The phosphorylation of TIF1 Ser473 was identified in HeLa and WEHI cells at interphase. Since Ser473 is located near the HP1 binding motif, an intriguing question arises what is the functional consequence, if any, of the phosphorylation of TIF1 Ser473 To elucidate the phosphorylation state of TIF1 Ser473 during cell cycle progression, Western blot ting but was performed with lysates from synchronized HeLa cells. Cells were synchronized to G1 S transition using Inhibitors,Modulators,Libraries double thymidine block. Phosphorylated TIF1 Ser473 was almost undetectable at the G1 S boundary under the thymidine block but the phospho rylation of TIF1 Ser473 was maximal Inhibitors,Modulators,Libraries 1 hour post release from the thymidine block, during early S phase. The level of phosphorylated TIF1 Ser473 decreased thereafter for 2 hours during the mid to late S phase cell cycle progression.
The level of total TIF1 remained relatively constant. The ele vated cyclin A and decreased cyclin B evidenced the G1 S to S cell cycle Inhibitors,Modulators,Libraries progression of thymidine release. HeLa cells were synchronized to prometaphase by noco dazole treatment. The level of phosphorylated TIF1 Ser473 was determined from HeLa extracts collected from 0 to 8 hours after nocodazole were removed. The phos anti phosphorylatedmonoclonal anti TIF1 antibodiesand rabbit phorylated TIF1 Ser473 level was highest during mitosis and decreased thereafter through G1 phase. The lower panel of Figure 2B shows the level of phosphorylated TIF1 Ser473 in relation to that of total TIF1 at each time point. The M phase marker cyclin B or phosphorylated histone H3S10 identify specific stages of mitotic release.
To further evaluate the Inhibitors,Modulators,Libraries phosphorylation state of TIF1 S473 from G1 to S phase, HeLa cells were serum starved for three days to arrest in G1 phase. The level of phospho rylated TIF1 Ser473 was almost non detectable Inhibitors,Modulators,Libraries in G1 phase, reached a maximum 2 hours after serum was added and declined thereafter. The gradual increase in the level of cyclin A or decrease in the level of cyclin B demonstrated the specific cell cycle stage from G1 to S. This dynamic, biphasic appearance of phos phorylated TIF1 Ser473 suggests that TIF1 may be involved in regulating the cell cycle by the switching on and off of Ser473 phosphorylation. Various cell cycle regulated proteins may exhibit changed activity when phosphorylation modulates their stability.
The total protein level of TIF1 remained approximately constant while the level of phosphorylated TIF1 Ser473 fluctuated throughout the cell cycle. To further demon strate this dynamic fluctuation of phosphorylated TIF1 Ser473 level, HeLa cells were immunostained using S473 antibody. The level of phosphorylated TIF1 Ser473 in the cells 2 hr after serum addition markedly exceeded that selleck chemicals llc in the serum starved cells. The pro tein level and distribution of TIF1 during G1 were the same as those of early S phase.