Abstract
The protein TIN2 is a member of telomere-binding protein complex that serves to cap and protect mammalian chromosome ends. As a number of proteins in this complex are phosphorylated in a cell cycle-dependent manner, we investigated whether TIN2 is modified by phosphorylation as well. We performed phospho-proteomic analysis of human TIN2, and identified two phosphorylated residues, serines 295 and 330. We demonstrated that both these sites were phosphorylated during mitosis in human cells, as detected by Phos-tag reagent and phosphorylation-specific antibodies. Phosphorylation of serines 295 and 330 appeared to be mediated, at least in part, by the mitotic kinase RSK2. Specifically, phosphorylation of TIN2 at both these residues was increased upon expression of RSK2 and reduced by an inhibitor of the RSK family of kinases. Moreover, RSK2 phosphorylated TIN2 in vitro. The identification of these specifically timed post-translational events during the cell cycle suggests a potential mitotic regulation of TIN2 by phosphorylation.
Highlights
The telomere-associated protein 1 interacting nuclear factor 2 (TIN2, TINF2, DKCA3) is a 40kDa protein integral to telomere function
TRF1 is phosphorylated at mitosis to allow sister telomere resolution [13], and the phosphorylation of TPP1 during G2/M phase is related to higher telomerase activity [14]
Phosphorylation of TIN2 on S295/T297 and/or S330 were detected in unbiased whole phospho-proteome analysis of HeLa cells throughout the cell cycle [17], HeLa cells treated with the drug rapamycin [18], and in nocodazole-arrested HeLa cells [19]
Summary
The telomere-associated protein 1 interacting nuclear factor 2 (TIN2, TINF2, DKCA3) is a 40kDa protein integral to telomere function. TIN2 is found in a complex with TRF1 and cohesins [9], the latter of which are functionally regulated by phosphorylation during mitosis [10,11,12]. TRF1 is phosphorylated at mitosis to allow sister telomere resolution [13], and the phosphorylation of TPP1 during G2/M phase is related to higher telomerase activity [14]. Given these observations, we explored the possibility that TIN2 may be phosphorylated, perhaps at mitosis
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