Figure 4: Phosphorylation of FZR1 by the CDK4-cyclin D kinase.

(a) Autoradiogram following SDS–PAGE of in vitro kinase assays. FLAG-tagged C. elegans CDK-4 (WT) or KD CDK-4 (KD) were immunopurified and incubated with ³²P-γ-ATP, and the purified GST protein alone, or GST-fused to either the LIN-35 pocket region, FZR-1 N terminus or FZR-1 C terminus. (b) Same as in a, but kinase assays were performed with FLAG-tagged human CDK4 (WT) or KD CDK4 (KD), in the presence of GST alone or GST-fused to the human FZR1 N terminus. The KD control showed some kinase signal in multiple experiments, probably through contaminating kinases. In the +PD lane, 0.9 μM of the CDK4/6 inhibitor PD-0332991 was added to the reaction with CDK4 (WT). Last lane (C) shows a negative control kinase assay, using anti-FLAG IP from cells transfected with empty vector (c). Illustration of the C. elegans LIN-35 protein showing the location of the two CDK-4 phosphorylated residues (P) in the spacer region between the pocket A and B domains. (d) Illustration of the C. elegans FZR-1 protein, indicating the positions of CDK-4 phosphorylated residues (P) and protein domains. The asterisk marks the he121 mutation. The C-box is located at a.a. 59–65, the 7 WD40 domains start at a.a. 388, the final two a.a. form the IR motif. (e) Illustration of the H. sapiens FZR1 protein, the positions of the CDK-4 phosphorylated residues (P) and protein domains are indicated. C- box: a.a. 43–55, the 7 WD40 domains start at a.a. 182, the final two a.a. form the IR motif. All kinase assays and mass spectrometric analysis of the C. elegans proteins were performed twice, mass spectrometry of human FZR1 once.