Figure 2.
Binding between Ana2 and the Sas4 G-box is required for Plk4-dependent phosphorylation of Ana2 and for enhanced Ana2-Sas4 association. (A) Linear maps of Sas4 constructs used in these experiments. PN2-3, tubulin-binding domain; MBD, microtubule-binding domain; CC, coiled-coil; L1, linker 1. The ABM harbors the indicated amino acid substitution (Bond et al., 2005; Cottee et al., 2013). (B) Sas4 G-box promotes hyperphosphorylation of Ana2 by Plk4. S2 cells were treated as described in Fig. 1 B. Anti-GFP IPs were then prepared from lysates, and Western blots of the inputs and IPs probed for GFP, V5, and myc. The hyperphosphorylated Ana2 isoform is clearly diminished in the presence of KD Plk4 or absence of the Sas4 G-box (Sas4-C). Note, G-box (lanes 9 and 10) expression is much higher than FL (lanes 3 and 4) Sas4 and thus binds proportionally more Ana2. (C) Graph shows the ratios of the band intensities of the upper (hyperphosphorylated) to the middle + lower Ana2 species for the indicated treatments (like lanes 3 vs. 4 and 9 vs. 10 in B). Asterisks mark significant differences between treatments: *, P < 0.05; **, P < 0.01. Error bars, SEM; n = 3 experiments. (D) Endogenous Ana2 does not coIP with Sas4 containing the ABM. S2 cells were depleted of endogenous Sas4 by targeting its UTR for 12 d. On days 4 and 8, cells were transfected with the indicated constructs and induced to express the next day for the duration of the experiment by the addition of 1 mM CuSO4. Anti-GFP IPs were then prepared from lysates, and Western blots of the inputs and IPs probed for GFP, Ana2, and α-tubulin. (E and F) An ABM in the Sas4 G-box disrupts hyperphosphorylation of transgenic (D) and endogenous (E) Ana2 by Plk4. S2 cells were depleted of Sas4 by RNAi targeting its UTR for 6 d. On day 4, cells were transfected with the indicated constructs and induced to express the next day for 24 h by the addition of 1mM CuSO4. Blots of cell lysates were probed with anti-GFP, V5, myc, Ana2, and α-tubulin. Note that hyperphosphorylated Ana2 is absent when endogenous Sas4 is replaced with Sas4-ABM. (G) Expression of Sas4-C (G-box) harboring the ABM prevents the electrophoretic shift in transgenic Ana2. S2 cells were treated as in A. Proteins were detected by immunoblotting cell lysates with anti-GFP, myc, V5, and α-tubulin.

Binding between Ana2 and the Sas4 G-box is required for Plk4-dependent phosphorylation of Ana2 and for enhanced Ana2-Sas4 association. (A) Linear maps of Sas4 constructs used in these experiments. PN2-3, tubulin-binding domain; MBD, microtubule-binding domain; CC, coiled-coil; L1, linker 1. The ABM harbors the indicated amino acid substitution (Bond et al., 2005; Cottee et al., 2013). (B) Sas4 G-box promotes hyperphosphorylation of Ana2 by Plk4. S2 cells were treated as described in Fig. 1 B. Anti-GFP IPs were then prepared from lysates, and Western blots of the inputs and IPs probed for GFP, V5, and myc. The hyperphosphorylated Ana2 isoform is clearly diminished in the presence of KD Plk4 or absence of the Sas4 G-box (Sas4-C). Note, G-box (lanes 9 and 10) expression is much higher than FL (lanes 3 and 4) Sas4 and thus binds proportionally more Ana2. (C) Graph shows the ratios of the band intensities of the upper (hyperphosphorylated) to the middle + lower Ana2 species for the indicated treatments (like lanes 3 vs. 4 and 9 vs. 10 in B). Asterisks mark significant differences between treatments: *, P < 0.05; **, P < 0.01. Error bars, SEM; n = 3 experiments. (D) Endogenous Ana2 does not coIP with Sas4 containing the ABM. S2 cells were depleted of endogenous Sas4 by targeting its UTR for 12 d. On days 4 and 8, cells were transfected with the indicated constructs and induced to express the next day for the duration of the experiment by the addition of 1 mM CuSO4. Anti-GFP IPs were then prepared from lysates, and Western blots of the inputs and IPs probed for GFP, Ana2, and α-tubulin. (E and F) An ABM in the Sas4 G-box disrupts hyperphosphorylation of transgenic (D) and endogenous (E) Ana2 by Plk4. S2 cells were depleted of Sas4 by RNAi targeting its UTR for 6 d. On day 4, cells were transfected with the indicated constructs and induced to express the next day for 24 h by the addition of 1mM CuSO4. Blots of cell lysates were probed with anti-GFP, V5, myc, Ana2, and α-tubulin. Note that hyperphosphorylated Ana2 is absent when endogenous Sas4 is replaced with Sas4-ABM. (G) Expression of Sas4-C (G-box) harboring the ABM prevents the electrophoretic shift in transgenic Ana2. S2 cells were treated as in A. Proteins were detected by immunoblotting cell lysates with anti-GFP, myc, V5, and α-tubulin.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal