Abstract
Cyclin-dependent kinase subunit (CKS) proteins interact with cyclin-dependent kinases (CDKs) with high affinity. Mammalian CKS1 and CKS2 bind CDK1 and CDK2 and partake in the control of cell cycle progression. We identified CKS-interacting proteins by affinity purification followed by mass spectrometry in the human lymphocytic cell line Ramos. Apart from known interactors, such as CDKs, we identified a novel CDK-dependent interaction between CKS proteins and the mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB bound both CKS1 and CKS2 and underwent CDK-dependent phosphorylation. mtSSB is known to participate in replication of mitochondrial DNA. We demonstrated that mitochondrial morphology and DNA integrity were compromised in cells depleted of both CKS proteins or that had inhibited CDK activity. These features are consistent with the hypothesis of CKS-dependent regulation of mtSSB function and support a direct role of cell cycle proteins in controlling mitochondrial DNA replication.
Highlights
Cyclin-dependent kinase subunit (CKS) proteins interact with cyclin-dependent kinases (CDKs) with high affinity
In agreement with published work [22, 23], CKS1 and CKS2 co-purified with CDK1, CDK2, and their respective activating cyclins, cyclin B and cyclin A (Table I)
Expression of mitochondrial singlestranded DNA-binding protein (mtSSB) is strictly regulated, and mtDNA content is directly proportional to the abundance of mtSSB mRNA [26]. We examined both the protein levels and expression of mtSSB mRNA to determine whether Cks knockdown cells have reduced mtSSB
Summary
CKS1, CKS2, CKS1E63Q, and CKS2E63Q were attached to Sepharose beads and used as baits. BSA coupled to the beads was used as control. Interactor proteins were pulled down from lysates of Ramos cells and identified by use of LC-MS/MS. Proteins binding to control beads were subtracted from identified proteins binding to baits. Proteins identified in three of four biological replicates are listed for CKS1 and CKS2. Proteins identified in two out of four experiments for CKS1E63Q and CKS2E63Q are not shown. Bait proteins NCBI accession number Expectation value (protein) SEQUEST score Sequence coverage Unique peptides
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