Plk4 phosphorylation induces a band-shift in Ana2 from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S1. Plk4 phosphorylation induces a shift in the electrophoretic mobility of Ana2. A. Incubation of Ana2 synthesized by coupled in vitro transcription and translation (IVTT) with active MBP-Plk4 in the presence of increasing ATP induces a distinct band-shift. B. The shift in mobility of Ana2-FLAG protein following its co-overexpression with active non-degradable Plk4 (Plk4ND) is abolished by treating the extract with λ-Phosphatase. C. Alanine substitutions in mass-spectrometry-identified phospho-sites with highest spectral count following in vitro phosphorylation, including those of the STAN motif, still retain the band-shift. D: Phosphorylation sites identified in Ana2 by mass-spectrometry. Serine or threonine residues identified following phosphorylation by MBP-Plk4 in vitro are highlighted in blue. Sites identified as phosphorylated in vivo on tagged-Ana2 (Protein A, FLAG, or GFP tags) purified from D.Mel-2 cells or early Drosophila embryos are highlighted in yellow. The STAN-motif is highlighted in grey. E. The phosphorylation-site responsible for the band shift is located in the N-terminal 280 amino acids of Ana2. Ana21-280, but not Ana2281-420 displays the band-shift in both the co-overexpression assay (upper) and the in vitro phosphorylation assay (lower). F. Band-shift assay using IVTT product of WT and non-phosphorylatable mutants of Ana2 for the sites highlighted in yellow within the N-terminal 280 amino-acid part. The band-shift is seen for each of these mutants

List of site-directed mutagenesis primers from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

List of site-directed mutagenesis primer

Expression levels in Ana2-Myc cell lines from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S4: Expression levels in Ana2-Myc cell lines: An immuno-blot showing comparable levels of expression between an Ana2-WT-Myc and an Ana2-S38A-Myc cell line. The same three cell lines are presented as in Fig. 6B: Untransfected D.Mel-2 cells (lane 1), pAct5-Ana2-WT-Myc (lane 2) and pAct5-Ana2-S38A-Myc (lane 3). Top panel: anti-Asl immuno-blot as a loading control. Bottom panel: anti-Ana2 immuno-blot, revealing endogenous Ana2 and overexpressed Ana2-Myc

GST-Ana2-S38A can bind Sas6 after Plk4 phosphorylation from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S3: GST-Ana2-S38A can bind Sas6 after Plk4 phosphorylation. Sas6 specifically interacts with both Ana2-WT and Ana2-S38A when the proteins are pre-phosphorylated by Plk4. GST, GST-tagged wild-type (GST-Ana2-WT) or the S38A substitution mutant (GST-Ana2-S38A) were treated with either MBP-Plk4 or MBP-Plk4KD and incubated in vitro with 35S-Met-labeled Sas6 produced in a coupled in vitro transcription/translation reaction. The resulting complex was analyzed by SDS-PAGE and autoradiography

Serine-38 of Ana2 is phosphorylated in vitro by Plk4 as identified by mass-spectrometry. from Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation

Supplementary Figure S2. Serine-38 of Ana2 is phosphorylated in vitro by Plk4 as identified by mass-spectrometry. Phospho-Mascot-interpreted fragmentation spectra of the peptide 13-LAPRP…EV (phospho-S-38) ILFG…SPR-65 with phosphorylated serine at the position 38 (highlighted red). Labelled peaks correspond to fragment ions. Despite the relatively long peptide, the fragmentation pattern unambiguously identifies the phosphorylation site