Asl-A.

FIGURE 2:

Asl-A phosphomutants are largely α-helical and self-oligomerize. (A) Abbreviated primary sequence of Asl-A indicating the 13 hydroxyl residues phosphorylated by Plk4 (bold) and mutated to nonphosphorylatable alanine (13A) or phosphomimetic aspartate/glutamate (13PM). Gray highlight indicates regions of predicted coiled-coil. (B) In the absence of endogenous Asl, expressed Asl-A-13A restores Plk4 self-destruction, whereas Asl-A-13PM has no effect on Plk4 protein levels. S2 cells were treated with Asl-C dsRNA for 6 d to deplete endogenous Asl without affecting transgenic Asl-A. On day 4, cells were transfected with inducible Plk4-GFP alone or with the indicated Asl-A-V5 construct and then induced to express the next day for 24 h. Immunoblots of lysates were probed with anti-GFP, V5, Asl, and α-tubulin (loading control). The graph shows the relative amounts of Plk4-GFP as determined by densitometry of the anti-GFP immunoblots, normalized to α-tubulin, and plotted relative to control (Cntrl). Error bars, SEM. n = 4 experiments. In all figures, asterisks indicate significance and error bars show SEM. (C) Asl-A mutants exhibit no significant differences of secondary structure. Circular dichroism analysis of Asl-A mutants suggests that Asl-A retains its α-helical structure even after phosphomimetic and nonphospho mutations are introduced. (D) Asl-A-WT, 13A, and 13PM constructs analyzed using SEC-MALS. Elution time from the Superdex 200 column is indicated on the x-axis in minutes. Normalized refractive index is indicated on the left y-axis (gray trace) and molecular mass is indicated on the right y-axis (black trace, kDa). Asl-A-WT elutes just after 20 min with an experimentally determined average molecular mass of 85.2 kDa, indicative of a stable dimeric state (calculated formula weights for Asl-A oligomeric states are monomer: 41.5 kDa; dimer: 83.0 kDa; trimer: 123.5 kDa; tetramer: 166.0 kDa; theoretical oligomeric masses are indicated by horizontal dashed lines). Control runs using bovine serum albumin (BSA; unpublished data) revealed that the globular BSA dimer (132 kDa) eluted at 25 min, suggesting that the Asl-A dimer, which has a slightly smaller molecular mass but elutes earlier, likely adopts an extended conformation. Asl-A-13A and 13PM constructs elute earlier than the Asl-A-WT protein and show higher experimentally determined molecular masses (110.9 and 100.6 kDa, respectively), suggesting that these mutant dimers may transiently self-associate into higher molecular weight complexes during the course of the gel filtration run. The vertical dashed line indicates the position of the elution peak for Asl-A-WT. (E) Asl-A phosphomutants retain the ability to homodimerize. S2 cells were RNAi-treated for 6 d to deplete Asl. On day 4, cells were cotransfected with the indicated GFP-tagged and V5-tagged Asl-A constructs and then induced to express the next day for 24 h. Samples were prepared by anti-GFP immunoprecipitation from cell lysates. Immunoblots were probed for GFP, V5, and α-tubulin.

FIGURE 3:

The phosphorylation state of Asl-A controls Plk4 activity. (A) Asl-A-13A enhances Slimb binding to Plk4, whereas Asl-A-13PM diminishes this interaction. Samples of anti-GFP immunoprecipitates were prepared from lysates of S2 cells treated as described in Figure 2B. Immunoblots were probed for GFP, V5, Slimb, and α-tubulin. (B, C) Graphs show relative amounts of Asl-A-V5 (B) or Slimb (C) bound to Plk4-GFP. Values were measured by densitometry of anti-V5 or anti-Slimb immunoblots of IP samples, normalized to the respective Plk4-GFP band intensity, and plotted relative to WT control. n = 3 experiments. ns, not significantly different. (D) Asl-A mutants modulate Plk4 autophosphorylation state. Samples of anti-GFP immunoprecipitates were prepared from lysates of S2 cells treated as described in Figure 2B except, in this experiment, cells were cotransfected with inducible Plk4-myc. Immunoblots were probed for GFP, myc, V5, and α-tubulin. Dashed lines mark Plk4-myc with different electrophoretic mobilities, indicating changes in phosphorylation state. (E) Asl-A-13PM suppresses Plk4-dependent Ana2 phosphorylation. Anti-GFP immunoprecipitates were prepared from lysates of S2 cells expressing GFP-Ana2, nondegradable Plk4-ND-myc, and the indicated Asl-V5 construct. Samples of anti-GFP immunoprecipitates were prepared from lysates of S2 cells treated as described in Figure 2B. Immunoblots were probed with anti-GFP to detect total GFP-Ana2 levels and with anti-pS318 to detect phospho-Ana2.

