Chk1 phosphorylation regulates interactions with Cul4A and DDB1. A, asynchronously growing HeLa cells expressing the indicated tagged proteins were cultured in the presence of vehicle (DMSO), 50 μM wortmannin, 10 mM caffeine or 1 μM Gö6976 for 2 h each. Cells were then exposed to 20 mM HU and 10 μM MG132 for 4 h prior to harvest. Flag₃ Chk1 was immunoprecipitated and precipitates were analyzed by Western blotting. Relative levels of DDB1 and Cul4 are indicated (n = 3). B, asynchronously growing HeLa cells were treated with control RNAi or RNAi specific for PP2A, Cul4A/DDB1 or PP2A/Cul4A/DDB1 for 48 h. Lysates were prepared and immunoblotted with antibodies specific for the indicated antibodies (left panel). The data from 4 independent experiments is presented graphically in the right panel. The data is presented as mean +/− standard deviation. P-values from Student’s t-test are shown when significantly different from control. Asterisks indicate significant p-values (* < 0.05; **<0.01)

Chk1 is stabilized in cells deficient for Cul4 and DDB1. A, B, asynchronously growing HeLa cells were cultured in the presence of the indicated siRNAs for 48 h followed by the addition of 100 μg/ml of CHX. Lysates were prepared at the indicated times after CHX addition. Proteins were resolved by SDS-PAGE and analyzed by Western blotting. A representative Western blot is shown in panel A and quantitation from 3 independent experiments is shown graphically in panel B. Standard deviation is shown as error bars along the y axis. C, D, same as in panels A, B except that cells were cultured in the presence of both CHX and 20 mM HU 48 h after siRNA transfection (n = 3).

Proteomic screen identifies DDB1 as a novel Chk1 interacting protein. A, lysates prepared from HU-treated HEK293 cells transfected with control plasmid or plasmid encoding Flag₃Chk1 were incubated with Anti-Flag M2-Agarose. Washed precipitates were then incubated in lysates prepared from HU-treated HEK293 cells. Bound proteins were resolved by SDS-PAGE and stained with Colloidal Coomassie Blue. The band denoted by an asterisk (*) was present in Flag₃ Chk1 but not control precipitates. This protein was excised from the gel and identified as DDB1 by mass spectrometry. B, endogenous Chk1 was immunoprecipitated from control cells or cells treated with 20 mM HU for 6 h. Cells were also treated with 10 μM MG132 for 2 h prior to harvest. Precipitates were resolved by SDS-PAGE and analyzed by Western blotting. Densitometry was performed to determine relative amounts of DDB1 and Cul4A in Chk1 immunoprecipitates (n = 3). C, HeLa cells expressing the indicated tagged proteins were cultured in the presence of 20 mM HU and 50 μM MG132 for 4 h. Flag₃Chk1 was immunoprecipitated and precipitates were analyzed by Western blotting. Densitometry was performed to quantitate relative amounts of DDB1 and Cul4A in Chk1 immunoprecipitates (n = 2). Relative levels of DDB1 and Cul4A are indicated.

Chk1 ubiquitination by Cul4A/DDB1. A, asynchronously growing HeLa cells transfected with RNAi targeting luciferase (control, lane 1), Cul4A (lane 2) or DDB1 (lane 3) were cultured in the presence of HU for 1h. Lysates were prepared and resolved directly by SDS-PAGE (A) or were incubated with a Cul4A-specific antibody to precipitate the Cul4A-containing E3 ligase for ubiquitination assays (B). B, Cul4A immunocomplexes were incubated alone (lane 1), with Flag₃Chk1 precipitates (lane 2) or with Flag₃Chk1 precipitates and purified E1 and E2 (lanes 3, 4, 5). Flag₃Chk1 precipitates were also incubated with purified E1 and E2, in the absence of Cul4A immunocomplexes (lane 6). Ubiquitination assays were performed in vitro and reaction products were resolved by SDS-PAGE followed by Western blotting with antibodies specific for Chk1 (top panel) or Flag (bottom panel) (n = 4). C, D, Asynchronously growing HeLa cells were co-transfected with plasmids encoding Flag vector or Flag₃Chk1, HA-ubiquitin and RNAi for 48 h and then treated with HU (20 mM) and MG132 (10 μM) for 4 h. Cell lysates were prepared and analyzed directly by Western blotting (panel C) or flag-tagged Chk1 precipitates were isolated prior to SDS-PAGE and subjected to Western blotting with antibodies specific for HA (top panel) and Flag (bottom panel) (n = 3).

Knockdown of Cul4A/DDB1 impairs Chk1 degradation and alters Chk1 function. A, asynchronously growing HeLa cells were transfected with the indicated siRNAs for 48 h and then treated with SN38 (1 μM) for 6 h. Cells lysates were prepared and subjected to Western blotting with antibodies specific for the indicated proteins (left panel). Compiled data from 3 independent experiments is presented graphically in the right panel. B, asynchronously growing HeLa cells were transfected with the indicated siRNAs for 24 h and then incubated with DMSO or 200 nM geldanamycin (GA) for another 24 h. Cells lysates were prepared and subjected to Western blotting with antibodies specific for the indicated proteins (left panel). Compiled data from 5 independent experiments is presented graphically in the right panel. C, Asynchronously growing HeLa cells transfected with the indicated siRNAs for 24 h were treated as described in B and then exposed to 10 Gy IR. In some cases, cells were also incubated with DMSO (vehicle) or Gö6976 (300 nM) and harvested after 9 h. Cells lysates were prepared and subjected to Western blotting with antibodies specific for the indicated proteins (left panel). Compiled data is presented graphically in the right panel. Relative levels of phistone-H3 were determined from 3 independent experiments. All of the graphs are presented as mean +/− standard deviation. P-values from Student’s t-test are shown when significantly different from control (* < 0.05; **<0.01).