Figure 1.
Plk4 activity enhances the interaction between Ana2 and Sas4, producing a hyperphosphorylated Ana2 species. (A) Linear map of Ana2 depicting functional domains and Plk4 phospho-sites. Ana2 is phosphorylated by Plk4 in an ordered pattern, first on NT residues T33/S38 (red) followed by STAN domain residues S318/S370/S373/S395 (green), and then central residues S63/T69/S150/T159/T242 (purple; McLamarrah et al., 2018). Sas4-binding domain (BD; Cottee et al., 2013), LC8 binding-sites (black boxes; Slevin et al., 2014), coiled coil (CC), and STAN domains are shown. (B–D) Ana2 binds Cep135 (B), Asl (C), and Sas4 (D), but coexpression of catalytically active ND Plk4 enhances only the association with Sas4. S2 cells were cotransfected with the indicated constructs and the next day induced to express for 24 h by the addition of 1 mM CuSO4. Anti-GFP IPs were then prepared from lysates, and Western blots of the inputs and 5% of the IPs probed for GFP, V5, myc, and α-tubulin. Note Ana2 presents as three different phospho-species (red lines in all figures) indicated as lower (nonphospho), middle (phospho), and upper (hyperphospho) species. Coexpression of active Plk4 (but not KD) and Sas4 generates a hyperphospho-Ana2 species (D). (E) Graphs show relative Ana2 intensities from IPs like D, lanes 3–5. Left: Ana2 intensities within a region of interest containing all Ana2 species were measured and normalized to the treatment lacking Plk4 expression. Right: Graph shows ratio of the upper (hyperphosphorylated) to the middle and lower Ana2 species for the three indicated treatments. Asterisks mark significant differences between treatments: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars, SEM; n = 3 (left panel) and 6 (right panel) experiments. (F) Coexpression of active Plk4 and Sas4 generates a hyperphosphorylated form of Ana2. Anti-GFP IPs were prepared from cell lysates expressing the indicated proteins and then were either mock-treated (lanes 1 and 2) or incubated with λ-phosphatase (λ-Ppase; lane 3) or λ-phosphatase plus phosphatase inhibitor cocktail (Phos. Inh; lane 4).

Plk4 activity enhances the interaction between Ana2 and Sas4, producing a hyperphosphorylated Ana2 species. (A) Linear map of Ana2 depicting functional domains and Plk4 phospho-sites. Ana2 is phosphorylated by Plk4 in an ordered pattern, first on NT residues T33/S38 (red) followed by STAN domain residues S318/S370/S373/S395 (green), and then central residues S63/T69/S150/T159/T242 (purple; McLamarrah et al., 2018). Sas4-binding domain (BD; Cottee et al., 2013), LC8 binding-sites (black boxes; Slevin et al., 2014), coiled coil (CC), and STAN domains are shown. (B–D) Ana2 binds Cep135 (B), Asl (C), and Sas4 (D), but coexpression of catalytically active ND Plk4 enhances only the association with Sas4. S2 cells were cotransfected with the indicated constructs and the next day induced to express for 24 h by the addition of 1 mM CuSO4. Anti-GFP IPs were then prepared from lysates, and Western blots of the inputs and 5% of the IPs probed for GFP, V5, myc, and α-tubulin. Note Ana2 presents as three different phospho-species (red lines in all figures) indicated as lower (nonphospho), middle (phospho), and upper (hyperphospho) species. Coexpression of active Plk4 (but not KD) and Sas4 generates a hyperphospho-Ana2 species (D). (E) Graphs show relative Ana2 intensities from IPs like D, lanes 3–5. Left: Ana2 intensities within a region of interest containing all Ana2 species were measured and normalized to the treatment lacking Plk4 expression. Right: Graph shows ratio of the upper (hyperphosphorylated) to the middle and lower Ana2 species for the three indicated treatments. Asterisks mark significant differences between treatments: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars, SEM; n = 3 (left panel) and 6 (right panel) experiments. (F) Coexpression of active Plk4 and Sas4 generates a hyperphosphorylated form of Ana2. Anti-GFP IPs were prepared from cell lysates expressing the indicated proteins and then were either mock-treated (lanes 1 and 2) or incubated with λ-phosphatase (λ-Ppase; lane 3) or λ-phosphatase plus phosphatase inhibitor cocktail (Phos. Inh; lane 4).

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