Casein kinase I (CK1) alpha (CK1alpha, official symbol CSNK1A1) phosphorylates GST-tagged recombinant human CDC25A on serine residues S79 and S82, with S79 serving as a priming site for S82 phosphorylation (Honaker and Piwnica-Worms 2010). Phosphorylation of CDC25A at S79 by CSNK1A1 is dependent on prior S76 phosphorylation but not on S88 phosphorylation (Honaker and Piwnica-Worms 2010). CSNK1A1-mediated phosphorylation of CDC25A on S82 is dependent on phosphorylation of S76 and S79 but not S88 (Honaker and Piwnica-Worms 2010). The study by Piao et al. 2011 confirmed that CHEK1-mediated phosphorylation of CDC25A at S76 is needed for CSNK1A1- or CSNK1E-mediated phosphorylation of CDC25A on S82. Enhanced phosphorylation of CDC25A on S82 but not S76, T80, S88 or S124 is observed in cells overproducing CSNK1A1 and reduced in cells depleted of CSNK1A1 or treated with CK1 inhibitors but not CK2 or CaMKII inhibitors (Honaker and Piwnica-Worms 2010). CK1 regulates CDC25A phosphorylation at S82 both in normal cycling cells and in cells experiencing genotoxic stress such as ionizing radiation (IR) (Honaker and Piwnica-Worms 2010). CK1epsilon (CSNK1E) kinase domain, as well as CSNK1A1 kinase domain, can directly phosphorylate full-length human CDC25A on S82, with CSNK1E kinase domain phosphorylating CDC25A at S82 more robustly than the kinase domain of CSNK1A1 (Piao et al. 2011). Endogenous CSNK1A1 and CSNK1E both co-immunoprecipitate with CDC25A (Piao et al. 2011).

A fusion protein between human CDC25A and firefly luciferase, CDC25A-FLuc, that enables direct real-time monitoring of CDC25A protein levels in cells, is stabilized in cells treated with CSNK1A1 siRNA or potent and selective CSKN1A1 inhibitor D4476, but not with inhibitor IC261, which shows higher selectivity for CK1delta (CSNK1D) and CK1epsilon (CSNK1E) (Honaker and Piwnica-Worms 2010). This contradicts the findings by Jin et al. 2003, which showed that CK1alpha or CK1 inhibition by CK1-7 and IC261 did not have a significant effect on CDC25A stability. D4476 inhibitor is more selective and potent against CK1alpha than CK1-7 and IC261 (Honaker and Piwnica-Worms 2010). In human embryonic kidney cell line HEK293, siRNA-mediated knockdown of CSNK1E dramatically increases CDC25A protein level while siRNA-mediated knockdown of CSNK1A1 increases CDC25A protein level moderately (Piao et al. 2011). In the human colon carcinoma cell line HCT116, however, CDC25A protein levels increase only upon siRNA-mediated knockdown of CSNK1A1 but not CSNK1E, suggesting that roles of CSNK1A1 and CSNK1E in regulating CDC25A levels are likely cell type-specific (Piao et al. 2011).

Knockdown of CSNK1A1 by siRNA leads to a 2-fold increase in the level of endogenous CDC25A protein level, including prolonged CDC25A half-life, while no CDC25A protein stabilization is observed upon siRNA-mediated knockdown of CSNK1D, CSNK1E or CSNK1G1 (Honaker and Piwnica-Worms 2010).

Cells overexpressing CSNK1A1 show increased association of BTRC subunit of the SCF-BTrCP complex with CDC25A (Honaker and Piwnica-Worms 2010). Point mutants of CDC25A, CDC25A S76A, CDC25A S79A, and CDC25A S82A, but not CDC25A T80A or CDC25A S88A, are severely impaired in their ability to bind to BTRC and exhibit an extended half-life (Honaker and Piwnica-Worms 2010). Another study showed that CDC25A phosphorylated by either CSNK1A1 or CSNK1E shows increased binding to BTRC (Piao et al. 2011). Knockdown of CSNK1E but not CSNK1A1 by siRNA in HEK293 cells reduces ubiquitination and increases half-life of CDC25A, but less so than the proteasome inhibition, suggesting that other kinases also contribute to CDC25A downregulation in HEK293 cells (Piao et al. 2011).