The circadian E3 ligase complex SCF^FBXL3+CRY targets TLK2

We recently demonstrated that the circadian clock component CRY2 is an essential cofactor in the SCF^FBXL3-mediated ubiquitination of c-MYC. Because our demonstration that CRY2 recruits phosphorylated substrates to SCF^FBXL3 was unexpected, we investigated the scope of this role by searching for additional substrates of FBXL3 that require CRY1 or CRY2 as cofactors. Here, we describe an affinity purification mass spectrometry (APMS) screen through which we identified more than one hundred potential substrates of SCF^FBXL3+CRY1/2, including the cell cycle regulated Tousled-like kinase, TLK2. Both CRY1 and CRY2 recruit TLK2 to SCF^FBXL3, and TLK2 kinase activity is required for this interaction. Overexpression or genetic deletion of CRY1 and/or CRY2 decreases or enhances TLK2 protein abundance, respectively. These findings reinforce the idea that CRYs function as co-factors for SCF^FBXL3, provide a resource of potential substrates, and establish a molecular connection between the circadian and cell cycle oscillators via CRY-modulated turnover of TLK2.