Here, we identified centrosomal P4

Here, we identified centrosomal P4.1-associated protein (CPAP), a human homologue of SAS-4, as a substrate of PLK2 whose activity oscillates during the cell cycle. cells named CEP192, centrosomal P4.1-associated protein (CPAP), and HsSAS-6, respectively (Nigg, 2007). Second, CEP192, CPAP, and HsSAS-6 are required for centriole duplication in human cells (Leidel et al, 2005; Zhu et al, 2008; Kohlmaier et al, 2009). There are no obvious homologues of ZYG-1 Amoxapine in the human genome, but PLK4 is speculated to be its functional homologue for the initiation of centriole duplication (Bettencourt-Dias et al, 2005; Kleylein-Sohn et al, 2007; Nigg, 2007). CPAP was initially identified as an interacting partner of Protein 4.1 R-135, and was reported to serve as a transcriptional coactivator that enhances both Stat-5-mediated and TNF–induced NF-B-mediated transcriptional activity (Peng et al, 2002; Koyanagi et al, 2005). However, CPAP is best known for maintaining centrosome integrity and normal spindle morphology during cell division (Cho et al, 2006). A study using immunodepletion suggested that CPAP may regulate microtubule nucleation at the centrosomes (Hung et al, 2000). Mutations of the gene are linked to autosomal recessive primary microcephaly, suggesting that CPAP is involved in the cell division of neuronal precursors to produce the proper number of neurons during brain development (Bond et al, 2005). As a human orthologue of SAS-4, the importance of CPAP in procentriole assembly is evident (Kleylein-Sohn et al, 2007; Kohlmaier et al, 2009; Tang et al, 2009). Overexpression of CPAP induces centriole elongation, suggesting that CPAP has a function in tethering microtubules for procentriole formation (Kohlmaier et al, 2009; Schmidt et al, 2009; Tang et al, 2009). However, it is still poorly understood how the biological activity of CPAP is regulated during procentriole assembly. In addition to PLK4, several human protein kinases are critical for centriole duplication and assembly (Habedanck et al, 2005; Kleylein-Sohn et al, 2007). For example, CDK2 is necessary for the initiation of centrosome duplication (Hinchcliffe and Sluder, 2002). A few candidate substrates have been identified as being necessary for the function of CDK2 in centrosome duplication. For example, CDK2 phosphorylates nucleophosmin/B23, MPS1, and CP110 (Okuda et al, 2000; Fisk and Winey, 2001; Chen et al, 2002). Phosphorylation of nucleophosmin/B23 results in its dissociation from the centrosome before the initiation of centrosome duplication (Okuda et al, 2000). MPS1 and NDR kinases are centrosomal kinases that are critical for centrosome duplication (Fisk et al, 2003; Hergovich et al, 2007). PLK2 is also crucial for centriole assembly, as knockdown of PLK2 results in defects in centriole duplication (Warnke et al, 2004). In this study, we tested the hypothesis in which the biological activities of centriole assembly components are controlled by protein kinases, whose activities oscillate during the cell cycle. In this manner, centriole assembly is coupled to the progression of the cell cycle. We report that PLK2 phosphorylates CPAP and controls its biological activity for Amoxapine Amoxapine centriole elongation. Results CPAP is phosphorylated by PLK2 in vitro To explore the function of PLK2 in centriole assembly, we carried out kinase assays with selected centrosomal proteins and identified CPAP as a specific substrate. Subsequent kinase assays with truncated forms of GST-CPAP narrowed down the PLK2 phosphorylation site(s) to within 563C613 residues (Figure 1A; Supplementary Figure S1ACD). To pinpoint the PLK2 phosphorylation Amoxapine site(s), we prepared point mutant proteins of GST-CPAP563C613 in which each serine or threonine was substituted to alanine. The phosphorylated form of wild-type GST-CPAP563C613 produced two radioactive bands of different sizes, suggesting that there are at least two phosphorylation sites in CPAP563C613 (Figure 1B). All GST-CPAP563C613 point mutants, except the S589A and S595A mutants, had two bands (Figure 1B). In fact, the S589A mutant showed only a lower band, whereas the S595A mutant showed only an upper band (Figure 1B and C). Furthermore, the double mutant (CPAPSSAA), in which both serine residues at positions 589 and 595 were substituted with alanine residues, was not phosphorylated (Figure 1C). We confirmed that full-length PLK2 didn’t phosphorylate GST-CPAP563C613 also,SSAA (Supplementary Rabbit polyclonal to ANKRD1 Amount S1E). These total results.