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. 2021 Nov 25;10:e68473. doi: 10.7554/eLife.68473

Figure 2. Phosphorylation of Thr143 and Thr149 in CycT1 contributes to its binding to CDK9.

(A) Threonine phosphorylation is detected in the full-length CycT1 protein. CycT1 was expressed in 293T cells untreated or treated with 5 nM or 1 μM okadaic acid, or 150 nM calyculin A (+/- signs on top). Co-IPs with CDK9 were then probed with anti-HA and anti-CDK9 antibodies in panels 1 and 2, with anti-phospho-threonine (pThr) antibodies in panels 3 and 4. Panels 5 and 6 contain input levels of CycT1 and CDK9 proteins. (B) Threonine phosphorylation is detected in CycT1(280). CycT1(280) protein was expressed in 293T cells untreated or treated with 5 nM or 1 μM okadaic acid, or 150 nM calyculin A (+/- signs on top). Co-IPs with CDK9 were then probed with anti-HA and anti-CDK9 antibodies in panels 1 and 2, with anti-pThr antibodies in panels 3 and 4. Panels 5 and 6 contain input levels of CycT1 and CDK9 proteins. (C) Thr143 and Thr149 are major phospho-threonine residues in CycT1(280). WT CycT1(280) or mutant CycT1(280)TT143149AA proteins were expressed in the presence of bortezomib and 1 μM okadaic acid in 293T cells. IPs with CycT1 were then probed with anti-pThr and anti-HA antibodies in panels 1 and 2. Panel 3 contains input levels of CycT1 proteins. (D) Thr143 and Thr149 are major phosphorylated residues in CycT1(192). WT CycT1(192) or mutant CycT1(192)TT143149AA proteins were expressed in the presence of bortezomib and/or 1 μM okadaic acid (+/- signs on top) in 293T cells. After IPs with anti-HA antibodies, IPed samples were subjected to SDS-PAGE, then phosphorylated proteins were detected by in-gel Phospho-Tag staining, with unphosphorylated BSA protein as the negative control. (E) Direct detection of Thr143/Thr149 phosphorylation by phosphopeptide mapping analysis. WT CycT1(192) or mutant CycT1(192)TT143149AA proteins were expressed in the presence of bortezomib and/or 1 μM okadaic acid (+/- signs on top) in 293T cells. After IP with anti-HA antibodies. IPed samples were digested by trypsin and subjected to SDS-PAGE, followed by silver staining (left panel) in-gel Phospho-Tag staining (right panel), using phosphorylated β-casein protein as the positive control and unphosphorylated BSA protein as the negative control.

Figure 2.

Figure 2—figure supplement 1. Thr143 and Thr149 are main phosphorylation sites in CycT1.

Figure 2—figure supplement 1.

(A) Thr143 and Thr149 are predicted to be potential phosphorylation sites. Potential phosphorylation sites between 124 aa. to 166 aa. in CycT1 were analyzed by the NetPhos 3.1 program. Threshold was set to 0.5 (default value), indicated by the pink line. Thr143 and Thr149 scored highest as potential phosphorylation sites. (B) Levels of phospho-tyrosine and phospho-serine residues are similar in WT CycT1(280) and mutant CycT1(280)TT143149AA proteins. WT CycT1(280) or mutant CycT1(280)TT143149AA proteins were expressed in the presence of bortezomib and okadaic acid in 293T cells, and IPed with anti-HA antibodies. IPed CycT1 proteins were then probed with anti-phospho-tyrosine (pTyr) and anti-phospho-serine (pSer) antibodies in panels 1 and 2. (C) Phosphorylation of WT CycT1(192) and mutant CycT1(192)TT143149AA proteins is also induced by high concentration of okadaic acid treatment. WT CycT1(192) or mutant CycT1(192)TT143149AA proteins were expressed in the absence or presence of okadaic acid and bortezomib (+/- signs on top) in 293T cells, and IPed with anti-HA antibodies, followed by western blotting (WB) with anti-HA antibodies by WB.