FIGURE 4:

Nonphospho Asl-A (13A) stimulates Plk4 activity in vitro. (A) Purified Plk4-Flag-His₆ was mixed with equimolar amounts of Asl-A and incubated with γ³²P-ATP. Reactions were sampled at 5, 30, and 60 min, and the samples resolved by SDS–PAGE. Top, Coomassie-stained gel; bottom, corresponding autoradiogram. Arrows indicate Plk4 bands presumably differing by phosphorylation state; the asterisk marks a contaminating band from an added reagent (a protease inhibitor) that migrates between the Plk4 bands. (B) Graphical representation of Plk4 autophosphorylation over time. y-Axis values are measurements of radioactivity in the Plk4 bands for each sample, expressed as the fold increase of a sample measurement over the Plk4 radioactivity present in the control (Plk4-only) at the initial (5 min) time point. n = 3 except Asl-A-13PM where n = 2. SEMs smaller than the radii of the graphing points are not plotted. (C) Asl-A-13A significantly increases Plk4 activity. The average values for Plk4 phosphorylation at the last time point (60 min) from the in vitro kinase assays shown in B are graphed. ns, not significant.

FIGURE 5:

Asl-A phosphomutants control kinase activity by manipulating Plk4 autoinhibition. (A) Asl-A-13PM expression causes centriole loss. The indicated Asl-GFP constructs were transfected into S2 cells and then induced the next day to express for 72 h. Cells were immunostained for PLP to mark centrioles. Significant centriole loss (less than two centrioles) occurs in cells expressing Asl-A-13PM. In contrast, centrioles are amplified (more than two centrioles) in cells expressing Asl-C, and centrioles are similarly amplified in cells coexpressing Asl-C and Asl-A-13PM. n = 3 experiments per construct (total 300 cells/construct). (B) Coexpressed Asl-A-13PM not only blocks the centriole amplification induced by Plk4 expression but causes significant centriole loss. Plk4-GFP/Asl-A-V5 dual-gene expression plasmids were transfected into S2 cells, and then were induced the next day to express for 72 h. Cells were immunostained for PLP to mark centrioles, and centriole numbers measured. n = 3 experiments per construct (total 300 cells/construct). (C) Regulation of Plk4 by Asl-A mutants occurs independently of Ana2. S2 cells were codepleted of endogenous Asl and Ana2 for 7 d. On day 5, Plk4-GFP and the indicated Asl-A-V5 constructs were cotransfected into cells and expression was induced the next day. Immunoblots of cell lysates were probed for Ana2, GFP, V5, and α-tubulin. (D) Asl-A-WT and Asl-A-13A can activate an autoinhibited Plk4-ΔPB3 mutant. S2 cells were transfected with Plk4-ΔPB3-GFP/Asl-A-V5 dual-gene expression plasmids, and samples were prepared as in B. n = 3 experiments per construct (total 300 cells/construct). (E) A Plk4 mutant incapable of autoinhibition is inhibited by Asl-A-13PM. S2 cells were transfected with Plk4-ΔL1-GFP/Asl-A-V5 dual-gene expression plasmids, and samples were prepared as in B. n = 3 experiments per construct (total 300 cells/construct).

FIGURE 6:

Plk4 phosphorylates its kinase domain and Asl-A, generating a state that inhibits kinase activity. (A) Atomic structure of the human Plk4 kinase domain (Wong et al., 2015). The residue, A226, is the human equivalent of Drosophila S228 within the C-loop. Human Plk4 autophosphorylates C-loop residue S232 in vitro (Sillibourne et al., 2010). (B) Asl-A-13PM does not inhibit Plk4-S228A from amplifying centrioles. Plk4-GFP/Asl-A-V5 dual-gene expression plasmids were transfected into cells and the next day were induced to express for 72 h. Centrioles were visualized with PLP immunostaining, and the number of centrioles per cell was counted. n = 3 experiments per construct (total 300 cells/construct). (C) Model: the phosphorylation state of Asl-A regulates Plk4 kinase activity by a multistep process of stimulation followed by negative feedback. 1, Initially, Plk4 is autoinhibited by its L1 domain, which masks its activation loop. 2, Nonphosphorylated Asl-A binds Plk4 and relieves autoinhibition to activate the kinase. 3, Homodimerized Plk4 phosphorylates itself (including its activation and C-loops) and, importantly, Asl-A. 4, Together, phospho-Asl-A and the phospho-C-loop recruit an unknown factor(s) to inactivate Plk4, perhaps by generating a complex that obstructs or distorts the active site.